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Conformational Regulation of Integrin Structure and Function
Abstract
Integrins are a structurally elaborate family of heterodimers that mediate divalent cation-dependent cell adhesion in a wide range of biological contexts. The inserted (I) domain binds ligand in the subset of integrins in which it is present. Its structure has been determined in two alternative conformations, termed open and closed. In striking similarity to signaling G proteins, rearrangement of a Mg(2+)-binding site is linked to large conformational movements in distant backbone regions. Mutations have been used to stabilize either the closed or open structures. These show that the snapshots of the open conformation seen only in the presence of a ligand or a ligand mimetic represent a high-affinity, ligand-binding conformation, whereas those of the closed conformation correspond to a low-affinity conformation. The C-terminal alpha-helix moves 10 A down the side of the domain in the open conformation. Locking in the conformation of the preceding loop is sufficient to increase affinity for ligand 9000-fold. This C-terminal "bell-rope" provides a mechanism for linkage to conformational movements in other domains. The transition from the closed to open conformation has been implicated in fast (<1 s) regulation of integrin affinity in response to activation signals from inside the cell. Recent integrin structures and functional studies reveal interactions between beta-propeller, I, and I-like domains in the headpiece, and a critical role for integrin EGF domains in the stalk region. These studies suggest that the headpiece of the integrin faces down toward the membrane in the inactive conformation and extends upward in a "switchblade"-like opening motion upon activation. These long-range structural rearrangements of the entire integrin molecule involving multiple interdomain contacts appear closely linked to conformational changes in the I domain, which result in increased affinity and competence for ligand binding.
... In both humans and mice, 18 α-subunits and eight β-subunits have now been identified; both participate in the formation of 24 non-covalently associated α/β heterodimer cell-surface receptors [10]. Several integrins, including the fibronectin receptor α5β1 integrin and the vitronectin receptor αVβ3 integrin, specifically recognize a surface-exposed arginyl-glycyl-aspartic acid (RGD) sequence in the cognate ligands in a Mg 2+ ion-dependent manner [11]. Recent investigations have revealed that the S-protein of SARS-CoV-2, but not other coronaviruses such as SARS-CoV and MERS-CoV, contains a surface-exposed RGD sequence [12][13][14]. ...
... The MLO-A5 cell line (mouse osteocytes) was obtained from Kerafast (Boston, MA, USA) and grown in MEM alpha (Thermo Fisher Scientific, Waltham, MA, USA) containing 5% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX, USA), 5% calf serum (Biowest, Riverside, MO, USA), and penicillin/streptomycin (Nacalai, Kyoto, Japan). A human monocytic cell line (THP-1) was purchased from ATCC (Manassas, VA, USA) and human lung epithelial cell lines (11)(12)(13)(14)(15)(16)(17)(18) were kindly provided by Dr. Yoshihiro Miyahara (Mie University Medical School, Mie, Japan). The human breast cancer line MDA-MB-231 was from ATCC. ...
... Relative expression of ACE2 to β-actin was determined by using the 2 -ΔCT method. In panel A, statistical significance between two groups (THP-1 vs. [11][12][13][14][15][16][17][18] was analyzed by a two-tailed unpaired t-test, while other comparisons were by one-way ANOVA. Results are shown as column scatterplots overlayed with the mean ± SEM of at least 3 independent experiments, in which each sample was triplicated. ...
The spike glycoprotein attached to the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to and exploits angiotensin-converting enzyme 2 (ACE2) as an entry receptor to infect pulmonary epithelial cells. A subset of integrins that recognize the arginyl–glycyl–aspartic acid (RGD) sequence in the cognate ligands has been predicted in silico to bind the spike glycoprotein and, thereby, to be exploited for viral infection. Here, we show experimental evidence that the β1 integrins predominantly expressed on human pulmonary epithelial cell lines and primary mouse alveolar epithelial cells bind to this spike protein. The cellular β1 integrins support adhesive interactions with the spike protein independently of ACE2, suggesting the possibility that the β1 integrins may function as an alternative receptor for SARS-CoV-2, which could be targeted for the prevention of viral infections.
... Integrins are cell adhesion receptors whose proper functioning plays a very significant role in diverse physiologic processes, such as embryonic development, cell proliferation, leukocyte adhesion and platelet aggregation [1]. As the name implies, integrins constitute the integral components of the plasma membrane, thereby integrating the extracellular and intracellular events of a living cell and transmitting the cellular signals bidirectionally [2]. ...
... Either the α or β subunit of integrins comprises an extracellular, globular domain (ectodomain), a single-pass transmembrane domain (TMD) and a short cytoplasmic tail (CT) (except for β4 integrin) [1]. The extracellular domain is responsible for interactions with integrin ligands that are present on cells (e.g., ICAM-1, VCAM-1 and MAdCAM-1), the extracellular matrix (ECM) (e.g., fibrinogen, collagen and laminin) and micro-organisms (e.g., β-glucan on Candida albicans) [5]. ...
... In immune cells, the inside-out signaling processes are initiated from cell-surface T-cell receptors or cytokine receptors. Upon high-affinity engagement of the activated integrin with its ligand counterpart, outside-in signals are transduced to the cytoplasm, leading to integrin clustering and modulation of intracellular pathways that are critical for altering cell spreading, migration, proliferation, differentiation and metabolism [1][2][3]7]. ...
Integrins represent the biologically and medically significant family of cell adhesion molecules that govern a wide range of normal physiology. The activities of integrins in cells are dynamically controlled via activation-dependent conformational changes regulated by the balance of intracellular activators, such as talin and kindlin, and inactivators, such as Shank-associated RH domain interactor (SHARPIN) and integrin cytoplasmic domain-associated protein 1 (ICAP-1). The activities of integrins are alternatively controlled by homotypic lateral association with themselves to induce integrin clustering and/or by heterotypic lateral engagement with tetraspanin and syndecan in the same cells to modulate integrin adhesiveness. It has recently emerged that integrins are expressed not only in cells but also in exosomes, important entities of extracellular vesicles secreted from cells. Exosomal integrins have received considerable attention in recent years, and they are clearly involved in determining the tissue distribution of exosomes, forming premetastatic niches, supporting internalization of exosomes by target cells and mediating exosome-mediated transfer of the membrane proteins and associated kinases to target cells. A growing body of evidence shows that tumor and immune cell exosomes have the ability to alter endothelial characteristics (proliferation, migration) and gene expression, some of these effects being facilitated by vesicle-bound integrins. As endothelial metabolism is now thought to play a key role in tumor angiogenesis, we also discuss how tumor cells and their exosomes pleiotropically modulate endothelial functions in the tumor microenvironment.
... revealed Fig 1D and E). Mammalian integrin I domains and TRAP I domains undergo structurally homologous conformational changes between open and closed conformations (Shimaoka et al, 2002;Song et al, 2012). Reshaping occurs in loops that surround a metal ion-dependent adhesion site (MIDAS), which holds a Mg 2+ that coordinates an acidic residue in ligands at the center of the ligand binding site. ...
... Reshaping occurs in loops that surround a metal ion-dependent adhesion site (MIDAS), which holds a Mg 2+ that coordinates an acidic residue in ligands at the center of the ligand binding site. Mammalian open and closed I domains have high and low affinity for ligand, respectively (Shimaoka et al, 2002). Alteration around the ligand binding site in the I domain is linked to pistoning of its a7-helix such that in the open state of TRAP an extensible b-ribbon appears between the I and TSR domains (Fig 1D and E). ...
Eukaryotic cell adhesion and migration rely on surface adhesins connecting extracellular ligands to the intracellular actin cytoskeleton. Plasmodium sporozoites are transmitted by mosquitoes and rely on adhesion and gliding motility to colonize the salivary glands and to reach the liver after transmission. During gliding, the essential sporozoite adhesin TRAP engages actin filaments in the cytoplasm of the parasite, while binding ligands on the substrate through its inserted (I) domain. Crystal structures of TRAP from different Plasmodium species reveal the I domain in closed and open conformations. Here, we probe the importance of these two conformational states by generating parasites expressing versions of TRAP with the I domain stabilized in either the open or closed state with disulfide bonds. Strikingly, both mutations impact sporozoite gliding, mosquito salivary gland entry, and transmission. Absence of gliding in sporozoites expressing the open TRAP I domain can be partially rescued by adding a reducing agent. This suggests that dynamic conformational change is required for ligand binding, gliding motility, and organ invasion and hence sporozoite transmission from mosquito to mammal.
... Interactions of some protein activators, e.g., talin, with CT of -subunits and membrane break this salt bridge, separate the -and -subunits, and the integrins switch to an extended conformation [159] of the and ectodomains that retains its low ligand affinity. Then, integrins interacting with extracellular ligands change to an open-extended, high-activity conformation [160]. It was observed [161] that after activation, integrins form ~100 nm clusters of ~50 integrins assisted in an early adhesion of cells. ...
... Interactions of some protein activators, e.g., talin, with CT of β-subunits and membrane break this salt bridge, separate the αand β-subunits, and the integrins switch to an extended conformation [159] of the α and β ectodomains that retains its low ligand affinity. Then, integrins interacting with extracellular ligands change to an open-extended, high-activity conformation [160]. It was observed [161] that after activation, integrins form~100 nm clusters of~50 integrins assisted in an early adhesion of cells. ...
Integrins are heterodimeric glycoproteins crucial to the physiology and pathology of many biological functions. As adhesion molecules, they mediate immune cell trafficking, migration, and immunological synapse formation during inflammation and cancer. The recognition of the vital roles of integrins in various diseases revealed their therapeutic potential. Despite the great effort in the last thirty years, up to now, only seven integrin-based drugs have entered the market. Recent progress in deciphering integrin functions, signaling, and interactions with ligands, along with advancement in rational drug design strategies, provide an opportunity to exploit their therapeutic potential and discover novel agents. This review will discuss the molecular modeling methods used in determining integrins' dynamic properties and in providing information toward understanding their properties and function at the atomic level. Then, we will survey the relevant contributions and the current understanding of integrin structure, activation, the binding of essential ligands, and the role of molecular modeling methods in the rational design of antagonists. We will emphasize the role played by molecular modeling methods in progress in these areas and the designing of integrin antagonists.
... Metal ion coordination in the I-domain of present in some α integrins plays a direct role regulating integrin activity by mediating changes in integrin conformation that increase binding affinity. On the other hand, metal ion coordination in the I-like domain of β integrins is less well defined and may be more important for the control of α/β heterodimers lacking an α I-domain, such as αvβ1 (Shimaoka et al., 2002;. As a result, changes in the extracellular concentration of divalent ions can promote the adoption of a specific conformation state. ...
... As a result, changes in the extracellular concentration of divalent ions can promote the adoption of a specific conformation state. Excess Mg 2+ and Mn 2+ can displace Ca 2+ within the I-domain and promote the adoption of open head group conformations and support ligand binding activity (Shimaoka et al., 2002;Luo et al., 2007;Tiwari et al., 2011). Interestingly, when cells are treated with excess Mn 2+ the affinity state achieved by compatible integrins is higher than that observed when integrins are activated by other means, providing further evidence for multiple different open integrin conformations (Luo et al., 2007;Ye et al., 2012). ...
Integrins are transmembrane proteins that are most typically thought of as integrating adhesion to the extracellular matrix with intracellular signaling and cell regulation. Traditionally, integrins are found at basolateral and lateral cell surfaces where they facilitate binding to the ECM and intercellular adhesion through cytosolic binding partners that regulate organization of actin microfilaments. However, evidence is accumulating that integrins also are apically localized, either endogenously or due to an exogenous stimulus. Apically localized integrins have been shown to regulate several processes by interacting with proteins such as connexins, tight junction proteins, and polarity complex proteins. Integrins can also act as receptors to mediate endocytosis. Here we review these newly appreciated roles for integrins localized to the apical cell surface.
... Yellow star indicates an antibody that detects total integrin population, blue star indicates an antibody that recognizes active conformers. Figure adapted from [1][2][3][4]. ...
... The current model for integrin activation has integrins adopting at least three different conformational states that relate to our understanding of activation. In general, a global conformational change from "bent" receptor to "upright-extended" receptor is accepted as required for integrin activation [4,[137][138][139]. Intermediate conformational states also exist, which occur when the integrin is partially bent [25], upright-extended without an open headpiece available for the ligand, or extended with fused transmembrane sequences [132]. ...
Integrins are transmembrane adhesion receptors that engage ligands in the extracellular matrix and cytoskeletal proteins to form multi-protein adhesive complexes. These complexes provide a platform for integrin signaling and a mechanical anchor between the cell and its environment. Integrin functionality is hypothesized to be sensitive to cell membrane composition, driven at least in part, by the tendency for the plasma membrane to compartmentalize into ordered and disordered domains resembling coexisting liquid phases observed in model membranes. In particular, past work suggests that adhesions are highly ordered membrane domains and that more integrins in an extended confirmation favor a more ordered membrane environment. This dissertation investigates how integrins associate with membrane order through newly developed super-resolution imaging techniques that are capable of directly observing the local enrichment and depletion of peptide markers of liquid-ordered and disordered domains. I quantify the local membrane environment surrounding β1 integrins by tabulating cross-correlations between antibody labeled β1 integrins and peptide markers. Through the use of conformational specific antibodies, I determine how the conformational state of integrins impacts its local membrane composition and how membrane perturbations alter β1 integrin activity. I find that β1 integrins co-localize with both markers of membrane order and disorder across the plasma membrane. I find that affinity for a membrane ordered probe depends on activation state and that β1 integrins from mature fibrillar adhesive complexes exhibit the strongest preference for membrane order. I determine that adhesions themselves are made up of integrins in at least two different conformational states, and active, extended conformation integrins do not exhibit a strong preference for either phase-like domain composition despite being confined to adhesions. Finally, I demonstrate that perturbing the plasma membrane composition with long-chain n-alcohols robustly affects the activation state of β1 integrins and the formation of adhesive complexes. The structure-function relationship of integrins has broad biological implications contributing to cell viability, growth, and motility. My work indicates that integrin association with membrane order is more subtle than previously proposed and that the subset of integrins in the active, extended conformation is less order preferring than the total integrin population. I show integrin activity is modulated by perturbations of membrane organization, and I conclude that integrin activation is indirectly impacted by membrane order, likely through the regulatory networks responsible for “inside-out” signaling.
