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Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin IIbβ3

¹ Department of Medicine, University of California, San Diego, La Jolla, CA 92093
² Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, England, UK
³ Cardiovascular Research Institute and ⁴ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143
⁵ Division of Hematology and Oncology and ⁶ Transplantation Biology Research Center, Massachusetts General Hospital, Boston, MA 02115

Correspondence to Sanford J. Shattil: sshattil{at}ucsd.edu

Platelet aggregation requires agonist-induced IIbβ3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered IIbβ3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates IIbβ3, which was measured with antibody PAC-1, indicating complete pathway reconstitution. Knockdown of Rap1–guanosine triphosphate–interacting adaptor molecule (RIAM), a Rap1 effector, blocks this response. In living cells, RIAM overexpression stimulates and RIAM knockdown blocks talin recruitment to IIbβ3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or β3 that disrupt their mutual interaction block both talin recruitment and IIbβ3 activation. However, one talin mutant (L325R) is recruited to IIbβ3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated IIbβ3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and IIbβ3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to IIbβ3 by RIAM mediates agonist-induced IIbβ3 activation, with implications for hemostasis and thrombosis.

Abbreviations used in this paper: BiFC, bimolecular fluorescence complementation; IRES, internal ribosomal entry site; PAR, protease-activated receptor; RIAM, Rap1-GTP–interacting adaptor molecule; shRNA, short hairpin RNA; VASP, vasodilator-stimulated phosphoprotein.

© 2008 Watanabe et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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