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ABSTRACT: A simple epithelium is the building block of all metazoans and a multicellular stage of a nonmetazoan. It comprises a closed monolayer of quiescent cells that surround a luminal space. Cells are held together by cell-cell adhesion complexes and surrounded by extracellular matrix. These extracellular contacts are required for the formation of a polarized organization of plasma membrane proteins that regulate the directional absorption and secretion of ions, proteins, and other solutes. While advances have been made in understanding how proteins are sorted to different plasma membrane domains, less is known about how cell-cell adhesion is regulated and linked to the development of epithelial cell polarity and regulation of homeostasis.
Progress in molecular biology and translational science 01/2013; 116C:3-23.
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Søren G F Rasmussen,
Brian T DeVree,
Yaozhong Zou,
Andrew C Kruse,
Ka Young Chung,
Tong Sun Kobilka,
Foon Sun Thian,
Pil Seok Chae,
Els Pardon,
Diane Calinski,
Jesper M Mathiesen,
Syed T A Shah,
Joseph A Lyons,
Martin Caffrey,
Samuel H Gellman,
Jan Steyaert,
Georgios Skiniotis, William I Weis,
Roger K Sunahara,
Brian K Kobilka
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Cheng Zhang,
Yoga Srinivasan,
Daniel H Arlow,
Juan Jose Fung,
Daniel Palmer,
Yaowu Zheng,
Hillary F Green,
Anjali Pandey,
Ron O Dror,
David E Shaw, William I Weis,
Shaun R Coughlin,
Brian K Kobilka
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ABSTRACT: Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2-Å-resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family.
Nature 12/2012; · 36.28 Impact Factor
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ABSTRACT: The Wnt/β-catenin pathway is highly regulated to insure the correct temporal and spatial activation of its target genes. In the absence of a Wnt stimulus, the transcriptional coactivator β-catenin is degraded by a multiprotein "destruction complex" that includes the tumor suppressors Axin and adenomatous polyposis coli (APC), the Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and the E3-ubiquitin ligase β-TrCP. The complex generates a β-TrCP recognition site by phosphorylation of a conserved Ser/Thr-rich sequence near the β-catenin amino terminus, a process that requires scaffolding of the kinases and β-catenin by Axin. Ubiquitinated β-catenin is degraded by the proteasome. The molecular mechanisms that underlie several aspects of destruction complex function are poorly understood, particularly the role of APC. Here we review the molecular mechanisms of destruction complex function and discuss several potential roles of APC in β-catenin destruction.
Cold Spring Harbor perspectives in biology 11/2012; · 9.40 Impact Factor
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ABSTRACT: We hypothesize that aspects of animal multicellularity originated before the divergence of metazoans from fungi and social amoebae. Polarized epithelial tissues are a defining feature of metazoans and contribute to the diversity of animal body plans. The recent finding of a polarized epithelium in the non-metazoan social amoeba Dictyostelium discoideum demonstrates that epithelial tissue is not a unique feature of metazoans, and challenges the traditional paradigm that multicellularity evolved independently in social amoebae and metazoans. An alternative view, presented here, is that the common ancestor of social amoebae, fungi, and animals spent a portion of its life cycle in a multicellular state and possessed molecular machinery necessary for forming an epithelial tissue. Some descendants of this ancestor retained multicellularity, while others reverted to unicellularity. This hypothesis makes testable predictions regarding tissue organization in close relatives of metazoans and provides a novel conceptual framework for studies of early animal evolution. Editor's suggested further reading in BioEssays Searching for Eve: Basal metazoans and the evolution of multicellular complexity Abstract.
BioEssays 08/2012; 34(10):833-40. · 4.95 Impact Factor
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ABSTRACT: Apical actomyosin activity in animal epithelial cells influences tissue morphology and drives morphogenetic movements during development. The molecular mechanisms leading to myosin II accumulation at the apical membrane and its exclusion from other membranes are poorly understood. We show that in the nonmetazoan Dictyostelium discoideum, myosin II localizes apically in tip epithelial cells that surround the stalk, and constriction of this epithelial tube is required for proper morphogenesis. IQGAP1 and its binding partner cortexillin I function downstream of α- and β-catenin to exclude myosin II from the basolateral cortex and promote apical accumulation of myosin II. Deletion of IQGAP1 or cortexillin compromises epithelial morphogenesis without affecting cell polarity. These results reveal that apical localization of myosin II is a conserved morphogenetic mechanism from nonmetazoans to vertebrates and identify a hierarchy of proteins that regulate the polarity and organization of an epithelial tube in a simple model organism.