... However, deriving selective and potent Abs against integrins remains challenging, because integrin heterodimers exist in multiple conformations 32,33 that are difficult to recapitulate with purified recombinant proteins. Furthermore, integrin conformations are influenced by cations and the local membrane environment, 34 and they are exquisitely controlled by 'inside-out' signaling, [34][35][36] which may also augment dynamic conformational states and modulate their functions in the extracellular environment. ...
... However, deriving selective and potent Abs against integrins remains challenging, because integrin heterodimers exist in multiple conformations 32,33 that are difficult to recapitulate with purified recombinant proteins. Furthermore, integrin conformations are influenced by cations and the local membrane environment, 34 and they are exquisitely controlled by 'inside-out' signaling, [34][35][36] which may also augment dynamic conformational states and modulate their functions in the extracellular environment. ...
Integrins are transmembrane multi-conformation receptors that mediate interactions with the extracellular matrix. In cancer, integrins influence metastasis, proliferation, and survival. Collagen-binding integrin-α11/β1, a marker of aggressive tumors that is involved in stroma-tumor crosstalk, may be an attractive target for anti-cancer therapeutic antibodies. We performed selections with phage-displayed synthetic antibody libraries for binding to either purified integrin-α11/β1 or in situ on live cells. The in-situ strategy yielded many diverse antibodies, and strikingly, most of these antibodies did not recognize purified integrin-α11/β1. Conversely, none of the antibodies selected for binding to purified integrin-α11/β1 were able to efficiently recognize native cell-surface antigen. Most importantly, only the in-situ selection yielded functional antibodies that were able to compete with collagen-I for binding to cell-surface integrin-α11/β1, and thus inhibited cell adhesion. In-depth characterization of a subset of in situ-derived clones as full-length immunoglobulins revealed high affinity cellular binding and inhibitory activities in the single-digit nanomolar range. Moreover, the antibodies showed high selectivity for integrin-α11/β1 with minimal cross-reactivity for close homologs. Taken together, our findings highlight the advantages of in-situ selections for generation of anti-integrin antibodies optimized for recognition and inhibition of native cell-surface proteins, and our work establishes general methods that could be extended to many other membrane proteins.
... In addition to this pro-inflammatory signaling pathway, the interaction of P. gingivalis and its fimbriae with CD14/TLR2 in monocytes/macrophages also stimulates proadhesive signaling that activates the ligand-binding capacity of the CD11b/CD18 integrin [103,104] (Fig. 2). The regulation of the binding affinity of β2 integrin from within the cell through proadhesive signaling is referred to as "inside-out signaling" [105]. Inside-out signaling may target cytoplasmic proteins to the integrin cytoplasmic tails causing high-affinity conformational changes on the ligand-binding domains of integrins [105]. ...
... The regulation of the binding affinity of β2 integrin from within the cell through proadhesive signaling is referred to as "inside-out signaling" [105]. Inside-out signaling may target cytoplasmic proteins to the integrin cytoplasmic tails causing high-affinity conformational changes on the ligand-binding domains of integrins [105]. ...
Purpose of Review
This review summarizes mechanisms by which Porphyromonas gingivalis interacts with community members and the host so that it can persist in the periodontium under inflammatory conditions that drive periodontal disease.
Recent Findings
Recent advances indicate that, in great part, the pathogenicity of P. gingivalis is dependent upon its ability to establish residence in the subgingival environment and to subvert innate immunity in a manner that uncouples the nutritionally favorable (for the bacteria) inflammatory response from antimicrobial pathways. While the initial establishment of P. gingivalis is dependent upon interactions with early colonizing bacteria, the immune subversion strategies of P. gingivalis in turn benefit co-habiting species.
Summary
Specific interspecies interactions and subversion of the host response contribute to the emergence and persistence of dysbiotic communities and are thus targets of therapeutic approaches for the treatment of periodontitis.
... Here we demonstrate that the flexibility of the Ad fiber allows the virus capsid to simultaneously bind CAR and ␣v integrins. Bending of the ␣v integrin has also been observed in its crystal structure (51) and may play an important role in its activation (24,33). In perhaps an analogous situation, the 4to 6-amino-acid insertion in the third to last beta repeat of the reovirus 1 tail imparts 1 with substantial flexibility and likely plays a similar role in orienting the 1 head for interaction with JAM-1 at the cell surface. ...
The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.
... An important aspect of integrins is their ability to transmit signals both "outside in" and inside out" that are transmitted through large conformational changes in the extracellular domains in the integrin [6,[19][20][21]. Although initially thought otherwise [18,19], it is now accepted that the subunits of inactive integrins are bent over between the thigh and calf domains of the α-subunit and in the third and fourth EGF-like regions of the β-subunit to produce a 135-degree downward angle [22,23]. Active integrins have an extended shape and there is separation of the legs of the α-and β-subunits (Fig. 4). ...
Integrins are a highly complex family of receptors that, when expressed on the surface of cells, can mediate reciprocal cell-to-cell and cell-to-extracellular matrix (ECM) interactions leading to assembly of integrin adhesion complexes (IACs) that initiate many signaling functions both at the membrane and deeper within the cytoplasm to coordinate processes including cell adhesion, migration, proliferation, survival, differentiation, and metabolism. All metazoan organisms possess integrins, and it is generally agreed that integrins were associated with the evolution of multicellularity, being essential for the association of cells with their neighbors and surroundings, during embryonic development and many aspects of cellular and molecular biology. Integrins have important roles in many aspects of embryonic development, normal physiology, and disease processes with a multitude of functions discovered and elucidated for integrins that directly influence many areas of biology and medicine, including mammalian pregnancy, in particular implantation of the blastocyst to the uterine wall, subsequent placentation and conceptus (embryo/fetus and associated placental membranes) development. This review provides a succinct overview of integrin structure, ligand binding, and signaling followed with a concise overview of embryonic development, implantation, and early placentation in pigs, sheep, humans, and mice as an example for rodents. A brief timeline of the initial localization of integrin subunits to the uterine luminal epithelium (LE) and conceptus trophoblast is then presented, followed by sequential summaries of integrin expression and function during gestation in pigs, sheep, humans, and rodents. As appropriate for this journal, summaries of integrin expression and function during gestation in pigs and sheep are in depth, whereas summaries for humans and rodents are brief. Because similar models to those illustrated in Fig. 1, 2, 3, 4, 5 and 6 are present throughout the scientific literature, the illustrations in this manuscript are drafted as Viking imagery for entertainment purposes.
... Integrin is a type of cell adhesion molecule. As mechanosensory, the integrin interacts with ECM or neighboring cells and receives signals from the environment to regulate cell adhesion, growth, differentiation, and apoptosis [1][2][3]. It consists of two subunits, α and β. ...
Integrin αIIbβ3 expresses on the plasma membrane of platelets, which can be activated by mechanical pulling force from its ligand. Herein, we performed all-atom molecular dynamics (MD) simulations on the full-length integrin embedded in the membrane with a ligand, fibronectin (FN), attaching to the integrin head. A pulling force was applied to the ligand in the MD simulations. The force applied on the ligand could transmit to the integrin head through the transient interaction, and have chances to induce the extension of the integrin. The loading rate affects the pulling force but has limited effects on the integrin extension. Our simulation showed the mechanical force could induce the conformational change of the membrane-embedded integrin.
... Previous structures of MIC2 fragments containing the VWA-TSR1 domains have revealed an unusually long TSR1 domain that associates with its preceding VWA domain in a potentially closed conformation 9 . The VWA domain plays a crucial role in binding to its ligand on the host cell surface, and its homologous domain in integrins, the inserted I domain has both a high-affinity open conformation and a low affinity closed conformation 10,11 . Both conformations have been defined in the Plasmodium TRAP orthologue, and the conformational change of TRAP has been suggested to be linked to stick-and-slip motility 12 . ...
Microneme protein 2 (MIC2) and MIC2-associated protein (M2AP) play crucial roles in the gliding motility and host cell invasion of Toxoplasma gondii. Complex formation between MIC2 and M2AP is required for maturation and transport from the microneme to the parasite surface. Previous studies showed that M2AP associates with the 6th TSR domain of MIC2 (TSR6), but the detailed interaction remains unclear. In this study, we report crystal structures of M2AP alone and in complex with TSR6. TSR domains have an unusually thin, long structure with a layer of intercalated residues on one side. The non-layered side of TSR6 with hotspot residue His-620 at the center binds to M2AP. Remarkably, we show that TSR6 residue Y602 is dynamic; it equilibrates between being part of the layer (the layered state) and in a flipped-out state in the absence of M2AP. However, when bound to M2AP, Y602 shifts to the flipped-out state. Our findings provide insights into the association and stabilization of MIC2-M2AP complex, and may be used to develop new therapies to prevent infections caused by this parasite.
... Previous structures of MIC2 fragments containing the VWA-TSR1 domains have revealed an unusually long TSR1 domain that associates with its preceding VWA domain in a potentially closed conformation [9]. The VWA domain plays a crucial role in binding to its ligand on the host cell surface, and its homologous domain in integrins, the inserted I domain has both a high-a nity open conformation and a low a nity closed conformation [10,11]. Both conformations have been de ned in the Plasmodium TRAP orthologue, and the conformational change of TRAP has been suggested to be linked to stick-and-slip motility [12]. ...
Microneme protein 2 (MIC2) and MIC2-associated protein (M2AP) play crucial roles in the gliding motility and host cell invasion of Toxoplasma gondii. Complex formation between MIC2 and M2AP is required for maturation and transport from the microneme to the parasite surface. Previous studies showed that M2AP associates with the 6th TSR domain of MIC2 (TSR6), but the detailed interaction remains unclear. In this study, we determined crystal structures of MA2P alone and in complex with TSR6. TSR domains have an unusually thin, long structure with a layer of intercalated residues on one side. The non-layered side of TSR6 with hotspot residue His-620 at the center binds to M2AP. Remarkably, we discovered that TSR6 residue Y602 is dynamic; it equilibrates between being part of the layer (the layered state) and in a flipped-out state in the absence of M2AP. However, when bound to M2AP, Y602 shifts to the flipped-out state. Our findings provide insights into the association and stabilization of MIC2-M2AP complex, and may be used to develop new therapies to prevent infections caused by this parasite.
... For endothelial cells, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are prominent examples, whereas for monocytes, selectins such as L-selectin and integrins such as β2-integrin (ITGB2) or α4-integrin (ITGA4) are highly expressed for this process [6][7][8]. Adhesion involving integrins is tightly controlled by ligand binding, which allows rapid switching from the inactive state (low affinity binding) to the active state (high affinity confirmation), which is also known as inside-out signaling [9][10][11]. The active state allows activation, blood was centrifuged at 2500× g for 15 min to isolate the plasma. ...
The interaction between monocytes and endothelial cells in inflammation is central to chemoattraction, adhesion, and transendothelial migration. Key players, such as selectins and their ligands, integrins, and other adhesion molecules, and their functions in these processes are well studied. Toll-like receptor 2 (TLR2), expressed in monocytes, is critical for sensing invading pathogens and initiating a rapid and effective immune response. However, the extended role of TLR2 in monocyte adhesion and migration has only been partially elucidated. To address this question, we performed several functional cell-based assays using monocyte-like wild type (WT), TLR2 knockout (KO), and TLR2 knock-in (KI) THP-1 cells. We found that TLR2 promotes the faster and stronger adhesion of monocytes to the endothelium and a more intense endothelial barrier disruption after endothelial activation. In addition, we performed quantitative mass spectrometry, STRING protein analysis, and RT-qPCR, which not only revealed the association of TLR2 with specific integrins but also uncovered novel proteins affected by TLR2. In conclusion, we show that unstimulated TLR2 influences cell adhesion, endothelial barrier disruption, migration, and actin polymerization.
... S3). In turn, the VWA domain contains a predicted MIDAS (metal ion-dependent adhesion site) motif, a discontinuous structural feature (Asp-x-Ser-x-Ser…Thr…Asp). In general, the coordination of a divalent metal ion (e.g., Ca 2+ /Mg 2+ /Mn 2+ ) at this site induces structural changes in VWA domains upon ligand binding that stabilize this adhesive domain in a high-affinity state for the ligand (32). For CglB, highly-conserved putative MIDAS residues map to D56, S58, S60, T182, and D211 (Fig. 2, B and C). ...
The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal-adhesion (bFA) sites to power gliding motility. Using total internal reflection fluorescence and force microscopies, we identify the von Willebrand A domain-containing outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling adhesin of the gliding transducer (Glt) machinery at bFAs. Biochemical and genetic analyses reveal that CglB localizes to the cell surface independently of the Glt apparatus; once there, it is recruited by the OM module of the gliding machinery, a heteroligomeric complex containing the integral OM β barrels GltA, GltB, and GltH, as well as the OM protein GltC and OM lipoprotein GltK. This Glt OM platform mediates the cell-surface accessibility and retention of CglB by the Glt apparatus. Together, these data suggest that the gliding complex promotes regulated surface exposure of CglB at bFAs, thus explaining the manner by which contractile forces exerted by inner-membrane motors are transduced across the cell envelope to the substratum.
... In addition, it is the α integrin subunit that binds the ECM to direct β integrin signaling, suggesting that the α integrin may be the more critical target in directing functions. 38,39 The importance of α7 integrin over β1 integrin has also been supported by previous studies in ASM cells 15,17 further strengthening this approach. ...
Asthma is a chronic inflammatory disorder of the lower airways characterized by modulation of airway smooth muscle (ASM) function. Infiltration of smooth muscle by inflammatory mediators is partially regulated by transmembrane integrins and the major smooth muscle laminin receptor α7β1 integrin plays a critical role in the maintenance of ASM phenotype. The goal of the current study was to investigate the role of α7 integrin in asthma using smooth muscle‐specific α7 integrin transgenic mice (TgSM‐Itgα7) using both acute and chronic OVA sensitization and challenge protocols that mimic mild to severe asthmatic phenotypes. Transgenic over‐expression of the α7 integrin in smooth muscle resulted in a significant decrease in airway resistance relative to controls, reduced the total number of inflammatory cells and substantially inhibited the production of crucial Th2 and Th17 cytokines in airways. This was accompanied by decreased secretion of various inflammatory chemokines such as eotaxin/CCL11, KC/CXCL3, MCP‐1/CCL2, and MIP‐1β/CCL4. Additionally, α7 integrin overexpression significantly decreased ERK1/2 phosphorylation in the lungs of TgSM‐Itgα7 mice and affected proliferative, contractile, and inflammatory downstream effectors of ERK1/2 that drive smooth muscle phenotype in the lung. Taken together, these results support the hypothesis that enhanced expression of α7 integrin in vivo inhibits allergic inflammation and airway resistance. Moreover, we identify ERK1/2 as a potential target by which α7 integrin signals to regulate airway inflammation. We conclude that identification of therapeutics targeting an increase in smooth muscle α7 integrin expression could serve as a potential novel treatment for asthma.
