Critical Residues of Integrin αIIb Subunit for Binding of αIIbβ3 (Glycoprotein IIb-IIIa) to Fibrinogen and Ligand-mimetic Antibodies (PAC-1, OP-G2, and LJ-CP3)

Kamata, Tamihiro; Irie, Atsushi; Tokuhira, Michihide; Takada, Yoshikazu

doi:10.1074/jbc.271.31.18610

Cited by 94 publications

(88 citation statements)

References 57 publications

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“…Enzymes with known ␤-propeller folds have their active sites at the top of the ␤-propeller, typically where adjacent loops run in opposite directions. Consistent with this hypothesis, the ␣ IIb residues, Gly-184 to Gly-193, located in one loop, and ␣ IIb Asp-224, located in a second loop, both predicted to be at the top of the ␤-propeller, have been implicated in ligand binding by site-directed mutagenesis (63,64). The identification of residues critical for ligand binding to ␣ 4 ␤ 1 and ␣ 5 ␤ 1 further supports the ␤-propeller model (65,66).…”

Section: Minireview: Ligand Binding To Integrins 21787supporting

confidence: 50%

Ligand Binding to Integrins

Plow¹,

Haas²,

Zhang³

et al. 2000

Journal of Biological Chemistry

1,227

1,063

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The "integrin" terminology was applied in a 1987 review article (1) to describe a family of structurally, immunochemically, and functionally related cell-surface heterodimeric receptors, which integrated the extracellular matrix with the intracellular cytoskeleton to mediate cell migration and adhesion. The three original ␤ subunits (␤ 1 , ␤ 2 , and ␤ 3 ) identified have now expanded to eight, and the number of ␣ subunits stands at 17. These subunits interact noncovalently in a restricted manner to form more than 20 family members. The diversity of integrins is expanded further by alternative splicing, post-translational modifications, and interactions with other cell-surface and intracellular molecules (2-4). The number of integrins and the remarkable breadth of their cellular distribution support the statement that the phenotype of virtually every cell is uniquely influenced by its display of integrins. Over the past 13 years, more than 14,000 scientific articles have dealt with various aspects of integrin biology and almost 1,000 have appeared in the Journal of Biological Chemistry. This article examines a central aspect of integrin biology: ligand recognition and the structural basis for this function. Ligand Repertoire and RecognitionRepertoire Considerations-A hallmark of the integrins is the ability of individual family members to recognize multiple ligands. Indeed, the extent of the integrin family pales in comparison with the number of their ligands. Table I summarizes the major extracellular ligands of integrins; the listing is undoubtedly incomplete. The list includes a large number of extracellular matrix proteins (bone matrix proteins, collagens, fibronectins, fibrinogen, laminins, thrombospondins, vitronectin, and von Willebrand factor), reflecting the primary function of integrins in cell adhesion to extracellular matrices. Many "counter-receptors" are ligands, reflecting the role of integrins in mediating cell-cell interactions. Included are numerous microorganisms, which utilize integrins to gain entry into cells. There are direct and multiple linkages between integrins and host defense systems, created by their recognition of hemostatic and complement factors. The preference of any given integrin among its ligands is determined by relative affinity, availability within a specific microenvironment, and the conformational state of the ligand, which controls exposure of its integrin recognition sequence.Integrin Recognition Sequences-A primary goal of many structure-function analyses in the integrin field has been the reduction of macromolecular ligands to minimal recognition sequences. This endeavor has been highly successful, and many bioactive amino acid sequences have been teased out of large extracellular matrix proteins (5). The prototypic example is the RGD sequence. RGD was originally identified as the sequence in fibronectin that engages the fibronectin receptor, integrin ␣ 5 ␤ 1 , but now is known to serve as a recognition motif in multiple ligands for several different integrins (see Ta...

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Section: Minireview: Ligand Binding To Integrins 21787supporting

confidence: 50%

Ligand Binding to Integrins

Plow¹,

Haas²,

Zhang³

et al. 2000

Journal of Biological Chemistry

1,227

1,063

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“…These mutants were expressed in CHO cells, and FITC-labeled Fbg binding to these cells was examined using FACS. As reported previously, the wild-type ␣IIb␤3 expressed in CHO cells is in a low affinity state and requires activation by the anti-␣IIb␤3 mAb PT25-2 to bind Fbg in the presence of 1 mM Ca 2ϩ /1 mM Mg 2ϩ (20). Cells expressing single Cys mutations, such as S367C, G382C, V332C, S551C, T564C, or S674C, bound Fbg in the presence of PT25-2, albeit slightly less than that observed in wild-type cells (data not shown).…”

Section: Bent Conformer Of ␣Iib␤3 Represents a Low Affinity Form-mentioning

confidence: 94%

“…Fibrinogen Binding Assay-FITC labeling of human Fbg was performed as described previously (20). Briefly, after adjusting the pH of human Fbg at 1 mg/ml in PBS to 8.5 using 5% Na 2 CO 3 , 1/100 volume of 10 mg/ml FITC in dimethyl sulfoxide (DMSO) was added and incubated at room temperature for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Structural Requirements for Activation in αIIbβ3 Integrin