Developmental cell 08/2012; 23(3):533-46. · 13.36 Impact Factor
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ABSTRACT: αE-catenin, an essential component of the adherens junction, interacts with the classical cadherin-β-catenin complex and with F-actin, but its precise role is unknown. αE-catenin also binds to the F-actin-binding protein vinculin, which also appears to be important in junction assembly. Vinculin and αE-catenin are homologs that contain a series of helical bundle domains, D1-D5. We mapped the vinculin-binding site to a sequence in D3a comprising the central two helices of a four-helix bundle. The crystal structure of this peptide motif bound to vinculin D1 shows that the two helices adopt a parallel, colinear arrangement suggesting that the αE-catenin D3a bundle must unfold in order to bind vinculin. We show that αE-catenin D3 binds strongly to vinculin, whereas larger fragments and full-length αE-catenin bind approximately 1,000-fold more weakly. Thus, intramolecular interactions within αE-catenin inhibit binding to vinculin. The actin-binding activity of vinculin is inhibited by an intramolecular interaction between the head (D1-D4) and the actin-binding D5 tail. In the absence of F-actin, there is no detectable binding of αE-catenin D3 to full-length vinculin; however, αE-catenin D3 promotes binding of vinculin to F-actin whereas full-length αE-catenin does not. These findings support the combinatorial or "coincidence" model of activation in which binding of high-affinity proteins to the vinculin head and tail is required to shift the conformational equilibrium of vinculin from a closed, autoinhibited state to an open, stable F-actin-binding state. The data also imply that αE-catenin must be activated in order to bind to vinculin.
Proceedings of the National Academy of Sciences 05/2012; 109(22):8576-81. · 9.68 Impact Factor
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ABSTRACT: Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled µ-opioid receptor (µ-OR) in the central nervous system. Here we describe the 2.8 Å crystal structure of the mouse µ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the µ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.
Nature 03/2012; 485(7398):321-6. · 36.28 Impact Factor
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Andrew C Kruse,
Jianxin Hu,
Albert C Pan,
Daniel H Arlow,
Daniel M Rosenbaum,
Erica Rosemond,
Hillary F Green,
Tong Liu,
Pil Seok Chae,
Ron O Dror,
David E Shaw, William I Weis,
Jürgen Wess,
Brian K Kobilka
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ABSTRACT: Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G(q/11)-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G(i/o)-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.
Nature 02/2012; 482(7386):552-6. · 36.28 Impact Factor
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Kazuko Haga,
Andrew C Kruse,
Hidetsugu Asada,
Takami Yurugi-Kobayashi,
Mitsunori Shiroishi,
Cheng Zhang, William I Weis,
Tetsuji Okada,
Brian K Kobilka,
Tatsuya Haga,
Takuya Kobayashi
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ABSTRACT: The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.
Nature 02/2012; 482(7386):547-51. · 36.28 Impact Factor
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ABSTRACT: A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β(2) adrenergic receptor (β(2)AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.
PLoS ONE 01/2012; 7(10):e46039. · 4.09 Impact Factor
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ABSTRACT: The opioid receptor family comprises three members, the µ-, δ- and κ-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The δ-opioid receptor (δ-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the µ-OR and κ-OR have recently been solved. Here we report the crystal structure of the mouse δ-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the µ-OR and κ-OR, the δ-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the δ-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.