... Integrin receptors are heterodimeric integral membrane proteins formed by different pairs of a and b subunits with different affinities for ECM proteins such as collagens, fibronectin, laminin, and cell-cell adhesion receptors such as ICAM and VCAM (19). On binding its partner molecule, integrin activates by extending the extracellular domains of both subunits and separating their cytoplasmic tails. ...
The cellular functions are regulated by a complex interplay of diffuse and local signals. Experimental work in cell physiology has led to recognize that understanding a cell's dynamics requires a deep comprehension of local fluctuations of cytosolic regulators. Macromolecular complexes are major determinants of local signaling. Multi-enzyme assemblies limit the diffusion restriction to reaction kinetics by direct exchange of metabolites. Likewise, close coupling of ion channels and transporters modulate the ion concentration around a channel mouth or transporter binding site. Extreme signal locality is brought about by conformational coupling between membrane proteins, as is typical of mechanotransduction. A paradigmatic case is integrin-mediated cell adhesion. Sensing the extracellular microenvironment and providing an appropriate response is essential in growth and development and has innumerable pathological implications. The process involves bidirectional signal transduction by complex supra-molecular structures that link integrin receptors to ion channels and transporters, growth factor receptors, cytoskeletal elements and other regulatory elements. The dynamics of such complexes is only beginning to be understood. A thoroughly studied example is the association between integrin receptors and the voltage-gated K+ channels Kv11.1. These channels are widely expressed in early embryos, where their physiological roles are poorly understood and apparently different from the shaping of action potential firing in the adult. Hints about these roles come from studies in cancer cells, where Kv11.1 is often overexpressed and appears to re-assume functions, such as controlling cell proliferation/differentiation, apoptosis and migration. Kv11.1 is implicated in these processes through its linking to integrin subunits.
... In addition, the release of Ca 2+ from the BG-containing ceramics (see Fig. 2c) would also possibly affect the activity of the integrins distributed on the cell membranes (it has well been demonstrated that integrins were Ca 2+ -sensitive and high [Ca 2+ ] o could inhibit the activity of integrins), 38,42 and thus weaken the assembly of the cytoskeleton proteins. 38,42,43 As regards a further decrease of the cytoskeleton tension in the GS-mediated cells, a possible explanation is that the released GS would interact with membrane proteins (GS has abundant amide groups), and therefore interfering with the adhesion of the cells on the mineralized surfaces. 44 Finally, the osteogenic commitment of MSCs mediated by the ceramics was assessed, and the corresponding results are ...
The construction of bone scaffolds to treat bone defects caused by infection is a major challenge because such bone scaffolds need to both prevent infection from recurring and regenerate bone defects. To this end, we constructed bioactive glass (BG)-coated, hierarchical porous tricalcium phosphate (TCP) ceramics as both bone scaffolds and drug delivery devices to treat S. aureus-infected bone defects. In vitro simulation experiments confirmed that the as-prepared porous ceramics could construct a dynamic microenvironment, in which the concentration of the released antibiotics and calcium ions always changed and an apatite layer could grow on the ceramic surfaces. In vitro experiments demonstrated that antibiotic-loaded ceramics possessed good cytocompatibility and the microenvironment established by the antibiotic-loaded ceramics could mediate the osteogenic commitment of mesenchymal stem cells (MSCs), and at the same time, the antibiotic-loaded ceramics presented excellent and relatively-lasting antibacterial activity. In vivo experiments demonstrated that the antibiotic-loaded ceramics not only could prevent the S. aureus-infected bone defects from recurring, but also could boost the endogenous regeneration of bone.
... These structures elucidate the role of the MIDAS 33,30,46,47 . The MIDAS undergoes a large structural change between a closed to an open form and is essential for PA-receptor interaction 48,47 . The residue D683 of PA coordinating the Mn 2+ cation in the MIDAS of the ANTXR receptors is crucial for PA-ANTXR binding. ...
Edema factor (EF), a major Bacillus anthracis toxin, is activated by host calmodulin (CaM) to produce supraphysiological concentrations of cyclic AMP (cAMP) thus perturbing intracellular signaling. The EF-CaM interaction induces conformational changes in an allosteric switch region of EF that lead to the formation of the catalytic site. Previous in silico studies targeting this switch region, complemented with experimental data, showed that thiophen ureidoacids (TUA) inhibit the enzyme catalytic activity. However, knowledge of the binding site and inhibition mode of TUA compounds are still lacking. Here, we characterize the interaction of the most active TUA compound (TUA-diCl) with EF, CaM and EF-CaM using biochemical assays coupled to biophysical methods and molecular modeling. We show that TUA-diCl interacts with EF, EF-CaM and unexpectedly with CaM. Mapping of the binding site by NMR, showed that TUA-diCl binds to the exposed hydrophobic patches of calcium loaded CaM, causing the compaction and changes in internal dynamics of the protein. Importantly, enzymatic, fluorescence and NMR data show that EF inhibition is due to the interaction of the compound with EF and is CaM-independent. Furthermore, competition experiments between TUA-diCl and the EF catalytic-site inhibitor 2’-MANT-3’-dATP, indicate that TUA-diCl is an allosteric inhibitor of EF. HDX-MS identifies a putative binding site of TUA-diCl on the helical domain of EF, a critical region for CaM insertion. Several possible binding pockets in the helical domain are analyzed in silico. TUA-diCl represents a new class of EF inhibitors with an allosteric mechanism, opening the way towards the design of innovative therapeutic compounds.
... A series of structural studies have shown that integrins have two allosteric conformations, and their activation is mediated by conformational changes that shift the heterodimer from a low-affinity (inactive) state to a high-affinity (active) state. 43,44 Integrin aggregation leads to the recruitment and phosphorylation of FAK, which provides a platform for various intermediate proteins (such as MAPK/ERK, JNK, Src kinase, and GTPases) to activate signaling pathways and mediate mechanical signal transduction (Fig. 3a). 45 For example, blockade of integrin αvβ3 in cultured mouse osteocytes (MLO-Y4 cells) impaired their chemosensitivity to laminar oscillatory fluid flow stimulus, resulting in disrupted COX-2 expression and PGE2 release. ...
Bone remodeling is a lifelong process that gives rise to a mature, dynamic bone structure via a balance between bone formation by osteoblasts and resorption by osteoclasts. These opposite processes allow the accommodation of bones to dynamic mechanical forces, altering bone mass in response to changing conditions. Mechanical forces are indispensable for bone homeostasis; skeletal formation, resorption, and adaptation are dependent on mechanical signals, and loss of mechanical stimulation can therefore significantly weaken the bone structure, causing disuse osteoporosis and increasing the risk of fracture. The exact mechanisms by which the body senses and transduces mechanical forces to regulate bone remodeling have long been an active area of study among researchers and clinicians. Such research will lead to a deeper understanding of bone disorders and identify new strategies for skeletal rejuvenation. Here, we will discuss the mechanical properties, mechanosensitive cell populations, and mechanotransducive signaling pathways of the skeletal system.
... The transition from a "bent" to an extended conformation is called "activation," which is reversible and rapid. This process involves two key mechanisms: the extension of the head and the separation of the legs, which are triggered by "inside-out" or "outside-in" signals (18,19). However, recent work clearly illustrated that integrins are vertically positioned on the cell membrane and exist in three main conformations: bent-closed (inactive), extended-closed (active, low affinity) and extended-open (active, high affinity) conformations (20). ...
Integrins, a family of adhesion molecules generally exist on the cell surface, are essential for regulating cell growth and its function. As a bi-directional signaling molecule, they mediate cell-cell and cell-extracellular matrix interaction. The recognitions of their key roles in many human pathologies, including autoimmunity, thrombosis and neoplasia, have revealed their great potential as a therapeutic target. This paper focuses on the activation of integrins, the role of integrins in tumorigenesis and progression, and advances of integrin-dependent tumor therapeutics in recent years. It is expected that understanding function and signaling transmission will fully exploit potentialities of integrin as a novel target for tumors.
... Zebrafish CD18 shared 49.8% identity and 64.4% similarity with human CD18. Since the extracellular I-like domain of mammalian CD18 is especially important for the regulation of ligand binding (57,58) we aligned the I-like domains and found that this domain was not only highly conserved between human and mouse (94.6% identity and 96.2% similarity) but also between human and zebrafish (65.4% identity and 75.8% similarity), suggesting that zebrafish CD18 may function similar to human CD18. Strikingly, the two conserved NXXF motifs with critical importance for the function of the cytoplasmic domain of CD18 in the mammalian system are present in zebrafish as well (Supplementary Figure 1). ...
Neutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the β 2 integrin family (CD11/CD18) are critically required for the initial neutrophil adhesion to the inflamed endothelium and several post-adhesion steps allowing their extravasation into the inflamed tissue. Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of β 2 integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae ( Danio rerio ) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo , the functional impact of β 2 integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the β subunit of β 2 integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2 , the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I.
... The integrin LFA1 (L2) plays major roles in these processes, and consequently, its activation must be tightly regulated. Accumulating evidence from structural and mutational studies of 1, 2, and 3 integrin heterodimers define three major states of integrin heterodimeric conformation based on global changes of the ectodomain and headpiece conformation associated with affinity changes: bent/ closed (low affinity), extended/closed (intermediate affinity), and extended/open (high affinity) (Fig. 1A) (4,5). In addition to the conformational and affinity changes, integrin clustering also supports adhesion through multivalent ligand binding by the low-and intermediate-affinity states (avidity) (6,7). ...
Integrin activation by the intracellular adaptor proteins talin1 and kindlin-3 is essential for lymphocyte adhesion. These adaptors cooperatively control integrin activation through bidirectional (inside-out and outside-in) activation signals. Using single-molecule measurements, we revealed the distinct dynamics of talin1 and kindlin-3 interactions with the integrin LFA1 (αLβ2) and their functions in LFA1 activation and LFA1-mediated adhesion. The kinetics of talin1 binding to the tail of the β2 subunit corresponded to those of LFA1 binding to its ligand ICAM1. ICAM1 binding induced transient interactions between the membrane-proximal cytoplasmic region of the β2 subunit with an N-terminal domain of kindlin-3, leading to disruption of the association between the integrin subunits (the α/β clasp) and unbending of the ectodomains of the α/β heterodimer. These conformational changes promoted high-affinity talin1 binding to the β2 tail that required the talin rod domain and the actomyosin cytoskeleton. Inside-out signaling induced by the GTPase Rap1 did not markedly stabilize the binding of talin1 and kindlin-3 to LFA1. In contrast, ligand-induced outside-in signaling, the stabilization of open LFA1 conformers, or shear force substantially altered the dynamics of talin1 and kindlin-3 association with LFA1 and enhanced both Rap1 and LFA1 activation. In migrating lymphocytes, asymmetrical distribution of talin1 and kindlin-3 correlated with the maturation of LFA1 from a low-affinity conformation at the leading edge to a high-affinity conformation in the adherent mid-body. Our results suggest that kindlin-3 spatiotemporally mediates a positive feedback circuit of LFA1 activation to control dynamic adhesion and migration of lymphocytes.
... While the same integrin heterodimer can have multiple extracellular binding partners, including collagen, laminin, fibronectin, and the CAM family of adhesion receptors, these binding partners may have unique binding sites on different integrins. In addition to their diversity of ligand binding, integrins respond to intracellular and extracellular activating signals through rapid conformational change (10)(11)(12). Further, integrins are also mechanosensitive and participate in catch-bonding, meaning that force will modulate their structure, binding, and downstream signaling (13)(14)(15). Integrins also form clusters in the membrane, which change in size as cells alter their speed (T cells) (2). ...
Integrins are transmembrane receptors associated with adhesion and migration and are often highly differentially expressed receptors amongst natural killer cell subsets in microenvironments. Tissue resident natural killer cells are frequently defined by their differential integrin expression compared to other NK cell subsets, and integrins can further localize tissue resident NK cells to tissue microenvironments. As such, integrins play important roles in both the phenotypic and functional identity of NK cell subsets. Here we review the expression of integrin subtypes on NK cells and NK cell subsets with the goal of better understanding how integrin selection can dictate tissue residency and mediate function from the nanoscale to the tissue environment.
... These heterodimeric molecules bind to a distinct array of ligands expressed on cells and/or deposited in the ECM in order to exert biological processes [2]. Depending on the cell types expressing integrins, integrin-mediated cell adhesion plays an important role in various biological phenomenon such as wound healing, host defense, inflammation, thrombosis and cancer metastasis [1,3]. The integrins αLβ2 (a.k.a. ...
Irisin, a myokine released from skeletal muscle, has recently been found to act as a ligand for the integrins αVβ5, αVβ1, and α5β1 expressed on mesenchymal cells, thereby playing an important role in the metabolic remodeling of the bone, skeletal muscle and adipose tissues. Although the immune-modulatory effects of irisin in chronic inflammation have been documented, its interactions with lymphocytic integrins have yet to be elucidated. Here, we show that irisin supports the cell adhesion of human and mouse lymphocytes. Cell adhesion assays using a panel of inhibitory antibodies to integrins have shown that irisin-mediated lymphocyte adhesion involves multiple integrins including not only α4β1 and α5β1, but also leukocyte-specific αLβ2 and α4β7. Importantly, mouse lymphocytic TK-1 cells that lack the expression of β1 integrins have exhibited αLβ2-and α4β7-mediated cell adhesion to irisin. Irisin has also been demonstrated to bind to purified recombinant integrin αLβ2 and α4β7 proteins. Thus, irisin represents a novel ligand for integrin αLβ2 and α4β7, capable of supporting lymphocyte cell adhesion independently of β1 integrins. These results suggest that irisin may play an important role in regulating lymphocyte adhesion and migration in the inflamed vasculature.
... Each β2 integrin contains a von Willebrand factor type A domain called the αI domain in the α subunit. This domain serves as a ligand binding domain, whereas other domains play regulatory roles [14]. Ligand binding to the αI domain is governed by its conformation. ...
