Amino acid residues in the alpha IIb subunit that are critical for ligand binding to integrin alpha IIbbeta 3 are clustered in the beta-propeller model.

Tetsuji Kamata,Kenneth K Tieu,Yoshikazu Takada,Atsushi Irie,Timothy A Springer

doi:10.1074/jbc.m107021200

Abstract

Several distinct regions of the integrin alpha(IIb) subunit have been implicated in ligand binding. To localize the ligand binding sites in alpha(IIb), we swapped all 27 predicted loops with the corresponding sequences of alpha(4) or alpha(5). 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 beta-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 Ca(2+)-binding predicted loops on the lower surface of the beta-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 beta-propeller toroid, centering on repeat 3.

Highlights

The integrin ␣IIb␤3 plays a critical role in primary hemostasis by mediating interactions between platelets and fibrinogen [1]
Residues that are critical for ligand binding and epitopes for function-blocking monoclonal antibodies1 have consistently been located in repeats 2– 4 of several integrin ␣ subunits, regardless of ligand specificity
We found that ligand binding was not affected by 19 of 27 swapping mutations using fibrinogen and ligand-mimetic monoclonal antibodies (mAbs) as ligands, effectively ruling out the possibility that a major ligand binding site is located in repeats 1, 6, and 7

Summary

Introduction

The integrin ␣IIb␤3 (glycoprotein IIb-IIIa, CD41/CD61) plays a critical role in primary hemostasis by mediating interactions between platelets and fibrinogen [1]. The second putative cation binding site of ␣IIb (residues 294 –314 in N-terminal repeat 5 of ␣IIb) can be chemically cross-linked to the fibrinogen ␥-chain C-terminal dodecapeptide (HHLGGAKQAGDV400–411) [4]. This peptide and antibodies against it have been shown to block binding of fibrinogen to ␣IIb␤3 [5]. We have recently localized epitopes for ligand-mimetic anti-␣IIb␤3 antibodies (OP-G2 and LJ-CP3) within repeats 2– 4 [16] These results do not rule out the possibility that ligands bind to other sequences of the ␣IIb subunit.

Methods

Results

Conclusion