Nature 01/2012; 485(7398):400-4. · 36.28 Impact Factor
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ABSTRACT: LDL receptor-related proteins 5 and 6 (LRP5/6) are coreceptors for Wnt growth factors, and also bind Dkk proteins, secreted inhibitors of Wnt signaling. The LRP5/6 ectodomain contains four β-propeller/EGF-like domain repeats. The first two repeats, LRP6(1-2), bind to several Wnt variants, whereas LRP6(3-4) binds other Wnts. We present the crystal structure of the Dkk1 C-terminal domain bound to LRP6(3-4), and show that the Dkk1 N-terminal domain binds to LRP6(1-2), demonstrating that a single Dkk1 molecule can bind to both portions of the LRP6 ectodomain and thereby inhibit different Wnts. Small-angle X-ray scattering analysis of LRP6(1-4) bound to a noninhibitory antibody fragment or to full-length Dkk1 shows that in both cases the ectodomain adopts a curved conformation that places the first three repeats at a similar height relative to the membrane. Thus, Wnts bound to either portion of the LRP6 ectodomain likely bear a similar spatial relationship to Frizzled coreceptors.
Developmental cell 11/2011; 21(5):862-73. · 13.36 Impact Factor
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ABSTRACT: Androgens have important roles in the development of the prostate gland and in prostate cancer. Since the finding that β-catenin is a cofactor of the androgen receptor (AR) and can augment AR signaling, several proteins have been found to affect AR signaling through their interaction with β-catenin. Here, we investigated inhibitor of β-catenin and T-cell factor (ICAT), a β-catenin binding protein that inhibits the canonical Wnt/β-catenin signaling pathway, in AR signaling. We demonstrated that expression of ICAT in two AR positive prostate cancer cell lines, LNCaP and LAPC4, augments ligand-dependent AR-mediated transcription. In contrast, short hairpin RNA knockdown of ICAT and β-catenin specifically blocks enhanced AR-mediated transcription by ICAT. Using both stable expression of ICAT and short hairpin RNA knockdown of ICAT expression approaches, we further showed that ICAT enhances expression of endogenous PSA and KLK2, two androgen response genes, and ligand-induced cell growth. In addition, we identified that ICAT and AR can form a ternary complex with β-catenin using in vitro glutathione S-transferase protein pulldown assays. Moreover, we detected the endogenous protein complex containing ICAT, AR, and β-catenin in prostate cancer cells using immunoprecipitation assays. Recruitment of endogenous ICAT onto the promoter region of the human PSA gene, an AR downstream target promoter, was also identified in LNCaP cells. Finally, using in vitro protein binding assays, we examined the effect of full-length and truncated ICAT on the AR-β-catenin interaction and observed that addition of full-length ICAT retained the interaction between β-catenin and AR proteins. Intriguingly, the truncated ICAT comprising the N-terminal helical domain showed a more pronounced effect on β-catenin binding to AR proteins. Our findings suggest a novel molecular mechanism underlying the cross talk between androgen and Wnt signaling pathways.
Molecular Endocrinology 09/2011; 25(10):1677-88. · 4.54 Impact Factor
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ABSTRACT: Studies of animal evolution often focus on sequence and transcriptional analysis, based on an assumption that the evolution of development is driven by changes in gene expression. We argue that biochemical and cell biological approaches are also required, because sequence-conserved proteins can have different biochemical, cellular, and developmental properties.
Developmental cell 07/2011; 21(1):32-4. · 13.36 Impact Factor
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Søren G F Rasmussen,
Brian T DeVree,
Yaozhong Zou,
Andrew C Kruse,
Ka Young Chung,
Tong Sun Kobilka,
Foon Sun Thian,
Pil Seok Chae,
Els Pardon,
Diane Calinski,
Jesper M Mathiesen,
Syed T A Shah,
Joseph A Lyons,
Martin Caffrey,
Samuel H Gellman,
Jan Steyaert,
Georgios Skiniotis, William I Weis,
Roger K Sunahara,
Brian K Kobilka
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ABSTRACT: G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the β(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.