A growing literature has shown that volatile anesthetics are promiscuous molecules targeting multiple molecules, some of which are critical for immunological functions. We focused on studies that delineated target molecules of volatile anesthetics on immune cells and summarized the effects of volatile anesthetics on immune functions. We also presented the perspectives of studying volatile anesthetics-mediated immunomodulation.
... Integrin-triggered intracellular signaling leads to cell division, survival, differentiation, and/or death, which are pivotal for vital phenomena (Meredith et al., 1993;Leone et al., 2005;Livshits et al., 2012). The activation of integrins before their adherence to cognate ligands, constitutes a variety of global, reversible, and cooperative conformational changes involving multiple structural domains (Shimaoka et al., 2002;Calderwood, 2004). An integrin's ligand-binding affinity is enhanced in response to inside-out signals derived from activated G-protein or via coupling with other receptors Carman and Springer, 2003;Gahmberg et al., 2009;Springer and Dustin, 2012). ...
Integrins are transmembrane proteins that mediate cellular adhesion and migration to neighboring cells or the extracellular matrix, which is essential for cells to undertake diverse physiological and pathological pathways. For integrin activation and ligand binding, bidirectional signaling across the cell membrane is needed. Integrins aberrantly activated under pathologic conditions facilitate cellular infiltration into tissues, thereby causing inflammatory or tumorigenic progressions. Thus, integrins have emerged to the forefront as promising targets for developing therapeutics to treat autoimmune and cancer diseases. In contrast, it remains a fact that integrin-ligand interactions are beneficial for improving the health status of different tissues. Among these ligands, irisin, a myokine produced mainly by skeletal muscles in an exercise-dependent manner, has been shown to bind to integrin αVβ5, alleviating symptoms under unfavorable conditions. These findings may provide insights into some of the underlying mechanisms by which exercise improves quality of life. This review will discuss the current understanding of integrin-ligand interactions in both health and disease. Likewise, we not only explain how diverse ligands play different roles in mediating cellular functions under both conditions via their interactions with integrins, but also specifically highlight the potential roles of the emerging ligand irisin in inflammation, cancer, and metabolic disease.
... Binding assays also show that soluble CD4 tetramers do not bind detectably to MHC-II-expressing cells, but that very weak binding can be detected when CD4 is coupled to streptavidin-coated beads (∼50,000 CD4s per bead) (Jönsson et al., 2016), emphasizing their profoundly weak binding. T-cell/APC adhesion in vivo more likely depends on the much stronger interactions of "professional" adhesion proteins, such as the integrins (Shimaoka et al., 2002) and small adhesion molecules comprising the CD2 subset of the IgSF . What, then, would be the physiological relevance of a very weak, monovalent interaction if not to increase overall T-cell/APC adhesion? ...
The T-cell coreceptors CD4 and CD8 have well-characterized and essential roles in thymic development, but how they contribute to immune responses in the periphery is unclear. Coreceptors strengthen T-cell responses by many orders of magnitude – beyond a million-fold according to some estimates – but the mechanisms underlying these effects are still debated. T-cell receptor (TCR) triggering is initiated by the binding of the TCR to peptide-loaded major histocompatibility complex (pMHC) molecules on the surfaces of other cells. CD4 and CD8 are the only T-cell proteins that bind to the same pMHC ligand as the TCR, and can directly associate with the TCR-phosphorylating kinase Lck. At least three mechanisms have been proposed to explain how coreceptors so profoundly amplify TCR signaling: (1) the Lck recruitment model and (2) the pseudodimer model, both invoked to explain receptor triggering per se, and (3) two-step coreceptor recruitment to partially triggered TCRs leading to signal amplification. More recently it has been suggested that, in addition to initiating or augmenting TCR signaling, coreceptors effect antigen discrimination. But how can any of this be reconciled with TCR signaling occurring in the absence of CD4 or CD8, and with their interactions with pMHC being among the weakest specific protein-protein interactions ever described? Here, we review each theory of coreceptor function in light of the latest structural, biochemical, and functional data. We conclude that the oldest ideas are probably still the best, i.e., that their weak binding to MHC proteins and efficient association with Lck allow coreceptors to amplify weak incipient triggering of the TCR, without comprising TCR specificity.
... Integrin activation induces rapid (i.e. within 1 s) conformational changes from a completely bent form with low ligand affinity to an intermediate or fully extended form with very high ligand affinity [74,76,77]. Most integrins typically bind a wide variety of ligands, making these receptors essential in many cell-cell and cell-matrix interactions [74]. ...
This review article describes the use of immune cells as potential candidates to deliver anti-cancer drugs deep within the tumor microenvironment. First, the rationale of using drug carriers to target tumors and potentially decrease drug-related side effects is discussed. We further explain some of the current limitations when using nanoparticles for this purpose. Next, a comprehensive step-by-step description of the migration cascade of immune cells is provided as well as arguments on why immune cells can be used to address some of the limitations associated with nanoparticle-mediated drug delivery. We then describe the benefits and drawbacks of using red blood cells, platelets, granulocytes, monocytes, macrophages, myeloid-derived suppressor cells, T cells and NK cells for tumor-targeted drug delivery. An additional section discusses the versatility of nanoparticles to load anti-cancer drugs into immune cells. Lastly, we propose increasing the circulatory half-life and development of conditional release strategies as the two main future pillars to improve the efficacy of immune cell-mediated drug delivery to tumors.
... Recently, the crystal structure of the aVp3 extracellular part was resolved alone (Xiong et al., 2001) and in the presence of the RGD sequence (Xiong et al., 2002; for review see Giancotti, 2003, Humphries et al., 2003bLiddington and Ginsberg, 2002;Shimaoka et al, 2002). The overall structure revealed a globular "head" composed of the aV and the p3 subunits, and two "leg"-like extensions connecting the "head" to the cell-surface. ...
Muskelin is an intracellular, kelch-repeat protein that is functionally involved in cell spreading on thrombospondin-1. The aim of this thesis project was to investigate the role of muskelin domains in the subcellular distribution and the regulation of the protein using cellular, biochemical, and bioinformatic approaches. Using bioinformatics to compare muskelin orthologues from Mus musculus, Homo sapiens, Rattus norvegicus, Danio rerio, Drosophila melanogaster, and Anopheles gambiae, highly conserved regions within muskelin were identified, with the combination of a discoidin domain, a LisH motif, a C-terminal to LisH motif, and six kelch repeats. A further bioinformatic analysis of the kelch repeat proteins in whole genomes from Homo sapiens, Drosophila melanogaster, and Anopheles gambiae, demonstrated that muskelin has a unique molecular architecture amongst the large family of kelch repeat proteins. EGFP-tagged forms of muskelin were prepared and used to study its cellular localisation and to explore possible regulatory mechanisms. EGFP-MK had a cytoplasmic distribution and also formed mobile, granular inclusion bodies in 25% of the transfected cells. Formation of the inclusion bodies was a specific property of EGFP-wild-type muskelin, because EGFP alone or EGFP-muskelin mutated at key residues within kelch repeat 4 (Y488A/V495A or G474S/G475S) or mutations in the discoidin domain (K132A/I133A/V134A) were equivalently expressed yet had a uniformly diffuse distribution. Because of the complex domain structure of muskelin, a rational view of domain boundaries has proven essential for successful expression of truncated forms. Truncated forms composed of the discoidin or the kelch repeats alone did not form inclusion bodies. In biochemical pull-down assays, muskelin was found to self-associate by a mechanism dependent on both the discoidin domain and the fourth kelch repeat. Mutations in kelch repeat 4 altered the sensitivity to limited proteolysis by proteinase K, suggesting that the protein conformation is altered. To begin to explore physiological factors that regulate the formation of inclusion bodies, the possible roles of five putative protein kinase C (PKC) phosphorylation sites that are conserved between muskelin species orthologues were evaluated in the cellular and biochemical assays. Activation of PKC decreased the formation of the inclusion bodies and resulted in phosphorylation of muskelin. Using a site-directed mutagenesis approach, two PKC phosphorylation sites critical for this activity were identified. Both of these sites are predicted to lie on the same face of the [beta]-propeller structure composed by the six kelch-repeats. Together, these studies have developed new evidence that muskelin is a multidomain protein with multiple protein-protein interaction sites.
... Because EGTA blocks both E-cad (cell-cell) and integrin (cellmatrix) signaling, we tested the effect of EGTA treatment in the presence of Mg 2+ (5 mM) or combined Mg 2+ (2.5 mM) and Mn 2+ (2.5 mM). Both Mg 2+ and Mn 2+ are known to specifically promote integrin-mediating signaling (Shimaoka et al., 2002). Addition of Mg 2+ did not rescue the effect of 20 min EGTA treatment (Fig. 5G,H; Fig. 5I, top), but did rescue the control pattern after 3 h recovery (Fig. 5I, bottom). ...
The pluripotent epiblast gives rise to all tissues and organs in the adult body. Its differentiation starts at gastrulation, when the epiblast generates mesoderm and endoderm germ layers through epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in vitro can adopt either mesenchymal or epithelial morphology. The relationship between epiblast cellular morphology and its pluripotency is not well understood. Here, using chicken epiblast and mammalian pluripotency stem cell (PSC) models, we show that PSCs undergo a mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and its subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible initiation of pluripotency exit, whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and that epiblast pluripotency is restricted to an intermediate cellular state residing between the fully mesenchymal and fully epithelial states.
... Sequence analysis indicates that these MIDAS residues are present in CLCA1 VWA (Whittaker and Hynes, 2002), and we have shown that this motif and the divalent cation are necessary for CLCA1 potentiation of TMEM16A (Sala-Rabanal et al., 2017). VWA domains are found in a functionally diverse range of proteins, including blood coagulation factors (von Willebrand factor), complement factors (Springer, 2006), signaling receptors (α integrins; Shimaoka et al., 2002), toxin receptors (anthrax toxin receptors; Lacy et al., 2004aLacy et al., , 2004bSantelli et al., 2004), extracellular matrix (collagen, Becker et al., 2014;and matrillin, Paulsson and Wagener, 2018), ion channel regulators or subunits (CLCAs, Patel et al., 2009;and Ca v 1.1 α2δ1, Dolphin, 2016), as well as in critical proteins in bacterial pathogens such as Plasmodium (Song et al., 2012) and Toxoplasma (Song and structural and functional features that are unique to distinct families of VWA domains (Becker et al., 2014;Song et al., 2012;Song and Springer, 2014;Springer, 2006). To date, no high-resolution structure has been determined for a VWA domain from the CLCA phylogenetic branch. ...
The secreted protein calcium-activated chloride channel regulator 1 (CLCA1) utilizes a von Willebrand factor type A (VWA) domain to bind to and potentiate the calcium-activated chloride channel TMEM16A. To gain insight into this unique potentiation mechanism, we determined the 2.0-Å crystal structure of human CLCA1 VWA bound to Ca2+. The structure reveals the metal-ion-dependent adhesion site (MIDAS) in a high-affinity "open" conformation, engaging in crystal contacts that likely mimic how CLCA1 engages TMEM16A. The CLCA1 VWA contains a disulfide bond between α3 and α4 in close proximity to the MIDAS that is invariant in the CLCA family and unique in VWA structures. Further biophysical studies indicate that CLCA1 VWA is preferably stabilized by Mg2+ over Ca2+ and that α6 atypically extends from the VWA core. Finally, an analysis of TMEM16A structures suggests residues likely to mediate interaction with CLCA1 VWA.
... Integrins are generally thought to have three major conformations: a bent, closed form, an extended form with the headpiece of the ectodomain still closed, and an extended form with the headpiece open and the α-and β-cytoplasmic tails a greater distance apart (Shimaoka et al., 2002;Li et al., 2017). The binding sites for many integrin ligands are cryptic-integrin activation causes a switch to the extended, open conformation and triggers exposure of these surfaces. ...
The advent of cell-cell and cell-extracellular adhesion enabled cells to interact in a coherent manner, forming larger structures and giving rise to the development of tissues, organs and complex multicellular life forms. The development of such organisms required tight regulation of dynamic adhesive structures by signaling pathways that coordinate cell attachment. Integrin-mediated adhesion to the extracellular matrix provides cells with support, survival signals and context-dependent cues that enable cells to run different cellular programs. One mysterious aspect of the process is how hundreds of proteins assemble seemingly spontaneously onto the activated integrin. An emerging concept is that adhesion assembly is regulated by autoinhibition of key proteins, a highly dynamic event that is modulated by a variety of signaling events. By enabling precise control of the activation state of proteins, autoinhibition enables localization of inactive proteins and the formation of pre-complexes. In response to the correct signals, these proteins become active and interact with other proteins, ultimately leading to development of cell-matrix junctions. Autoinhibition of key components of such adhesion complexes—including core components integrin, talin, vinculin, and FAK and important peripheral regulators such as RIAM, Src, and DLC1—leads to a view that the majority of proteins involved in complex assembly might be regulated by intramolecular interactions. Autoinhibition is relieved via multiple different signals including post-translation modification and proteolysis. More recently, mechanical forces have been shown to stabilize and increase the lifetimes of active conformations, identifying autoinhibition as a means of encoding mechanosensitivity. The complexity and scope for nuanced adhesion dynamics facilitated via autoinhibition provides numerous points of regulation. In this review, we discuss what is known about this mode of regulation and how it leads to rapid and tightly controlled assembly and disassembly of cell-matrix adhesion.
Integrin receptors are heterodimeric surface receptors that play multiple roles regarding cell–cell communication, signaling, and migration. The four members of the β2 integrin subfamily are composed of an alternative α (CD11a–d) subunit, which determines the specific receptor properties, and a constant β (CD18) subunit. This review aims to present insight into the multiple immunological roles of integrin receptors, with a focus on β2 integrins that are specifically expressed by leukocytes. The pathophysiological role of β2 integrins is confirmed by the drastic phenotype of patients suffering from leukocyte adhesion deficiencies, most often resulting in severe recurrent infections and, at the same time, a predisposition for autoimmune diseases. So far, studies on the role of β2 integrins in vivo employed mice with a constitutive knockout of all β2 integrins or either family member, respectively, which complicated the differentiation between the direct and indirect effects of β2 integrin deficiency for distinct cell types. The recent generation and characterization of transgenic mice with a cell-type-specific knockdown of β2 integrins by our group has enabled the dissection of cell-specific roles of β2 integrins. Further, integrin receptors have been recognized as target receptors for the treatment of inflammatory diseases as well as tumor therapy. However, whereas both agonistic and antagonistic agents yielded beneficial effects in animal models, the success of clinical trials was limited in most cases and was associated with unwanted side effects. This unfavorable outcome is most probably related to the systemic effects of the used compounds on all leukocytes, thereby emphasizing the need to develop formulations that target distinct types of leukocytes to modulate β2 integrin activity for therapeutic applications.
