ABSTRACT: Upon vascular injury, platelets initiate interaction with exposed subendothelial matrices through various receptors such as
glycoprotein (GP) Ib/IX/V complex, α2β1 integrin, and GPVI/FcRγ. Although these interactions cannot sustain stable platelet
thrombus formation by themselves, they ultimately lead to the activation of αIIbβ3 integrin (GPIIb-IIIa complex [GPIIb-IIIa]),
the most abundant receptor in platelets. The αIIbβ3 integrin plays a central role in primary hemostasis by serving as a receptor
for fibrinogen and von Willebrand factor (vWf). It establishes a stable interaction with vWf bound to the extracellular matrices
and uses fibrinogen as a bridging molecule in platelet aggregate formation. The αIIbβ3 integrin also plays an important role
in the pathogenesis of thrombosis. Over the past decades, a tremendous amount of effort has been made to elucidate the ligand-binding
mechanisms of αIIbβ3, in part because of its clinical significance. Most of the studies have relied on biochemical analyses
of purified αIIbβ3 or recombinant proteins generated in vitro. With the lack of actual 3-dimensional structure, molecular
modeling has provided a useful framework for interpreting such experimental data on structure-function correlation of integrin
molecules. However, it has also generated disagreement between different models. The aim of this minireview is to summarize
the past efforts as well as the recent accomplishments in elucidating the structure/function of αIIbβ3. Finally, we will try
to explain all those experimental data using the recently published crystal structure of the extracellular domains of the
αVβ3 heterodimeric complex.
International Journal of Hematology 04/2012; 74(4):382-389. · 1.27 Impact Factor
ABSTRACT: Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended
conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas
another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity
is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant αIIbβ3 integrins that
are constrained specifically in either a bent or an extended conformation. These mutant αIIbβ3 integrins were expressed in
mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction
of artificial disulfide bridges in the β-head/β-tail interface. Cells expressing bent integrins all failed to bind fibrinogen
unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the α-genu, where the integrin legs fold backward. Among these
mutants, activation was maximized in one integrin with an N-glycosylation site located behind the α-genu. This extension-induced activation was completely blocked when the swing-out
of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and
high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain.
Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like
movement of the integrin legs.
Journal of Biological Chemistry 12/2010; 285(49):38428-38437. · 4.77 Impact Factor
ABSTRACT: Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant αIIbβ3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant αIIbβ3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the β-head/β-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the α-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the α-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.
Journal of Biological Chemistry 09/2010; 285(49):38428-37. · 4.77 Impact Factor
ABSTRACT: The affinity of integrin-ligand interaction is regulated extracellularly by divalent cations and intracellularly by inside-out signaling. We report here that the extracellular, membrane-proximal alpha/beta stalk interactions not only regulate cation-induced integrin activation but also play critical roles in propagating inside-out signaling. Two closely related integrins, alphaIIbbeta3 and alphaVbeta3, share high structural homology and bind to similar ligands in an RGD-dependent manner. Despite these structural and functional similarities, they exhibit distinct responses to Mn(2+). Although alphaVbeta3 showed robust ligand binding in the presence of Mn(2+), alphaIIbbeta3 showed a limited increase but failed to achieve full activation. Swapping alpha stalk regions between alphaIIb and alphaV revealed that the alpha stalk, but not the ligand-binding head region, was responsible for the difference. A series of alphaIIb/alphaV domain-swapping chimeras were constructed to identify the responsible domain. Surprisingly, the minimum component required to render alphaIIbbeta3 susceptible to Mn(2+) activation was the alphaV calf-2 domain, which does not contain any divalent cation-binding sites. The calf-2 domain makes interface with beta epidermal growth factor 4 and beta tail domain in three-dimensional structure. The effect of calf-2 domain swapping was partially reproduced by mutating the specific amino acid residues in the calf-2/epidermal growth factor 4-beta tail domain interface. When this interface was constrained by an artificially introduced disulfide bridge, the Mn(2+)-induced alphaVbeta3-fibrinogen interaction was significantly impaired. Notably, a similar disulfide bridge completely abrogated fibrinogen binding to alphaIIbbeta3 when alphaIIbbeta3 was activated by cytoplasmic tail truncation to mimic inside-out signaling. Thus, disruption/formation of the membrane-proximal alpha/beta stalk interface may act as an on/off switch that triggers integrin-mediated bidirectional signaling.
Journal of Biological Chemistry 08/2005; 280(26):24775-83. · 4.77 Impact Factor
ABSTRACT: Chemical or enzymic reduction/oxidation of integrin cysteine residues (e.g. by reducing agents and protein disulphide isomerase) may be a mechanism for regulating integrin function. It has also been proposed that unique cysteine residues in the integrin beta3 subunit are involved in the regulation of alphaIIbbeta3. In the present study, we studied systematically the role of disulphide bonds in beta3 on the ligand-binding function of alphaIIbbeta3 by mutating individual cysteine residues of beta3 to serine. We found that the disulphide bonds that are critical for alphaIIbbeta3 regulation are clustered within the EGF (epidermal growth factor) domains. Interestingly, disrupting only a single disulphide bond in the EGF domains was enough to activate alphaIIbbeta3 fully. In contrast, only two (of 13) disulphide bonds tested outside the EGF domains activated alphaIIbbeta3. These results suggest that the disulphide bonds in the EGF domains should be intact to keep alphaIIbbeta3 in an inactive state, and that there is no unique cysteine residue in the EGF domain critical for regulating the receptor. The cysteine residues in the EGF domains are potential targets for chemical or enzymic reduction.