Nature 07/2011; 477(7366):549-55. · 36.28 Impact Factor
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ABSTRACT: The plakin protein family serves to connect cell-cell and cell-matrix adhesion molecules to the intermediate filament cytoskeleton. Desmoplakin (DP) is an integral part of desmosomes, where it links desmosomal cadherins to the intermediate filaments. The 1056-amino-acid N-terminal region of DP contains a plakin domain common to members of the plakin family. Plakin domains contain multiple copies of spectrin repeats (SRs). We determined the crystal structure of a fragment of DP, residues 175-630, consisting of four SRs and an inserted SH3 domain. The four repeats form an elongated, rigid structure. The SH3 domain is present in a loop between two helices of an SR and interacts extensively with the preceding SR in a manner that appears to limit inter-repeat flexibility. The intimate intramolecular association of the SH3 domain with the preceding SR is also observed in plectin, another plakin protein, but not in α-spectrin, suggesting that the SH3 domain of plakins contributes to the stability and rigidity of this subfamily of SR-containing proteins.
Journal of Molecular Biology 06/2011; 409(5):800-12. · 4.00 Impact Factor
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ABSTRACT: A fundamental characteristic of metazoans is the formation of a simple, polarized epithelium. In higher animals, the structural integrity and functional polarization of simple epithelia require a cell-cell adhesion complex that contains a classical cadherin, the Wnt-signaling protein β-catenin and the actin-binding protein α-catenin. We show that the non-metazoan Dictyostelium discoideum forms a polarized epithelium that is essential for multicellular development. Although D. discoideum lacks a cadherin homolog, we identify an α-catenin ortholog that binds a β-catenin-related protein. Both proteins are essential for formation of the epithelium, polarized protein secretion, and proper multicellular morphogenesis. Thus, the organizational principles of metazoan multicellularity may be more ancient than previously recognized, and the role of the catenins in cell polarity predates the evolution of Wnt signaling and classical cadherins.
Science 03/2011; 331(6022):1336-9. · 31.20 Impact Factor
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Daniel M. Rosenbaum,
Cheng Zhang,
Joseph A. Lyons,
Ralph Holl,
David Aragao,
Daniel H. Arlow,
G. F. Rasmussen,
Hee-Jung Choi,
Brian T. DeVree,
Roger K. Sunahara,
Pil Seok Chae,
Samuel H. Gellman,
Ron O. Dror,
David E. Shaw, William I. Weis,
Martin Caffrey,
Peter Gmeiner,
Brian K. Kobilka
Nature 01/2011; 469(7329):236-240. · 36.28 Impact Factor
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ABSTRACT: Langerin mediates the carbohydrate-dependent uptake of pathogens by Langerhans cells in the first step of antigen presentation to the adaptive immune system. Langerin binds to an unusually diverse number of endogenous and pathogenic cell surface carbohydrates, including mannose-containing O-specific polysaccharides derived from bacterial lipopolysaccharides identified here by probing a microarray of bacterial polysaccharides. Crystal structures of the carbohydrate-recognition domain from human langerin bound to a series of oligomannose compounds, the blood group B antigen, and a fragment of β-glucan reveal binding to mannose, fucose, and glucose residues by Ca(2+) coordination of vicinal hydroxyl groups with similar stereochemistry. Oligomannose compounds bind through a single mannose residue, with no other mannose residues contacting the protein directly. There is no evidence for a second Ca(2+)-independent binding site. Likewise, a β-glucan fragment, Glcβ1-3Glcβ1-3Glc, binds to langerin through the interaction of a single glucose residue with the Ca(2+) site. The fucose moiety of the blood group B trisaccharide Galα1-3(Fucα1-2)Gal also binds to the Ca(2+) site, and selective binding to this glycan compared to other fucose-containing oligosaccharides results from additional favorable interactions of the nonreducing terminal galactose, as well as of the fucose residue. Surprisingly, the equatorial 3-OH group and the axial 4-OH group of the galactose residue in 6SO(4)-Galβ1-4GlcNAc also coordinate Ca(2+), a heretofore unobserved mode of galactose binding in a C-type carbohydrate-recognition domain bearing the Glu-Pro-Asn signature motif characteristic of mannose binding sites. Salt bridges between the sulfate group and two lysine residues appear to compensate for the nonoptimal binding of galactose at this site.
Journal of Molecular Biology 01/2011; 405(4):1027-39. · 4.00 Impact Factor