Introduction:
Exosomes, a type of extracellular vesicles, are effective tools for delivering small-molecule drugs and biological therapeutics into cells and tissues. Surface modifications with targeting ligands ensure precise delivery to specific cells, minimizing accumulation in healthy organs and reducing the side effects. This is a rapidly growing area in drug delivery research and this review aims to comprehensively discuss the recent advances in the field.
Area covered:
Recent studies have presented compelling evidence supporting the application of exosomes as efficient delivery vehicles that escape endosome trapping, achieving effective in vivo delivery in animal models. This review provides a systemic discussion on the exosome-based delivery technology, with topics covering exosome purification, surface modification, and targeted delivery of various cargos ranging from siRNAs, miRNAs, and proteins, to small molecule drugs.
Expert opinion:
Exosome-based gene and drug delivery has low toxicity and low immunogenicity. Surface modifications of the exosomes can effectively avoid endosome trapping and increase delivery efficiency. This exciting technology can be applied to improve the treatments for a wide variety of diseases.
Regulatory T cells (Treg) are essential for the maintenance of peripheral tolerance. Treg dysfunction results in diverse inflammatory and autoimmune diseases with life-threatening consequences. β2-integrins (CD11a-d/CD18) play important roles in the migration of leukocytes into inflamed tissues and cell signaling. Of all β2-integrins, T cells, including Treg, only express CD11a/CD18, termed lymphocyte function-associated antigen 1 (LFA-1), on their surface. In humans, loss-of-function mutations in the common subunit CD18 result in leukocyte adhesion deficiency type-1 (LAD-1). Clinical symptoms vary depending on the extent of residual β2-integrin function, and patients may experience leukocytosis and recurrent infections. Some patients can develop autoimmune diseases, but the immune processes underlying the paradoxical situation of immune deficiency and autoimmunity have been scarcely investigated. To understand this complex phenotype, different transgenic mouse strains with a constitutive knockout of β2-integrins have been established. However, since a constitutive knockout affects all leukocytes and may limit the validity of studies focusing on their cell type-specific role, we established a Treg-specific CD18-floxed mouse strain. This mini-review aims to delineate the role of LFA-1 for the induction, maintenance, and regulatory function of Treg in vitro and in vivo as deduced from observations using the various β2-integrin-deficient mouse models.
Cell adhesion molecules mediate cell-cell or cell-matrix interactions, some of which are mechanical sensors, such as integrins. Emerging evidence indicates that anthrax toxin receptor 1 (ANTXR1), a newly identified cell adhesion molecule, can also sense extracellular mechanical signals such as hydrostatic pressure and extracellular matrix (ECM) rigidity. ANTXR1 can interact with ECM through connecting intracellular cytoskeleton and ECM molecules (just like integrins) to regulate numerous biological processes, such as cell adhesion, cell migration or ECM homeostasis. Although with high structural similarity to integrins, its functions and downstream signal transduction are independent from those of integrins. In this perspective, based on existing evidence in literature, we analyzed the structural and functional evidence that ANTXR1 can act as a potential sensor for extracellular mechanical cues. To our knowledge, this is the first in-depth overview of ANTXR1 from the perspective of mechanobiology. STATEMENT OF SIGNIFICANCE: An overview of ANTXR1 from the perspective of mechanobiology; An analysis of mechanical sensitivity of ANTXR1 in structure and function; A summary of existing evidence of ANTXR1 as a potential mechano-sensor.
Comprehensive Summary
Controlling the conformational transition of polypeptides under slightly acidic environment is challenging. Herein, we report a class of pH‐responsive helix‐to‐coil conformationally transitionable polypeptides (pCTPs) by simply conjugating tertiary amine groups (TAs) to polylysine. Their conformation is highly dependent on the charge state of TAs, showing a helical structure when most TAs are deprotonated and a non‐helical structure when majority of TAs are protonated. The conformational transition pH can be modulated by tuning the hydrophobicity of TAs and the incorporation of hydrophobic monomers at a pH range of 7.2 to 6.0. Such pCTP showed a vesicle‐to‐micelle transition when their conformation transformed from helix to coil, facilitating controlled drug release. Our study provided an approach to control the conformational transition of polypeptides under slightly acidic condition.
In multicellular organisms, cell adhesion is a pivotal physiological process which is essential for cell–cell communications, cell migration, and interactions with extracellular matrix. Integrins, a family of large hetero-dimeric type I membrane proteins, are known for driving cell adhesion functions. Among 24 different integrins, four β2 integrins, αL β2, αM β2, αX β2 and αD β2, are specific for cell adhesion and migration of leukocytes. Many cytosolic proteins interact with short cytosolic tails (CTs) of β2 and other integrins which are essential in bi-directional signaling processes. Further, phosphorylation of CTs of integrins regulates binding of intra-cellular proteins and signaling systems. In this review, recent advances in structures and interactions of multi-protein complexes of integrin tails, with a focus on β2 integrin, and cytosolic proteins are discussed along with a proposed future direction.
Integrins are transmembrane receptors that play a critical role in many biological processes which can be therapeutically modulated using integrin blockers, such as peptidomimetic ligands. This work aimed to develop new potential β1 integrin antagonists using modeled receptors based on the aligned crystallographic structures and docked with three lead compounds (BIO1211, BIO5192, and TCS2314), widely known as α4β1 antagonists. Lead-compound complex optimization was performed by keeping intact the carboxylate moiety of the ligand, adding substituents in two other regions of the molecule to increase the affinity with the target. Additionally, pharmacokinetic predictions were performed for the ten best ligands generated, with the lowest docking interaction energy obtained for α4β1 and BIO5192. Results revealed an essential salt bridge between the BIO5192 carboxylate group and the Mg2+ MIDAS ion of the integrin. We then generated more than 200 new BIO5192 derivatives, some with a greater predicted affinity to α4β1. Furthermore, the significance of retaining the pyrrolidine core of the ligand and increasing the therapeutic potential of the new compounds is emphasized. Finally, one novel molecule (1592) was identified as a potential drug candidate, with appropriate pharmacokinetic profiles, similar dynamic behavior at the integrin interaction site compared with BIO5192, and a higher predicted affinity to VLA-4.
Neutrophils are the most abundant leukocytes in humans and are critical for innate immunity and inflammation. Integrins are critical for neutrophil functions, especially for their recruitment to sites of inflammation or infections. Integrin conformational changes during activation have been heavily investigated but are still not fully understood. Many regulators, such as talin, Rap1-interacting adaptor molecule (RIAM), Rap1, and kindlin, are critical for integrin activation and might be potential targets for integrin-regulating drugs in treating inflammatory diseases. In this review, we outline integrin activation regulators in neutrophils with a focus on the above critical regulators, as well as newly discovered modulators that are involved in integrin activation.
The ability of integrins on the cell surface to mediate cell adhesion to the extracellular matrix ligands is regulated by intracellular signaling cascades. During this signaling process, the talin (TLN) recruited to integrin cytoplasmic tails plays the critical role of the major adaptor protein to trigger integrin activation. Thus, intracellular levels of TLN are thought to determine integrin-mediated cellular functions. However, the epigenetic regulation of TLN expression and consequent modulation of integrin activation remain to be elucidated. Bioinformatics analysis led us to consider miR-200c-3p as a TLN1-targeting miRNA. To test this, we have generated miR-200c-3p-overexpressing and miR-200c-3p-underexpressing cell lines, including HEK293T, HCT116, and LNCaP cells. Overexpression of miR-200c-3p resulted in a remarkable decrease in the expression of TLN1, which was associated with the suppression of integrin-mediated cell adhesion to fibronectin. In contrast, the reduction in endogenous miR-200c-3p levels led to increased expression of TLN1 and enhanced cell adhesion to fibronectin and focal adhesion plaques formation. Moreover, miR-200c-3p was found to target TLN1 by binding to its 3′-untranslated region (UTR). Taken together, our data indicate that miR-200c-3p contributes to the regulation of integrin activation and cell adhesion via the targeting of TLN1.
This study isolated antibacterial probiotic strains from sour shrimp, a Vietnamese traditional fermented food, and optimized culturing conditions for the chosen strain. Strain V101 was chosen for optimization experiments for its high antagonistic activity and production of strong antibacterial compounds against indicator harmful bacteria – Escherichia coli, and Staphylococcus aureus. Molecular identification of 16S rRNA sequencing showed that V101 strain was Lactobacillus paracasei. Optimization of culturing conditions found that this strain produces the highest biomass and acid accumulation on modified MRS at pH 5.5; temperature 37 °C in 60 h. Under optimum condition, biomass and acid production increased 76.61% and 10.69% respectively, and antibacterial activities against E. coli improved up to 20%. Pilot fermentation of L. paracasei V101 on production Vietnamese pickles, sour shrimp showed that the fermentation process was quicker, tastier, and fermented products could be preserved longer. These findings will be a potential prerequisite for further study on L. paracasei V101 as a strain not only good for food fermentation and preserving but also a helpful probiotic bacteria bring benefits to human health.
The major platelet integrin αIIbβ3 is polymorphic at residue 33, and the Pro³³ isoform exhibits prothrombotic character. We explored via which pathways the Leu³³/Pro³³ polymorphism modulates integrin αIIbβ3 mediated outside-in signaling. We have investigated Src Y418 phosphorylation in human platelets adherent on fibrinogen in the presence or absence of divalent cation Mn²⁺ and of the soluble form of the CD40 ligand (sCD40L). Whereas HPA-1a/1a platelets did not show Src activation on 10 µg/ml immobilized fibrinogen even after 40 min, we observed a significant Src phosphorylation increase in the HPA-1b/1b isoform. Mn²⁺ alone also increased the Src activity in the platelets and addition of Mn²⁺ to platelets adherent on fibrinogen resulted in a synergism of Src Y418 phosphorylation in both isoforms, whereas after 40 min adhesion HPA-1b/1b platelets demonstrated a substantially (sixfold) higher Src kinase activation than HPA-1a/1a. Our observation that in the presence of Mn²⁺ the Src pY418 phosphorylation increases in both isoforms was independent of the fibrinogen concentration suggesting a dominant role of the Mn²⁺. Whereas sCD40L, another αIIbβ3 ligand, did not enhance the Src activity in HPA-1a/1a platelets adherent on fibrinogen, we observed a threefold Src kinase activation in HPA-1b/1b platelets in the presence of sCD40L.
Integrins are a family of 24 adhesion receptors which are both widely-expressed and important in many pathophysiological cellular processes, from embryonic development to cancer metastasis. Hence, integrin inhibitors are valuable research tools which may have promising therapeutic uses. Here, we focus on the four collagen-binding integrins α1β1, α2β1, α10β1 and α11β1. TC-I-15 is a small molecule inhibitor of α2β1 that inhibits platelet adhesion to collagen and thrombus deposition, and obtustatin is an α1β1-specific disintegrin that inhibits angiogenesis. Both inhibitors were applied in cellular adhesion studies, using synthetic collagen peptide coatings with selective affinity for the different collagen-binding integrins and testing the adhesion of C2C12 cells transfected with each. Obtustatin was found to be specific for α1β1, as described, whereas TC-I-15 is shown to be non-specific, since it inhibits both α1β1 and α11β1 as well as α2β1. TC-I-15 was 100-fold more potent against α2β1 binding to a lower-affinity collagen peptide, suggestive of a competitive mechanism. These results caution against the use of integrin inhibitors in a therapeutic or research setting without testing for cross-reactivity.
Cardiovascular diseases including blood vessel disorders represent a major cause of death globally. The essential roles played by local and systemic vascular inflammation in the pathogenesis of cardiovascular diseases have been increasingly recognized. Vascular inflammation triggers the aberrant activation of endothelial cells, which leads to the functional and structural abnormalities in vascular vessels. In addition to humoral mediators such as pro-inflammatory cytokines and prostaglandins, the alteration of physical and mechanical microenvironment – including vascular stiffness and shear stress – modify the gene expression profiles and metabolic profiles of endothelial cells via mechano-transduction pathways, thereby contributing to the pathogenesis of vessel disorders. Notably, connexins and integrins crosstalk each other in response to the mechanical stress, and, thereby, play an important role in regulating the mechano-transduction of endothelial cells. Here, we provide an overview on how the inter-play between connexins and integrins in endothelial cells unfold during the mechano-transduction in vascular inflammation.
The immune system normally functions to mitigate infectious and neoplastic risk. In the absence of immunosuppression, the transplantation of allogeneic tissue constitutes a challenge as allorecognition triggers injurious effector mechanisms culminating in allograft destruction. This chapter will focus on the basic mechanisms of allorecognition, costimulation, T cell amplication, effector mechanisms, innate immunity and antibody production.
A novel gene ( CAS2 ) specifically expressed during appressorium formation was isolated from Colletotrichum gleosporioides using Differential Display RT-PCR. CAS2 comprises 368 deduced amino acid residues and is 50% identical to a hypothetical protein from Chaetomium globosum . ProtFun 2.2 server analysis predicted that Cas2 functions as a transport and binding protein. Based on putative transmembrane domain prediction software (HMMTOP), Cas2 protein is composed of five alpha-helical transmembrane domains with a very short external N-terminus tail and long internal C-terminus. ExPASy ScanProsite analysis showed the presence of integrin beta chain cysteine-rich domain, N-myristoylation site, EGF-like domain, 2Fe-2S ferredoxins, iron-sulfur binding region, VWFC domain, fungal hydrophobins signature, membrane lipoprotein lipid attachment site and Janus-faced atracotoxin (J-ACTX) family signature in CAS2 protein. Mutants with deleted CAS2 were not significantly different in terms of vegetative growth, conidiation, and appressoria production compared to wild type. However, the cas2 mutant produced multipolar germination, a feature which distinguishes it from wild type strain. Interestingly, the mutant is non-virulent to mango fruits, indicating that CAS2 may encode proteins that function as novel virulence factors in fungal pathogens.