Kamata

Handa

Ito

et al. 2010

Journal of Biological Chemistry

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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 ␣IIb␤3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant ␣IIb␤3 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 ␤-head/␤-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 ␣-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the ␣-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.Integrin-mediated bidirectional signaling is closely associated with the structural rearrangement of integrin itself. During inside-out signaling, talin has been shown to bind to the ␤ cytoplasmic tail and to disrupt the endogenous interaction between the ␣ and ␤ cytoplasmic tails (1, 2). The dissociation of the two tails induces a structural rearrangement of the extracellular domains increasing the affinity to the ligand. During outside-in signaling, ligand binding in turn induces the structural rearrangement of the extracellular domains. This structural change propagates through the plasma membrane to separate the cytoplasmic tails, providing binding sites for numerous cytoplasmic proteins (3). Thus, the two structures flanking the plasma membrane affect each other. This structural rearrangement can be detected using a group of monoclonal antibodies (mAbs) that bind preferentially to the ligand-bound form (4). These anti-LIBS 2 (ligand-induced binding site) mAbs have been used not only to investigate the activation status of a specific integrin but also to activate it (5). However, without information on the actual three-dimensional structure, it is impossible to determine the specific conformation recognized by each anti-LIBS mAb.The first observation of the actual three-dimensional structure of integrin was made using conventional electron ...

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“…In non-I-domain integrin ␣ subunits, particularly the ␣ IIb subunit, several ligand-binding sites have been identified by the characterization of naturally occurring mutations in patients with Glanzmann thrombasthenia as well as mutagenesis analyses. [15][16][17][18][19] However, ligand-binding regions of ␣ v remain elusive. To clarify the critical regions for ligand binding in the ␣ v subunit, we focused on the predicted W3 4-1 loop (C142-C155) and W3 2-3 loop (G172-G181) and investigated their role in ligand binding by alanine-scanning mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

Ligand binding to integrin αvβ3requires tyrosine 178 in the αv subunit

Honda

Tomiyama

Pampori

et al. 2001

Blood

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Integrin ␣ v ␤ 3 has been implicated in angiogenesis and other biological processes. However, the ligand-binding sites in ␣ v , a non-I-domain ␣ subunit, remain to be identified. Recently in ␣ IIb , the other partner of the ␤ 3 subunit, several discontinuous residues important for ligand binding were identified in the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alaninescanning mutagenesis in 293 cells was used to investigate the role of these loops (cys- teine IntroductionIntegrins are a large family of ␣␤ heterodimeric adhesion receptors that is often subdivided into groups based on 8 known integrin ␤ subunits. 1 The ␤ 3 -integrin subfamily is composed of ␣ v ␤ 3 , originally identified as the vitronectin receptor, and ␣ IIb ␤ 3 , a plateletspecific receptor for fibrinogen and von Willebrand factor. ␣ IIb ␤ 3 plays a crucial role in platelet aggregation, normal hemostasis, and pathological thrombus formation. 2 On the other hand, ␣ v ␤ 3 is expressed in a number of tissues, including platelets, endothelial cells, vascular smooth muscle cells, and osteoclasts, and it plays a key role in angiogenesis and bone resorption. 3,4 The ␣ IIb and ␣ v subunits are homologous and 36% identical in primary amino acid sequence, 5 and most ligands that bind to ␣ IIb ␤ 3 , including fibrinogen, von Willebrand factor, and vitronectin, also bind to ␣ v ␤ 3 . However, there are some distinctive features between these 2 integrins. 3 First, the ␣ IIb subunit has been found only in combination with ␤ 3 , whereas ␣ v can associate with at least 5 ␤ subunits (␤ 1 , ␤ 3 , ␤ 5 , ␤ 6 , and ␤ 8 ). 6 Second, some ligands, such as osteopontin, matrix metalloproteinase-2, and adenovirus penton base, bind to ␣ v ␤ 3 but not to ␣ IIb ␤ 3 . Third, treatment of ␣ IIb ␤ 3 with ethylenediamine tetraacetic acid (EDTA) at 37°C dissociates the complex into its individual subunits; ␣ v ␤ 3 remains a heterodimer. Finally, the ligand-binding function of ␣ v ␤ 3 , but not ␣ IIb ␤ 3 , is suppressed by calcium (Ca 2ϩ ). 7 Generally, all integrins require divalent cations for ligand recognition, and multiple residues important for ligand binding have been identified on both ␣ and ␤ subunits. 8 The N-terminal region of integrin ␣ subunits has 7 repeats of homologous sequences of about 60 amino acid residues. Some integrin ␣ subunits (eg, ␣ 2 , ␣ L , and ␣ M ) contain an inserted domain of about 200 amino acids residues (the I-domain) between the second and the third repeats in the ␣ subunit, which is critically involved in ligand binding. 9,10 The crystal structure of the I-domain has been determined, and the metal ion-dependent adhesion site (MIDAS) motif that contributes to cation binding, as well as ligand binding, has been clarified. 11 Interestingly, a MIDAS-like motif essential for the ligand-binding function was also identified in integrin ␤ subunits. 12,13 On the other hand, integrin ␣ subunits, such as ␣ v , ␣ IIb , and ␣ 4 , do not have the I-domain. The structural basis for the i...

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Critical Residues of Integrin αIIb Subunit for Binding of αIIbβ3 (Glycoprotein IIb-IIIa) to Fibrinogen and Ligand-mimetic Antibodies (PAC-1, OP-G2, and LJ-CP3)

Cited by 94 publications

References 57 publications

Ligand Binding to Integrins

Ligand Binding to Integrins

Structural Requirements for Activation in αIIbβ3 Integrin

Ligand binding to integrin αvβ3requires tyrosine 178 in the αv subunit

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