Ligand binding to integrin αvβ3requires tyrosine 178 in the αv subunit

Abstract

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...

Missense mutations in the β3 subunit have a different impact on the expression and function between αIIbβ3 and αvβ3

“…45 In addition, we recently clarified the difference in the ligand binding sites between ␣IIb and ␣v. 34 In this study, we newly demonstrate that Ser162Leu and Arg216Gln mutations show a different effect on ligand binding between 2 ␤ 3 integrins. Consistent with the reports by Newman's group, 32,33 we showed that Ser162Leu and Arg216Gln mutations impaired the stability of the complex between ␣IIb and ␤ 3 , as evidenced by the fact that the binding of complex-specific MoAb AP2 was markedly impaired compared with that of the ␣IIb-specific MoAb TP80.…”

“…However, our previous study showed that the expression of these endogenous ␣v integrins appeared to be low compared with exogenous ␣ v ␤ 3 . 34 Indeed, the Leu117Trp ␤ 3 mutation severely impaired the expression of ␣v subunits as well as ␣ v ␤ 3 , suggesting that the bulk of expressed ␣v integrins is ␣ v ␤ 3 in these conditions ( Figure 5). …”

“…Although the Arg216Gln/Leu292Ser mutation completely abolished ␣ IIb ␤ 3 expression, this mutation did not impair ␣ v ␤ 3 expression at all. Because 293 cells possess endogenous ␣v␤1 and ␣v␤5, 34,35 LM142 (anti-␣v) may detect these ␣v integrins. However, our previous study showed that the expression of these endogenous ␣v integrins appeared to be low compared with exogenous ␣ v ␤ 3 .…”

Functional and structural correlations of individual αIIbβ3 molecules

“…For example, although Ca 2ϩ or Mg 2ϩ are required for ligand binding to integrins, neither by themselves activate integrins, whereas Mn 2ϩ , at millimolar concentrations, induces integrin-mediated cell adhesion, 4,9,31,32 binding of isolated integrins to immobilized ligands, 12,33,34 and binding of soluble ligands to integrins on cell surfaces. [35][36][37] The crystal structure of the extracellular portion of the integrin ␣v␤3 revealed that it contains 8 divalent cation-binding sites. 14 Four sites were located in the ␤-propeller domain of ␣v, 1 at the ␣ subunit genu (knee), and 3 in the ␤3 ␤A domain.…”

Proinflammatory Activities of S100: Proteins S100A8, S100A9, and S100A8/A9 Induce Neutrophil Chemotaxis and Adhesion

“…Mac-1 (CD11b/CD18) and ␣ V ␤ 3 (CD51/CD61) are two integrins expressed by neutrophils known to bind fibrinogen (48,49). To ‫ء‬ , p Ͻ 0.01, Student's t test (S100A8/A9 in both chambers vs S100A8/A9 in lower chamber only).…”