Cell–cell adhesion molecules have critically important role in the early events of reproduction including gamete transport, sperm–oocyte interaction, embryonic development, and implantation. Major adhesion molecules involved in reproduction include cadherins, integrins, and disintegrin and metalloprotease domain‐containing (ADAM) proteins. ADAMs on the surface of sperm adhere to integrins on the oocyte in the initial stages of sperm–oocyte interaction and fusion. Cadherins act in early embryos to organize the inner cell mass and trophectoderm. The trophoblast and uterine endometrial epithelium variously express cadherins, integrins, trophinin, and selectin, which achieve apposition and attachment between the elongating conceptus and uterine epithelium before implantation. An overview of the major cell–cell adhesion molecules is presented and this is followed by examples of how adhesion molecules help us to shape early reproductive events. The argument is made that a deeper understanding of adhesion molecules and reproduction will inform new strategies that improve the embryo survival and increase the efficiency of natural mating and assisted breeding in cattle. Cell‐cell adhesion molecules have important roles in the organization of tissues and organs during development and in the maintenance of cellular and tissue integrity throughout life. Adhesion molecules are particularly important in early reproductive events including gamete transport, fertilization, embryonic development, and implantation. A deeper understanding of adhesion molecules in early reproduction should lead to new strategies to increase the proportion of embryos that establish a pregnancy.
Integrin adhesion receptors participate in two-way transfer of information across the plasma membrane. For example, cytoplasmic events, such as activation of protein kinase C, cause an increase in the fibrinogen (Fg) binding affinity of the extracellular domain of integrin alpha(IIb)beta3 (''inside-out signaling''). Conversely, ligand binding to alpha(IIb)beta3 results in the generation of intracellular signals. We used anti-LIBS2, an anti-beta3 monoclonal antibody, to understand potential mechanisms of this bidirectional signaling. Anti-LIBS2 bound to alpha(IIb)beta3 with low affinity (K(d) = 7.4 muM), and mimicked inside-out signaling by promoting Fg binding. The affinity of anti-LIBS2 binding was increased 20-fold (K(d) = 326 nm) by addition of an Fg-mimetic synthetic peptide, RGDS. Thus, anti-LIBS2 and ligands (Fg and Fg-mimetic peptides) bind cooperatively to integrin alpha(IIb)beta3, indicating a functional linkage between the ligand-binding site and the antibody-binding site. The anti-LIBS2-binding site was mapped by its binding to proteolytic and recombinant fragments of the beta3 subunit. The epitope was located within an 89-residue region immediately adjacent to the transmembrane domain and 400 residues carboxyl-terminal to the known ligand-binding site(s). Electron microscope images of rotary shadowed ternary complexes of Fg, anti-LIBS2, and alpha(IIb)beta3 revealed that the ligand-binding site and anti-LIBS2 epitope are separated by about 16 nm. This indicates that propagated long distance conformational changes can occur in alpha(IIb)beta3. Such changes are likely to be involved in the bidirectional signaling function of this integral membrane protein.
The complete analysis of a cDNA clone encoding the mouse integrin beta-7 subunit that was isolated from a lambda-gt10 cDNA library prepared from interleukin-4-activated mouse spleen B cells is reported. The 805-amino acid sequence deduced from the cDNA revealed a signal peptide, a 704-amino acid extracellular domain with eight N-glycosylation sites, a transmembrane domain, and a 61-amino acid intracellular domain. Several structural features were conserved with other integrin beta-chains including the four cysteine-rich epidermal growth factor-like repeat sequences in the extracellular domain. Comparative analysis of the mouse and human beta-7 subunits revealed 87% sequence identity with conservation of three potential intracellular tyrosine phosphorylation sites. The cDNA hybridized to two mRNA transcripts of 3 and 2 kilobases (kb) in size. The 3-kb transcript, which is considered the productive form, was found in T and B leukemic cell lines, a macrophage line, and a mastocytoma cell line. Surprisingly, the smaller 2-kb transcript, which seems unlikely to encode a complete beta-7 protein, was found expressed in epithelial cells cultured from mouse skin. The integrin beta-7 subunit displays sequence identity with the beta-subunit of the M290 antigen found expressed almost exclusively on intraepithelial lymphocytes in the small intestine suggesting that beta-7 may play an adhesive role in intraepithelial lymphocytes immuno-surveillance of the gut mucosa. The cellular distribution of beta-7 transcripts is more extensive than the T-lineage-restricted distribution of the M290 antigen, indicating that beta-7 may be the common beta-subunit for more than one receptor or that translation of beta-7 transcripts varies in different cell types.
We screened monoclonal antibodies to cell-surface proteins and selected an antibody, called 6H1, that recognizes a putative integrin-associated protein. The 6H1 monoclonal antibody (mAb) indirectly coprecipitated α3β1 and/or α6β1, but not α2β1, or α5β1 from Brij 96 detergent lysates of multiple cell lines. Large scale purification using the 6H1 mAb yielded a single protein of 45-60 kDa with an amino-terminal sequence that exactly matched CD63. Confirming that the 6H1 mAb recognized the CD63 protein, 6H1 and a known anti-CD63 mAb yielded identical coprecipitation results and identical colocalization into lysosomal granules containing cathepsin D. Furthermore, we used an established anti-CD63 mAb to detect this protein in an α3β1 immunoprecipitate, and also we observed VLA-3 and CD63 colocalization in cellular “footprints.” Notably, the cytoplasmic domain of α3 was neither required nor sufficient for CD63 association, suggesting that it occurred elsewhere within the α3β1 complex. Knowledge of these specific CD63-α3β1 and CD63-α6β1 biochemical associations should lead to critical insights into the specialized functions of α3β1, α6β1, and CD63.
Integrin αIIbβ3, a platelet fibrinogen receptor, is critically involved in thrombosis and hemostasis. However, how ligands interact with αIIbβ3 has been controversial. Ligand-mimetic anti-αIIbβ3 antibodies (PAC-1, LJ-CP3, and OP-G2) contain the RGD-like RYD sequence in their CDR3 in the heavy chain and have structural and functional similarities to native ligands. We have located binding sites for ligand-mimetic antibodies in αIIb and β3 using human-to-mouse chimeras, which we expect to maintain functional integrity of αIIbβ3. Here we report that these antibodies recognize several discontinuous binding sites in both the αIIb and β3 subunits; these binding sites are located in residues 156–162 and 229–230 of αIIb and residues 179–183 of β3. In contrast, several nonligand-mimetic antibodies (e.g. 7E3) recognize single epitopes in either subunit. Thus, binding to several discontinuous sites in both subunits is unique to ligand-mimetic antibodies. Interestingly, these binding sites overlap with several (but not all) of the sequences that have been reported to be critical for fibrinogen binding (e.g. N-terminal repeats 2–3 but not repeats 4–7, of αIIb). These results suggest that ligand-mimetic antibodies and probably native ligands may make direct contact with these discontinuous binding sites in both subunits, which may constitute a ligand-binding pocket.
Integrins can be expressed in at least three functional states (i.e. latent, active, and ligand-occupied). However, the molecular bases for the transitions between these states are unknown.
In the present study, changes in the accessibility of several β1 epitopes (e.g.N29, B44, and B3B11) were used to probe activation-related conformational changes. Dithiothreitol or Mn2+ activation of integrin-mediated adhesion in the human B cell line, IM9, resulted in a marked increase in the exposure of
the B44 epitope, while N29 expression levels were most sensitive to dithiothreitol treatment. These results contrasted with
the epitope expression patterns of spontaneously adherent K562 cells, where N29 was almost fully accessible and B44 was low.
Addition of a soluble ligand resulted in a marked increase in B44 levels, suggesting that this antibody detected a ligand-induced
binding site. The N29 epitope was mapped to a cysteine-rich region near the NH2 terminus of the integrin chain, thus defining a novel regulatory site.
These studies indicate that the activation of integrin function by different stimuli may involve related but nonidentical
conformations. Both Mn2+ and dithiothreitol appear to induce localized conformational changes that mimic a ligand-occupied receptor. This differs
from the “physiologically” activated integrins on K562 cells that display a marked increase in overall epitope accessibility
without exposure of the ligand-induced binding site epitopes. The increased exposure of the N29 site on K562 cells may indicate
a role for this region in the regulation of integrin function.
A central characteristic of integrin adhesion receptors is their capacity to become activated, thereby enhancing their affinity
for ligands. Here, we report the identification of a discrete site within the I domain of integrin αMβ2, which modulates the adhesive activity of this receptor. Based upon the crystal structure, this region is composed of two
short and spatially proximal loops, E162QLKKSKTL and Q190NNPNPRS. Mutations in these loops yield receptors which support spontaneous cell adhesion to fibrinogen, whereas mutation
of an adjacent region and wild-type receptors require activation to adhere to this substrate. An activating monoclonal antibody
enhanced the adhesive activity of one but not the other loop mutants, suggesting that the activation states of these two mutant
receptors were not identical. Given that similar I domains exist in several other integrin α subunits and non-integrin proteins,
and possibly in all integrin β subunits, these two loop segments may represent a universal target for controlling integrin
activation and the function of other I domain-containing proteins. In support of this hypothesis, several naturally occurring
mutations that activate von Willebrand factor map to the same loops of its I(A) domain.
Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alpha IIb or 3 allowed normal expression, heterodimerization, processing and surface exposure of alpha IIb(GFP)beta3 and aIIb beta3(GFP) receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alpha IIb beta3 capping revealed an intense autofluorescent cap corresponding to unlabelled immuno-clustered GFP-tagged aIIb beta3. GFP-tagged aIIb beta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(PAK) tyrosine phosphorylation similar to wild-type alpha IIb beta3 (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alpha IIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alpha IIb beta3 receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alpha IIb beta3 during the early stages of cell attachment and spreading, starting with alpha IIb beta3 clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alpha IIb beta3 in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alpha IIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the 3 integrin cytoplasmic tail, rather than the alpha IIb subunit, plays a major role in alpha IIb beta3 affinity modulation. With the successful direct visualization of functional aIIb beta3 receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.
Integrin αIIbβ3 mediates platelet aggregation and “outside-in” signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to αIIbβ3 function, αIIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified αIIb subunits were expressed with β3 in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25–50% of that observed when αIIbβ3 affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of αIIbβ3 also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72Syk and fibrinogen-dependent phosphorylation of pp125FAK, even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in αIIbβ3 function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of αIIbβ3 in platelets.
Integrin b subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membraneycytosol to the ligand-binding headpiece. Se- quence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat bound- aries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connec- tivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor- like modules (Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. & Krissansen, G. W. (1999) Genomics 56, 169 -178). N-terminally truncated b2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 1 3 were expressed in bacteria and refolded. The module 2 1 3 fragment was as reactive with three mAbs to activation epitopes as a b2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.
Integrins are heterodimeric (α, β) cell adhesion receptors. We demonstrate that point mutations in the cytoplasmic domains
of both the α and β subunits promote constitutive signaling by the integrin αβ. By generating charge reversal mutations, we show these “activating” mutations may act by disrupting a potential salt bridge
between the membrane-proximal portions of the α and β subunit cytoplasmic domains. Thus, the modulation of specific interactions
between the α and β subunit cytoplasmic domains may regulate transmembrane signaling through integrins. In addition, these
activating mutations induce dominant alterations in cellular behavior, such as the assembly of the extracellular matrix. Consequently,
somatic mutations in integrin cytoplasmic domains could have profound effects in vivo on integrin-dependent functions such as matrix assembly, cell migration, and anchorage-dependent cell growth and survival.
We have determined the high resolution crystal structure of the I domain from the α-subunit of the integrin α2β1, a cell surface
adhesion receptor for collagen and the human pathogen echovirus-1. The domain, as expected, adopts the dinucleotide-binding
fold, and contains a metal ion-dependent adhesion site motif with bound Mg2+ at the top of the β-sheet. Comparison with the crystal structures of the leukocyte integrin I domains reveals a new helix
(the C-helix) protruding from the metal ion-dependent adhesion site face of the domain which creates a groove centered on
the magnesium ion. Modeling of a collagen triple helix into the groove suggests that a glutamic acid side chain from collagen
can coordinate the metal ion, and that the C-helix insert is a major determinant of binding specificity. The binding site
for echovirus-1 maps to a distinct surface of the α2-I domain (one edge of the β-sheet), consistent with data showing that
virus and collagen binding occur by different mechanisms. Comparison with the homologous von Willebrand factor A3 domain,
which also binds collagen, suggests that the two domains bind collagen in different ways.
Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated
by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin α2β1 and a Staphylococcus aureus adhesin reveal a “trench” on the surface of both of these proteins. This trench can accommodate a collagen triple-helical
structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R.
C. (1997) J. Biol. Chem. 272, 28512–28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A.,
DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 4, 833–838). We report here the crystal structure of the α subunit I domain from the α1β1 integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked.
Furthermore, we compare the collagen-binding mechanisms of the human α1 integrin I domain and the A domain from the S. aureus collagen adhesin, Cna. Although the S. aureus and human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective
ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their
affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen
via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from
a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands
that contain repeating structural motifs.
von Willebrand Factor (vWF) is a multimeric protein that mediates platelet adhesion to exposed subendothelium at sites of
vascular injury under conditions of high flow/shear. The A1 domain of vWF (vWF-A1) forms the principal binding site for platelet
glycoprotein Ib (GpIb), an interaction that is tightly regulated. We report here the crystal structure of the vWF-A1 domain
at 2.3-Å resolution. As expected, the overall fold is similar to that of the vWF-A3 and integrin I domains. However, the structure
also contains N- and C-terminal arms that wrap across the lower surface of the domain. Unlike the integrin I domains, vWF-A1
does not contain a metal ion-dependent adhesion site motif. Analysis of the available mutagenesis data suggests that the activator
botrocetin binds to the right-hand face of the domain containing helices α5 and α6. Possible binding sites for GpIb are the
front and upper surfaces of the domain. Natural mutations that lead to constitutive GpIb binding (von Willebrand type IIb
disease) cluster in a different site, at the interface between the lower surface and the terminal arms, suggesting that they
disrupt a regulatory region rather than forming part of the primary GpIb binding site. A possible pathway for propagating
structural changes from the regulatory region to the ligand-binding surface is discussed.
The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2-A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the insertion (I)-domain on the alpha chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.
The 3.0-A structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding to Plasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A' strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.
