Identification of a Talin-binding Site in the Integrin β3 Subunit Distinct from the NPLY Regulatory Motif of Post-ligand Binding Functions: THE TALIN N-TERMINAL HEAD DOMAIN INTERACTS WITH THE MEMBRANE-PROXIMAL REGION OF THE β3 CYTOPLASMIC TAIL

Sonali Patil,Arom Jedsadayanmata,June D Wencel-Drake,Wei Wang,Irina Knezevic,Stephen C.-T Lam

doi:10.1074/jbc.274.40.28575

Sonali Patil, Arom Jedsadayanmata + Show 4 more

Open Access

https://doi.org/10.1074/jbc.274.40.28575

Copy DOI

Abstract

Following platelet aggregation, integrin alpha(IIb)beta(3) becomes associated with the platelet cytoskeleton. The conserved NPLY sequence represents a potential beta-turn motif in the beta(3) cytoplasmic tail and has been suggested to mediate the interaction of beta(3) integrins with talin. In the present study, we performed a double mutation (N744Q/P745A) in the integrin beta(3) subunit to test the functional significance of this beta-turn motif. Chinese hamster ovary cells were co-transfected with cDNA constructs encoding mutant beta(3) and wild type alpha(IIb). Cells expressing either wild type (A5) or mutant (D4) alpha(IIb)beta(3) adhered to fibrinogen; however, as opposed to control A5 cells, adherent D4 cells failed to spread, form focal adhesions, or initiate protein tyrosine phosphorylation. To investigate the role of the NPLY motif in talin binding, we examined the ability of the mutant alpha(IIb)beta(3) to interact with talin in a solid phase binding assay. Both wild type and mutant alpha(IIb)beta(3), purified by RGD affinity chromatography, bound to a similar extent to immobilized talin. Additionally, purified talin failed to interact with peptides containing the AKWDTANNPLYK sequence indicating that the talin binding domain in the integrin beta(3) subunit does not reside in the NPLY motif. In contrast, specific binding of talin to peptides containing the membrane-proximal HDRKEFAKFEEERARAK sequence of the beta(3) cytoplasmic tail was observed, and this interaction was blocked by a recombinant protein fragment corresponding to the 47-kDa N-terminal head domain of talin (rTalin-N). In addition, RGD affinity purified platelet alpha(IIb)beta(3) bound dose-dependently to immobilized rTalin-N, indicating that an integrin-binding site is present in the talin N-terminal head domain. Collectively, these studies demonstrate that the NPLY beta-turn motif regulates post-ligand binding functions of alpha(IIb)beta(3) in a manner independent of talin interaction. Moreover, talin was shown to bind through its N-terminal head domain to the membrane-proximal sequence of the beta(3) cytoplasmic tail.

Highlights

Following platelet aggregation, integrin ␣IIb␤3 becomes associated with the platelet cytoskeleton
The NPLY Motif in the ␤3 Cytoplasmic Tail Is Essential for ␣IIb␤3-mediated Post-ligand Binding Functions—By using the Chou and Fasman and Gor II methods to predict protein secondary structures, it has previously been reported that the N744PLY sequence represents a potential ␤-turn motif in the cytoplasmic tail of the integrin ␤3 subunit [41]
The N744Q/P745A ␤3 mutant construct was cotransfected with a wild type ␣IIb construct into Chinese hamster ovary (CHO)-K1 cells, and stable clonal lines expressing the mutant receptor were developed for functional studies

Summary

Introduction

Integrin ␣IIb␤3 becomes associated with the platelet cytoskeleton. RGD affinity purified platelet ␣IIb␤3 bound dose-dependently to immobilized rTalin-N, indicating that an integrin-binding site is present in the talin N-terminal head domain These studies demonstrate that the NPLY ␤-turn motif regulates post-ligand binding functions of ␣IIb␤3 in a manner independent of talin interaction. Since the corresponding region in the ␤1 cytoplasmic tail has been implicated in talin binding function [27, 28], we examined the ability of the purified mutant receptor to interact with talin in a solid phase binding assay In these experiments, we found that the N744Q/P745A mutation in ␤3 blocked post-ligand binding functions but did not affect ␣IIb␤3 binding to talin. Results of our study show that the N-terminal head domain of talin interacts with the membrane-proximal region of the ␤3 cytoplasmic sequence

Methods

Results

Conclusion