Identification of a Talin-binding Site in the Integrin β3 Subunit Distinct from the NPLY Regulatory Motif of Post-ligand Binding Functions

doi:10.1074/jbc.274.40.28575

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*

From the ^‡Department of Pharmacology and ^¶School of Biomedical and Health Information Sciences, the University of Illinois, Chicago, Illinois 60612

Abstract

Following platelet aggregation, integrin α_IIbβ₃ becomes associated with the platelet cytoskeleton. The conserved NPLY sequence represents a potential β-turn motif in the β₃ cytoplasmic tail and has been suggested to mediate the interaction of β₃integrins with talin. In the present study, we performed a double mutation (N744Q/P745A) in the integrin β₃ subunit to test the functional significance of this β-turn motif. Chinese hamster ovary cells were co-transfected with cDNA constructs encoding mutant β₃ and wild type α_IIb. Cells expressing either wild type (A5) or mutant (D4) α_IIbβ₃ 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 α_IIbβ₃ to interact with talin in a solid phase binding assay. Both wild type and mutant α_IIbβ₃, 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 β₃ subunit does not reside in the NPLY motif. In contrast, specific binding of talin to peptides containing the membrane-proximal HDRKEFAKFEEERARAK sequence of the β₃ 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 α_IIbβ₃ 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 β-turn motif regulates post-ligand binding functions of α_IIbβ₃ 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 β₃cytoplasmic tail.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants HL-41793 (to S. C.-T. L.) and HL-52755 (to J. D. W.-D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Both authors made equal contributions.
↵‖ Supported by an Established Investigator Award from the American Heart Association and Genentech. To whom correspondence should be addressed: Dept. of Pharmacology (M/C 868), University of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612. Tel.: 312-413-5928; Fax: 312-996-1225; E-mail: sclam@uic.edu.
↵2 S. Patil, A. Jedsadayanmata, J. D. Wencel-Drake, W. Wang, I. Knezevic, and S. C.-T. Lam, unpublished observations.
Abbreviations:

CHO cells

Chinese hamster ovary cells

mAb

monoclonal antibody

FITC

fluorescein isothiocyanate

PCR

polymerase chain reaction

DMEM

Dulbecco’s modified Eagle’s medium

BSA

bovine serum albumin

PBS

phosphate-buffered saline

FAK

focal adhesion kinase

ELISA

enzyme-linked immunosorbent assay

PAGE

polyacrylamide gel electrophoresis

RACE

rapid amplification of cDNA ends
- Received March 9, 1999.
- Revision received July 20, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.