Cooperative Role of the Membrane-proximal and -distal Residues of the Integrin β3 Cytoplasmic Domain in Regulation of Talin-mediated αIIbβ3 Activation

Takaaki Hato,Jun Yamanouchi,Tatsushiro Tamura,Yoshihiro Yakushijin,Ikuya Sakai,Masaki Yasukawa

doi:10.1074/jbc.m707246200

Abstract

Integrin cytoplasmic tails regulate integrin activation that is required for high affinity binding with ligands. The interaction of the integrin beta subunit tail with a cytoplasmic protein, talin, largely contributes to integrin activation. Here we report the cooperative interaction of the beta3 membrane-proximal and -distal residues in regulation of talin-mediated alpha IIb beta3 activation. Because a chimeric integrin, alpha IIb beta3/beta1, in which the beta3 tail was replaced with the beta1 tail was constitutively active, we searched for the residues responsible for integrin activation among the residues that differed between the beta3 and beta1 tails. Single amino acid substitutions of Ile-719 and Glu-749 in the beta3 membrane-proximal and -distal regions, respectively, with the corresponding beta1 residues or alanine rendered alphaIIbbeta3 constitutively active. The I719M/E749S double mutant had the same ligand binding activity as alpha IIb beta3/beta1. These beta3 mutations also induced alphaVbeta3 activation. Conversely, substitution of Met-719 or Ser-749 in the beta1 tail with the corresponding beta3 tail residue (M719I or S749E) inhibited alpha IIb beta3/beta1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type alpha IIb beta3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced alpha IIb beta3 activation. These results suggest that the beta3 membrane-proximal and -distal residues cooperatively regulate talin-mediated alpha IIb beta3 activation.

Highlights

Platelet integrin ␣IIb␤3 is a transmembrane receptor that mediates platelet adhesion and aggregation [1, 2]. ␣IIb␤3 exists in a low affinity state in resting platelets and requires activation for high affinity binding with soluble ligands [1, 2]
Mutation of the Ile-719 and Glu-749 Residues in the ␤3 Tail Activates ␤3 Integrins—To identify the residues critical for regulation of ␣IIb␤3 activation in the ␤3 tail, we focused on amino acids in the ␤3 tail that differed from those in the ␤1 tail as the ␣IIb␤3/␤1 chimeric integrin, in which the ␤3 tail is replaced with the ␤1 tail, has been reported to be constitutively active [24]
Expressed on the surface of Chinese hamster ovary (CHO) cells by cotransfection of The expression of ␣IIb␤3 mutants on the cell surface was mutant ␤3 and wild-type ␣IIb cDNAs, and surface expression 93–136% that of the wild-type ␣IIb␤3, indicating almost equivof ␣IIb␤3 was determined by flow cytometry using a non-func- alent expression of the mutated integrins (Fig. 2A)

Summary

Introduction

Platelet integrin ␣IIb␤3 is a transmembrane receptor that mediates platelet adhesion and aggregation [1, 2]. ␣IIb␤3 exists in a low affinity state in resting platelets and requires activation for high affinity binding with soluble ligands [1, 2]. We report the cooperative interaction of the ␤3 membrane-proximal and -distal residues in regulation of talin-mediated ␣IIb␤3 activation. Determine whether the mutations of the ␤3 tail responsible for ␣IIb␤3 activation give rise to ␣V␤3 activation, recombinant ␣V␤3 was expressed on the surface of CHO cells by transfection with wild-type ␣V and mutant ␤3 cDNAs. The surface expression of mutant ␣V␤3 was comparable with that of wildtype ␣V␤3.

Results

Conclusion