Molecular Requirements for Assembly and Function of a Minimized Human Integrin αIIbβ3

Abstract

Integrin subunit compatibility within and between species plays a major role in heterodimer assembly and ligand specificity. As an example, human ␣ IIb pairs only with human ␤ 3 and does not assemble a heterodimer with ␤ 3 from other species. We use interspecies subunit chimeras to identify molecular requirements for subunit compatibility and show that species-restricted heterodimer assembly depends on a unique hexapeptide VGSDNH in an extended loop of the hypothetical human ␤ 3 MIDAS domain. This allows us to express ␣ IIb (1-233) and ␤ 3 (111-318) as a soluble, mini-integrin that retains RGD-dependent ligand recognition. Thus, in the case of one integrin, ␣ IIb ␤ 3 , the molecular requirements for integrin subunit compatibility and ligand recognition are intimately related.

Probing Conformational Changes in the I-like Domain and the Cysteine-rich Repeat of Human β3 Integrins following Disulfide Bond Disruption by Cysteine Mutations

“…These data are in accordance with previous studies showing that ␤ 3 subunit residues 217-298 and 324 -366 are involved in ␣/␤ heterodimerization (50). Also, amino acids in ␤ 3 necessary for the selective ␣ IIb ␤ 3 subunit compatibility have been identified in a short sequence encompassing residues 275-280 (40,51).…”

“…More importantly, the sequence within this Cys 177 -Cys 184 loop appears to be part of the discontinuous antigenic sites recognized by some ligand-mimetic monoclonal antibodies, and amino acid substitutions in this sequence prevent ligand-mimetic monoclonal antibody as well as fibrinogen binding (38). On the other hand, the Cys 232 -Cys 273 bond, which does not exist in the ␤ 4 integrin subunit, is in close proximity to the hexapeptide 275 VGSDNH 280 in the ␤ 3 subunit that confers species restricted heterodimer assembly to ␣ IIb ␤ 3 (40). And finally, Cys 598 is located in the conserved motif II of the fourth CRR and is part of a small tryptic fragment of the fourth CRR (residues 581-600) that has recently been identified to contain unpaired cysteines (10).…”

Expression of a structural domain of the β2 subunit essential for αMβ2 ligand recognition

“…It has yet to be determined whether these multiple interactive sites participate in ligand recognition in a cooperative or additive manner. The expression of minimized integrins, as recently shown by McKay and colleagues for ␣ IIb ␤ 3 [58], will undoubtedly help define the three-dimensional structural requirement of ligand recognition. Our results help limit the minimal structural domains of the ␤ 2 integrin essential for function.…”

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

“…It has been demonstrated that the hypothetical human ␤ 3 metal ion-dependent adhesion site domain is critical for heterodimer assembly with human ␣IIb and ligand binding function. 42,43 Moreover, the hexapeptide sequence 275Val-Gly-Ser-Asp-Asn-His280 within the ␤ 3 metal ion-dependent adhesion site domain appears necessary for species-restricted heterodimer formation. 43 Our present data demonstrated that the His280Pro mutation at the sixth residue of the unique hexapeptide did not impair either assembly of pro␣IIb and ␤ 3 or ligand binding function.…”

“…42,43 Moreover, the hexapeptide sequence 275Val-Gly-Ser-Asp-Asn-His280 within the ␤ 3 metal ion-dependent adhesion site domain appears necessary for species-restricted heterodimer formation. 43 Our present data demonstrated that the His280Pro mutation at the sixth residue of the unique hexapeptide did not impair either assembly of pro␣IIb and ␤ 3 or ligand binding function. In pulse chase studies, very little pro␣IIb was processed into mature ␣IIb, suggesting that at least a portion of this mutant pro␣ IIb ␤ 3 was retained and degraded within endoplasmic reticulum.…”