The role of fibrinogen as a cofactor for platelet aggregation was examined by measuring the binding of 125I-labeled human fibrinogen to gel-filtered human platelets both before and after platelet stimulation by ADP and epinephrine. Platelet stimulation by ADP resulted in the rapid, reversible binding of fibrinogen to receptors on the platelet surface. Fibrinogen binding increased as the concentration of ADP was increased from 0.1 to 2 microM, reaching a plateau at higher ADP concentrations. Binding occurred only after platelet stimulation and in the presence of divalent cations. However, fibrinogen binding did not occur to ADP-stimulated platelets from three patients with Glanzmann's thrombasthenia. Analysis of fibrinogen binding as a function of increasing fibrinogen concentration demonstrated that maximal platelet stimulation exposed approximately or equal to 45,000 binding sites per platelet with a dissociation constant of 80--170 nM. These fibrinogen binding parameters were essentially the same whether ADP or epinephrine was the platelet-stimulating agent. Thus, these studies demonstrate that platelet stimulation by ADP and epinephrine exposes a limited number of fibrinogen receptors on the platelet surface. Furthermore, these data suggest that the fibrinogen molecules bound to the platelet as a consequence of platelet stimulation are directly involved in the platelet aggregation response.
The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), is a calcium-dependent heterodimer that binds fibrinogen, von Willebrand factor, and fibronectin after platelet activation. We examined GPIIb-IIIa alone and bound to these ligands by electron microscopy after rotary shadowing with platinum/tungsten. We found, as observed previously, that in the presence of detergent and 2 mM Ca2+, GPIIb-IIIa consists of an 8 x 12-nm globular head with two 18-nm flexible tails extending from one side. We also found that in the presence of EDTA, GPIIb-IIIa dissociates into two similar comma-shaped subunits, each containing a portion of the globular head and a single tail. Using monoclonal antibodies to GPIIb, GPIIIa, and the GPIIb-IIIa heterodimer, we found that the tails contained the carboxyl termini of each subunit, while the nodular head was composed of amino-terminal segments of both subunits. Electron microscopy of GPIIb-IIIa bound to fibrinogen revealed a highly specific interaction of the nodular head of GPIIb-IIIa with the distal end of the trinodular fibrinogen molecule and with the tails of GPIIb-IIIa extended laterally at an angle of approximately 98 degrees with respect to the long axis of fibrinogen. When a GPIIb-IIIa was bound to each end of a single fibrinogen, the tails were oriented to opposite sides of fibrinogen, enabling fibrinogen to bridge two adjacent platelets. Electron microscopy of GPIIb-IIIa bound to fibronectin revealed GPIIb/IIIa-binding sites approximately two-thirds of the distance from the amino terminus of each end of the fibronectin molecule, while GPIIb-IIIa was found to bind to von Willebrand factor protomers along a rod-like region near the central nodule of the molecule.
Integrins are cell-surface heterodimers formed by the association of one alpha- and one beta-subunit. Glycoprotein IIIa (GPIIIa or beta 3 subunit) is the common beta-subunit of the beta 3 subfamily of integrins, which, when associated with glycoprotein IIb (GPIIb), constitutes the receptor for fibrinogen and other adhesive proteins at the platelet surface (the GPIIb-IIIa complex) and, when associated with the alpha v-subunit, constitutes the vitronectin receptor present in several cell types. Protein chemical analysis of GPIIIa allows us to define the following structural domains: the cysteine-rich and proteinase-resistant N-terminal domain (GPIIIa 1-62); the adhesive-protein-binding domain (GPIIIa 101-422); the cysteine-rich and proteinase-resistant core (GPIIIa 423-622); and the C-terminal domain comprising an extracellular subdomain (GPIIIa 623-692), a transmembrane subdomain (GPIIIa 693-721), and a cytoplasmic subdomain (GPIIIa 722-762). We also assign unambiguously the disulphide bonds within the N-terminal, the fibrinogen-binding and the C-terminal domains, and the two long-range disulphide bonds which join the N-terminus to the proteinase-resistant core (Cys5-Cys435) and the fibrinogen-binding domain to the extracellular side of the C-terminal domain (Cys406-Cys655). In addition, we propose three alternative models for the arrangement of the disulphide bonds within the core and of the disulphide bonds joining the core to the extracellular side of the C-terminal domain, consistent with our experimental findings, favouring temporarily that which imposes less steric hindrance for the formation of these disulphide bonds. On the basis of this information and on the highly conserved overall structure observed in the beta-subunits of the integrin family known so far, except in beta 4, we propose to extend the cysteine-pairing pattern and the structural domains outlined here for GPIIIa to all the beta-subunits of the integrin family.
Despite the identification and characterization of several distinct ligands for the leukocyte integrin (CD11/CD18) family of adhesion receptors, little is known about the structural regions on these molecules that mediate ligand recognition. In this report, we use alpha subunit chimeras of Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), and an extended panel of newly generated and previously characterized mAbs specific to the alpha chain of Mac-1 to map the binding sites for four distinct ligands for Mac-1: iC3b, fibrinogen, ICAM-1, and the as-yet uncharacterized counter-receptor responsible for neutrophil homotypic adhesion. Epitopes of mAbs that blocked ligand binding were mapped with the chimeras and used to localize the ligand recognition sites because the data obtained from functional assays with the Mac-1/p150,95 chimeras were not easily interpreted. Results show that the I domain on the alpha chain of Mac-1 is an important recognition site for all four ligands, and that the NH2-terminal and perhaps divalent cation binding regions but not the COOH-terminal segment may contribute. The recognition sites in the I domain appear overlapping but not identical as individual Mac-1-ligand interactions are distinguished by the discrete patterns of inhibitory mAbs. Additionally, we find that the alpha subunit NH2-terminal region and divalent cation binding region, despite being separated by over 200 amino acids of the I domain, appear structurally apposed because three mAbs require the presence of both of these regions for antigenic reactivity, and chimeras that contain the NH2 terminus of p150,95 require the divalent cation binding region of p150,95 to associate firmly with the beta subunit.
The integrins are a family of receptors involved in both cell-matrix and cell-cell interactions. They are heterodimers consisting of an a subunit non-covalently associated with a $\beta$ subunit. Each integrin heterodimer forms a transmembrane complex with the extracellular domain interacting with a ligand and the intracellular domain interacting with cytoskeletal elements. As a way to identify the functional domains on the $\beta\sb1$ subunit, the epitopes for a panel of chicken $\beta\sb1$-specific mAbs that interfere with particular integrin functions were determined. The epitopes for one set of mAbs that block ligand binding were mapped toward the N-terminal 200 residues. Residues 141 to 160 may fit into an $\alpha$-helix and a peptide of this sequence perturbed the binding between the $\beta\sb1$ integrin and fibronectin. A second set of mAbs that either block ligand binding, alter ligand specificity or induce $\alpha/\beta$ subunit dissociation were mapped to a region near the transmembrane segment. These data suggest a portion of $\beta\sb1$ ligand binding domain rests within the first 200 residues and a regulatory domain, that affects ligand binding through secondary conformational changes, resides in a region close to the transmembrane domain. To investigate the structural basis of the functional linkage between these two domains, cysteine residues implicated in long-range disulfide bond formation were mutated and the functional effects examined. The results showed that subtle structural changes of the $\beta\sb1$ subunit affect events subsequent to ligand binding, including sub-cellular localization, matrix assembly, cell migration and signal transduction.
We show that the adhesion of T lymphoid cells to immobilized fibronectin can be increased by two distinct mechanisms. The first is by increasing the affinity of the fibronectin receptor/ligand interaction using the anti-beta 1 integrin monoclonal antibody 8A2. The second is by treating the cells with phorbol 12-myristate 13-acetate (PMA), which alters events that occur after receptor occupancy (e.g., cell spreading) without affecting receptor affinity. The effects of these two mechanisms on adhesion in the presence of physiological concentrations of soluble fibronectin suggest that they have different biological consequences. Under these conditions, the net effect of increasing the affinity of the fibronectin receptors is to decrease cell adhesion, whereas the increase in adhesion induced by PMA is unaffected. This suggests that the high affinity receptors are not primarily available for cell adhesion under these circumstances, and that they have an alternative function. We further show that high affinity binding of soluble fibronectin can be induced by either differentiation of the monocytic cell line THP-1 or by cross-linking the T cell receptor complexes on the T lymphoid cell line HUT-78. The differentiated monocytic cells express two populations of fibronectin receptors: a minority in a high affinity state, and the majority in a low affinity state. Thus they will both continue to adhere in the presence of physiological concentrations of soluble fibronectin and bind significant amounts of soluble fibronectin at the cell surface.
We report the 1.8-Å crystal structure of the CD11a I-domain with bound manganese ion. The CD11a I-domain contains binding sites for intercellular adhesion molecules 1 and 3 and can exist in both low- and high-affinity states. The metal-bound form reported here is likely to represent a high-affinity state. The CD11a I-domain structure reveals a strained hydrophobic ridge adjacent to the bound metal ion that may serve as a ligand-binding surface and is likely to rearrange in the absence of bound metal ion. The CD11a I-domain is homologous to domains found in von Willebrand factor, and mapping of mutations found in types 2a and 2b von Willebrand disease onto this structure allows consideration of the molecular basis of these forms of the disease.
Chemokines trigger rapid integrin-dependent lymphocyte arrest to vascular endothelium. We show that the chemokines SLC, ELC, and SDF-1α rapidly induce lateral mobility and transient increase of affinity of the β2 integrin LFA-1. Inhibition of phosphatidylinositol 3-OH kinase (PI(3)K) activity blocks mobility but not affinity changes and prevents lymphocyte adhesion to ICAM-1 immobilized at low but not high densities, suggesting that mobility enhances the frequency of encounters between high-affinity integrin and ligand but that at higher ligand density affinity changes are sufficient for arrest. Thus, chemokines trigger, through distinct signaling pathways, both a high-affinity state and lateral mobility of LFA-1 that can coordinately determine the vascular arrest of circulating lymphocytes under physiologic conditions.
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We have determined the high resolution crystal structure of the A domain from the a chain of integrin CR3. The domain adopts a classic α/β “Rossmann” fold and contains an unusual Mg2+ coordination site at its surface. One of the coordinating ligands is the glutamate side chain from another A domain molecule. We suggest that this site represents a general metal ion-dependent adhesion site (MIDAS) for binding protein ligands. We further propose that the subunits of integrins contain a MIDAS motif within a modified A domain. Our crystal structure will allow reliable models to be built for other members of the A domain superfamily and should facilitate development of novel adhesion modulatory drugs.
The cysteine-rich region (CRR) of the integrin β subunits is organised into four repeating elements. By expression of a panel of truncated β2 subunits, and CRR segments fused to the C-terminal end of a CD4 soluble fragment, the segment required for the expression of two monoclonal antibody conformational epitopes was determined. This segment, E482–Q574, contains 16 cysteines representing two repeating units. We have thus defined the CRR unit motif of ‘xC---C---C---CxCxxCxC---Cx’, where ‘x’ represents a single residue, and ‘---’ represents a stretch of four to 14 residues.
The α1β1 integrin is a major cell surface receptor for collagen. Ligand binding is mediated, in part, through a ∼200 amino acid inserted ‘I’-domain contained in the extracellular part of the integrin α chain. Integrin I-domains contain a divalent cation binding (MIDAS) site and require cations to interact with integrin ligands. We have determined the crystal structure of recombinant I-domain from the rat α1β1 integrin at 2.2 Å resolution in the absence of divalent cations. The α1 I-domain adopts the dinucleotide binding fold that is characteristic of all I-domain structures that have been solved to date and has a structure very similar to that of the closely related α2β1 I-domain which also mediates collagen binding. A unique feature of the α1 I-domain crystal structure is that the MIDAS site is occupied by an arginine side chain from another I-domain molecule in the crystal, in place of a metal ion. This interaction supports a proposed model for ligand-induced displacement of metal ions. Circular dichroism spectra determined in the presence of Ca2+, Mg2+ and Mn2+ indicate that no changes in the structure of the I-domain occur upon metal ion binding in solution. Metal ion binding induces small changes in UV absorption spectra, indicating a change in the polarity of the MIDAS site environment.
The interaction between leukocyte function-associated antigen-1 (LFA-1), a member of the beta (2)-integrin family of adhesion molecules, and intracellular adhesion molecule ICAM-1 (cd54) is thought to play a critical role in the inflammatory process. On the basis of an anilino diaryl sulfide screening lead 1, in combination with pharmacophore analysis of other screening hits, we have identified an adjacent binding pocket. Subsequently, a p-ethenylcarbonyl linker was discovered to be optimal for accessing this binding site. Solution-phase parallel synthesis enabled rapid optimization of the cinnamides for this pocket. In conjunction with fine-tuning of the diaryl substituents, we discovered a novel series of potent, nonpeptide inhibitors of LFA-1/ICAM-1 interaction, exemplified by A-286982 (28h), which has IC50 values of 44 and 35 nM in an LFA-1/ICAM-1 binding assay and LFA-l-mediated cellular adhesion assay, respectively.
We used integrin alphaLbeta2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins. mAbs to the alphaL I domain and beta2 I-like domain inhibit adhesion of wild-type alphaLbeta2 to intercellular adhesion molecule-1. However, with alphaLbeta2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and (ii) that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, alphaLbeta2 containing a locked open I domain was completely resistant to inhibition by mAbs to the beta2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type alphaLbeta2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by Mn(2+), as measured with mAb m24, which we map here to the beta2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or Mn(2+)-induced exposure of the KIM127 epitope in the beta2 stalk region. Furthermore, locked open I domains, in alphaLbeta2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in Mg(2+) and Mn(2+). These results suggest that Mn(2+) activates alphaLbeta2 by binding to a site other than the I domain, most likely the I-like domain of beta2.
We introduced disulfide bonds to lock the integrin L2 I domain in predicted open, ligand binding or closed, nonbinding conformations. Transfectants expressing L2 heterodimers containing locked-open but not locked-closed or wild-type I domains constitutively adhered to intercellular adhesion molecule-1 (ICAM-1) substrates. Locking the I domain closed abolished constitutive and activatable adhesion. The isolated locked-open I domain bound as well as the activated L2 heterodimer, and binding was abolished by reduction of the disulfide. Lovastatin, which binds under the conformationally mobile C-terminal alpha-helix of the I domain, inhibited binding to ICAM-1 by alphaLbeta2 with wild-type, but not locked-open I domains. These data establish the importance of conformational change in the alphaL I domain for adhesive function and show that this domain is sufficient for full adhesive activity.