Biochemical Journal 04/2004; 378(Pt 3):1079-82. · 4.90 Impact Factor
ABSTRACT: The alpha(L) I (inserted or interactive) domain of integrin alpha(L)beta(2) undergoes conformational changes upon activation. Recent studies show that the isolated, activated alpha(L) I domain is sufficient for strong ligand binding, suggesting the beta(2) subunit to be only indirectly involved. It has been unclear whether the activity of the alpha(L) I domain is regulated by the beta(2) subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169-176) in the beta(2) I domain with a corresponding beta(3) sequence, or mutating Lys(174) to Thr, constitutively activates alpha(L)beta(2) binding to ICAM-1. These mutants do not require Mn(2+) for ICAM-1 binding and are insensitive to the inhibitory effect of Ca(2+). We have also localized a component of the mAb 24 epitope (a reporter of beta(2) integrin activation) in the CPNKEKEC sequence. Glu(173) and Glu(175) of the beta(2) I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon beta(2) integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the alpha(L) I domain did not eliminate the mAb 24 epitope. This confirms that the alpha(L) I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the beta(2) subunit I domain. These results suggest that the CPNKEKEC sequence of the beta(2) I domain is involved in regulating the alpha(L) I domain.
The Journal of Immunology 04/2002; 168(5):2296-301. · 5.79 Impact Factor
ABSTRACT: 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.
Journal of Biological Chemistry 11/2001; 276(47):44275-44283. · 4.77 Impact Factor
ABSTRACT: 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.
Journal of Biological Chemistry 03/2000; 275(11):7795-7802. · 4.77 Impact Factor
ABSTRACT: A docking model of the α2 I-domain and collagen has been proposed based on their crystal structures (Emsley, J., King, S., Bergelson, J., and Liddington,
R. C. (1997) J. Biol. Chem. 272, 28512–28517). In this model, several amino acid residues in the I-domain make direct contact with collagen (Asn-154,
Asp-219, Leu-220, Glu-256, His-258, Tyr-285, Asn-289, Leu-291, Asn-295, and Lys-298), and the protruding C-helix of α2 (residues 284–288) determines ligand specificity. Because most of the proposed critical residues are not conserved, different
I-domains are predicted to bind to collagen differently. We found that deleting the entire C-helix or mutating the predicted
critical residues had no effect on collagen binding to whole α2β1, with the exception that mutating Asn-154, Asp-219, and His-258 had a moderate effect. We performed further studies and found
that mutating the conserved surface-exposed residues in the metal ion-dependent adhesion site (MIDAS) (Tyr-157 and Gln-215)
significantly blocks collagen binding. We have revised the docking model based on the mutagenesis data. In the revised model,
conserved Tyr-157 makes contact with collagen in addition to the previously proposed Asn-154, Asp-219, His-258, and Tyr-285
residues. These results suggest that the collagen-binding I-domains (e.g. α1, α2, and α10) bind to collagen in a similar fashion.
Journal of Biological Chemistry 11/1999; 274(45):32108-32111. · 4.77 Impact Factor
ABSTRACT: We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the IIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)-289 to Ser (TCT) mutation (F289S) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gln (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on IIbβ3 surface expression, the wild-type IIb cDNA or mutant IIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type β3 cDNA. Flow cytometric analysis using an anti-IIbβ3 complex antibody revealed that 50.6% of CHO cells with wild-type IIbβ3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with IIb(F289S)β3, IIb(E324K)β3 and IIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the IIb subunit may hamper surface expression of the IIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.
British Journal of Haematology 07/1998; 102(3):829 - 840. · 4.94 Impact Factor
ABSTRACT: Integrins mediate signal transduction through interaction with multiple cellular or extracellular matrix ligands. Integrin
αvβ3 recognizes fibrinogen, von Willebrand factor, and vitronectin, while αvβ1 does not. We studied the mechanisms for defining
ligand specificity of these integrins by swapping the highly diverse sequences in the I domain-like structure of the β1 and
β3 subunits. When the sequence CTSEQNC (residues 187–193) of β1 is replaced with the corresponding CYDMKTTC sequence of β3,
the ligand specificity of αvβ1 is altered. The mutant (αvβ1–3-1), like αvβ3, recognizes fibrinogen, von Willebrand factor,
and vitronectin (a gain-of-function effect). The αvβ1–3-1 mutant is recruited to focal contacts on fibrinogen and vitronectin,
suggesting that the mutant transduces intracellular signals on adhesion. The reciprocal β3–1-3 mutation blocks binding of
αvβ3 to these multiple ligands and to LM609, a function-blocking anti-αvβ3 antibody. These results suggest that the highly
divergent sequence is a key determinant of integrin ligand specificity. Also, the data support a recent hypothetical model
of the I domain of β, in which the sequence is located in the ligand binding site.
Journal of Biological Chemistry 08/1997; 272(32):19794-19800. · 4.77 Impact Factor
ABSTRACT: Integrins are a family of α/β heterodimers of cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions. Both α and β subunits have a large extracellular domain and a short cytoplasmic domain. The α subunit has seven sequence repeats of 60–70 residues in its N-terminal region. The β-propeller model, in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetry axis, has been proposed as a structural model of these seven repeats. Several predicted loops critical for ligand binding have been identified in the upper face of the proposed β-propeller model. Several α subunits (e.g., α2, αL and αM) have I-domains of about 200 residues inserted between their second and third repeats. These I-domains adopt a Rossman-fold structure and have major ligand and cation binding sites (the MIDAS site) on their surfaces. The β subunit has an I-domain-like structure in its N-terminal region. This structure includes multiple sequences/conserved oxygenated residues critical for ligand binding (e.g., Asp-119 in β3), and non-conserved residues critical for ligand specificities. Several “activation-dependent” epitopes have been identified in the Cys-rich (stalk) region of β1. It has yet to be determined how these multiple ligand binding sites in the α and β subunits are involved in ligand binding, and how conformational changes on activation/ligand occupancy relate to signal transduction.