We have used immunoprecipitation with mAbs to probe folding during biosynthesis of the beta 2 integrin subunit of lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) before and after association with the alpha L subunit. An evolutionarily conserved region is present in the beta 2 subunit between amino acid residues 102 and 344. mAbs to one subregion before the conserved region, and two subregions after the conserved domain, immunoprecipitated both the unassociated beta '{}2 precursor and mature alpha L/beta 2 complex, suggesting portions of these subregions are folded before association with alpha L. An activating mAb to the C-terminal cysteine-rich region, KIM127, preferentially bound to the unassociated beta subunit, suggesting that it may bind to an epitope that is in an alpha beta interface in unactivated LFA-1. By contrast, mAbs to five different epitopes in the conserved region did not react with unassociated beta '{}2 precursor, suggesting that this region folds after alpha L association and is intimately associated with the alpha L subunit in the alpha L/beta 2 complex. mAbs to two different epitopes that involve the border between the conserved region and the C-terminal segment, were fully or partially reactive with the beta '{}2 precursor, suggesting that this region is partially folded before association with alpha L. The findings suggest that the conserved region is a distinct folding and hence structural unit, and is intimately associated with the alpha subunit.
The cysteine-rich repeats in the stalk region of integrin β subunits appear to convey signals impinging on the cytoplasmic domains to the ligand-binding headpiece of integrins. We have examined the functional properties of mAbs to the stalk region and mapped their epitopes, providing a structure-function map. Among a panel of 14 mAbs to the β2 subunit, one, KIM127, preferentially bound to αLβ2 that was activated by mutations in the cytoplasmic domains, and by Mn2+. KIM127 also bound preferentially to the free β2 subunit compared with resting αLβ2. Activating β2 mutations also greatly enhanced binding of KIM127 to integrins αMβ2 and αXβ2. Thus, the KIM127 epitope is shielded by the α subunit, and becomes reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2, MEM48, and KIM185, activated αLβ2 and bound equally well to resting and activated αLβ2, differentially recognized resting αMβ2 and αXβ2, and bound fully to activated αMβ2 and αXβ2. The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and MEM48 bind to overlapping epitopes involving residues 534, 536, 541, 543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185 maps near the end of cysteine-rich repeat 4. The nonactivating mAbs, 6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344–432 and 432–487, respectively. We thus define five different β2 stalk subregions, mAb binding to which correlates with effect on activation, and define regions in an interface that becomes exposed upon integrin activation.
The Ribbons software interactively displays molecular models, analyzes crystallographic results, and creates publication-quality images. The ribbon drawing popularized by Richardson is featured in this software. Spacefilling and ball-and-stick representations, dot and triangular surfaces, density map contours, and text are also supported. Atomic coordinates in Protein Data Bank (PDB) format are input. Output may be produced in the inventor/virtual reality modeling language (VRML) format. The VRML format has become the standard for three-dimensional interaction on the World Wide Web. The on-line manual is presented in hypertext markup language suitable for viewing with a standard Web browser. The examples give the flavor of the software system. Nearly 100 commands are available to create primitives and output. Examples include creating spheres colored by residue type, fitting a cylinder to a helix, and making a Ramachandran plot. The user essentially creates a small database of American Standard Code for Information Interchange (ASCII) files. This provides extreme flexibility in customizing the display.
Integrins are adhesion molecules that convey signals both to and from the cytoplasm across the plasma membrane. In resting cells, integrins in a low affinity state can be activated by 'inside-out signaling', in which signals affecting integrin heterodimer cytoplasmic domains cause a conformational change in the integrin ligand-binding headpiece connected to the membrane by two long, approximately 16 nm stalks. Here we demonstrate a mechanism for conveying a conformational change over the long distance from the plasma membrane to the headpiece. We prepared soluble, alpha5beta1 integrin heterodimer extracellular fragments in which interactions between alpha- and beta-subunit cytoplasmic domains were replaced with an artificial clasp. Release of this C-terminal clasp by specific protease cleavage resulted in an approximately 14 nm separation of the stalks coupled to increased binding to fibronectin. This activation did not require any associated molecules or clustering and was observed with physiological concentrations of divalent cations. These findings suggest that the overall mechanism for integrin inside-out activation involves the spatial separation of the cytoplasmic and/or transmembrane domains.
We have determined the crystal structure of a complex between the I domain of integrin α2β1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin–ligand recognition.
Several distinct regions of the integrin αIIb subunit have been implicated in ligand binding. To localize the ligand binding sites in αIIb, we swapped all 27 predicted loops with the corresponding sequences of α4 or α5. 19 of the 27 swapping mutations had no effect on binding to both fibrinogen and ligand-mimetic antibodies (e.g. LJ-CP3), suggesting that these regions do not contain major ligand binding sites. In contrast, swapping the remaining 8 predicted
loops completely blocked ligand binding. Ala scanning mutagenesis of these critical predicted loops identified more than 30
discontinuous residues in repeats 2–4 and at the boundary between repeats 4 and 5 as critical for ligand binding. Interestingly,
these residues are clustered in the predicted β-propeller model, consistent with this model. Most of the critical residues
are located at the edge of the upper face of the propeller, and several critical residues are located on the side of the propeller
domain. None of the predicted loops in repeats 1, 6, and 7, and none of the four putative Ca2+-binding predicted loops on the lower surface of the β-propeller were important for ligand binding. The results map an important
ligand binding interface at the edge of the top and on the side of the β-propeller toroid, centering on repeat 3.
Integrin αIIbβ3, a platelet fibrinogen receptor, is critically involved in thrombosis and hemostasis. However, how ligands interact with
αIIbβ3 has been controversial. Ligand-mimetic anti-αIIbβ3 antibodies (PAC-1, LJ-CP3, and OP-G2) contain the RGD-like RYD sequence in their CDR3 in the heavy chain and have structural
and functional similarities to native ligands. We have located binding sites for ligand-mimetic antibodies in αIIb and β3 using human-to-mouse chimeras, which we expect to maintain functional integrity of αIIbβ3. Here we report that these antibodies recognize several discontinuous binding sites in both the αIIb and β3 subunits; these binding sites are located in residues 156–162 and 229–230 of αIIb and residues 179–183 of β3. In contrast, several nonligand-mimetic antibodies (e.g. 7E3) recognize single epitopes in either subunit. Thus, binding to several discontinuous sites in both subunits is unique
to ligand-mimetic antibodies. Interestingly, these binding sites overlap with several (but not all) of the sequences that
have been reported to be critical for fibrinogen binding (e.g. N-terminal repeats 2–3 but not repeats 4–7, of αIIb). These results suggest that ligand-mimetic antibodies and probably native ligands may make direct contact with these discontinuous
binding sites in both subunits, which may constitute a ligand-binding pocket.
To establish a structure and function map of the β2 integrin subunit, we mapped the epitopes of a panel of β2 monoclonal antibodies
including function-blocking, nonblocking, and activating antibodies using human/mouse β2 subunit chimeras. Activating antibodies
recognize the C-terminal half of the cysteine-rich region, residues 522–612. Antibodies that do not affect ligand binding
map to residues 1–98 and residues 344–521. Monoclonal antibodies to epitopes within a predicted I-like domain (residues 104–341)
strongly inhibit LFA-1-dependent adhesion. These function-blocking monoclonal antibodies were mapped to specific residues
with human → mouse knock-out or mouse → human knock-in mutations. Combinatorial epitopes involving residues distant in the
sequence provide support for a specific alignment between the β-subunit and I domains that was used to construct a three-dimensional
model. Antigenic residues 133, 332, and 339 are on the first and last predicted α-helices of the I-like domain, which are
adjacent on its “front.” Other antigenic residues in β2 and in other integrin β subunits are present on the front. No antigenic
residues are present on the “back” of the domain, which is predicted to be in an interface with other domains, such as the
α subunit β-propeller domain. Most mutations in the β2 subunit in leukocyte adhesion deficiency are predicted to be buried
in the β2 subunit I-like domain. Two long insertions are present relative to α-subunit I-domains. One is tied down to the
back of the I-like domain by a disulfide bond. The other corresponds to the “specificity-determining loop” defined in β1 and
β3 integrins and contains the antigenic residue Glu175 in a disulfide-bonded loop located near the “top” of the domain.
The leukocyte integrin αXβ2 (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. αXβ2 is more resistant to activation than other β2 integrins and is inactive in transfected cells. However, when human αX is paired with chicken or mouse β2, αXβ2 is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the
N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked
by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats.
Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of
the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface
that restrains the human αX/human β2 integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that
activate ligand binding.
The αM subunit of integrin Mac-1 contains several distinct regions in its extracellular segment. The N-terminal region has
been predicted to fold into a β-propeller domain composed of seven β-sheets each about 60 amino acid residues long, with the
I-domain inserted between β-sheets 2 and 3. The structure of the C-terminal region is unknown. We have used monoclonal antibodies
(mAbs) as probes to study the dependence of the structure of different regions of the αM subunit on association with the β2
subunit in the αM/β2 heterodimer. All of the mAbs to the I-domain immunoprecipitated the unassociated αM precursor and reacted
with the αM subunit expressed alone on the surface of COS cells. By contrast, four mAbs to the β-propeller domain did not
react with the unassociated αM precursor nor with the uncomplexed αM subunit expressed on COS cell surface. The four mAbs
were mapped to three subregions in three different β-sheets, making it unlikely that each recognized an interface between
the α and β subunits. These results suggest that folding of different β-propeller subregions is coordinate and is dependent
on association with the β2 subunit. The segment C-terminal to the β-propeller domain, residues 599–1092, was studied with
nine mAbs. A subset of four mAbs that reacted with the αM/β2 complex but not with the unassociated αM subunit were mapped
to one subregion, residues 718–759, and five other mAbs that recognized both the unassociated and the complexed αM subunit
were localized to three other subregions, residues 599–679, 820–882, and 943–1047. This suggests that much of the region C-terminal
to the β-propeller domain folds independently of association with the β2 subunit. Our data provide new insights into how different
domains in the integrin α and β subunits may interact.
A recombinant peptide encoding the CD11b A domain bound 54Mn2+ with a high affinity. Other divalent cations, including Mg2+, Zn2+, Ni2+, Co2+, and Cd2+, but not Ca2+ or Ba2+ competed effectively for Mn2+ binding. Amino acid substitutions within two conserved and noncontiguous regions in the recombinant peptide abolished 54Mn2+ binding. When these substitutions were introduced independently in complement receptor type 3 (CR3), each abolished the metal-dependent binding of the receptor to the major C3 opsonin iC3b, without impairing subunit association or surface expression of the receptor. These findings identify an unsuspected and novel metal-binding site within the A domain of CR3 that is required for metal-dependent ligand binding and also identify a good target for designing drugs aimed at countering the inflammatory potential of this key receptor.
Integrins can be expressed in at least three functional states (i.e. latent, active, and ligand-occupied). However, the molecular bases for the transitions between these states are unknown. In the present study, changes in the accessibility of several beta1 epitopes (e.g. N29, B44, and B3B11) were used to probe activation-related conformational changes. Dithiothreitol or Mn2+ activation of integrin-mediated adhesion in the human B cell line, IM9, resulted in a marked increase in the exposure of the B44 epitope, while N29 expression levels were most sensitive to dithiothreitol treatment. These results contrasted with the epitope expression patterns of spontaneously adherent K562 cells, where N29 was almost fully accessible and B44 was low. Addition of a soluble ligand resulted in a marked increase in B44 levels, suggesting that this antibody detected a ligand-induced binding site. The N29 epitope was mapped to a cysteine-rich region near the NH2 terminus of the integrin chain, thus defining a novel regulatory site. These studies indicate that the activation of integrin function by different stimuli may involve related but nonidentical conformations. Both Mn2+ and dithiothreitol appear to induce localized conformational changes that mimic a ligand-occupied receptor. This differs from the "physiologically" activated integrins on K562 cells that display a marked increase in overall epitope accessibility without exposure of the ligand-induced binding site epitopes. The increased exposure of the N29 site on K562 cells may indicate a role for this region in the regulation of integrin function.
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The complete analysis of a cDNA clone encoding the mouse integrin beta 7 subunit that was isolated from a lambda gt10 cDNA library prepared from interleukin-4-activated mouse spleen B cells is reported. The 805-amino acid sequence deduced from the cDNA revealed a signal peptide, a 704-amino acid extracellular domain with eight N-glycosylation sites, a transmembrane domain, and a 61-amino acid intracellular domain. Several structural features were conserved with other integrin beta chains including the four cysteine-rich epidermal growth factor-like repeat sequences in the extracellular domain. Comparative analysis of the mouse and human beta 7 subunits revealed 87% sequence identity with conservation of three potential intracellular tyrosine phosphorylation sites. The cDNA hybridized to two mRNA transcripts of 3 and 2 kilobases (kb) in size. The 3-kb transcript, which is considered the productive form, was found in T and B leukemic cell lines, a macrophage line, and a mastocytoma cell line. Suprisingly, the smaller 2-kb transcript, which seems unlikely to encode a complete beta 7 protein, was found expressed in epithelial cells cultured from mouse skin. The integrin beta 7 subunit displays sequence identity with the beta subunit of the M290 antigen found expressed almost exclusively on intraepithelial lymphocytes in the small intestine suggesting that beta 7 may play an adhesive role in intraepithelial lymphocytes immunosurveillance of the gut mucosa. The cellular distribution of beta 7 transcripts is more extensive than the T-lineage-restricted distribution of the M290 antigen, indicating that beta 7 may be the common beta subunit for more than one receptor or that translation of beta 7 transcripts varies in different cell types.
Intercellular adhesion molecule 1 (ICAM-1, CD54) binds to the integrin LFA-1 (CD11a/CD18), promoting cell adhesion in immune and inflammatory reactions. ICAM-1 is also subverted as a receptor by the major group of rhinoviruses. Electron micrographs show that ICAM-1 is a bent rod, 18.7 nm long, suggesting a model in which the five immunoglobulin-like domains are oriented head to tail at a small angle to the rod axis. ICAM-1 sequences important to binding LFA-1, rhinovirus, and four monoclonal antibodies were identified through the characterization of chimeric ICAM-1 molecules and mutants. The amino-terminal two immunoglobulin-like domains of ICAM-1 appear to interact conformationally. Domain 1 of ICAM-1 contains the primary site of contact for both LFA-1 and rhinovirus; the presence of domains 3-5 markedly affects the accessibility of the binding site for rhinovirus and less so for LFA-1. The binding sites appear to be distinct but overlapping; rhinovirus binding also differs from LFA-1 binding in its lack of divalent cation dependence. Our analysis suggests that rhinoviruses mimic LFA-1 in binding to the most membrane-distal, and thus most accessible, site of ICAM-1.