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Published online
doi:10.1083/jcb.200803094
The Journal of Cell Biology, Vol. 181, No. 7, 1211-1222
The Rockefeller University Press, 0021-9525 $30.00
© Watanabe et al.
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Article

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



Naohide Watanabe1, Laurent Bodin1, Manjula Pandey1, Matthias Krause2, Shaun Coughlin3,4, Vassiliki A. Boussiotis5,6, Mark H. Ginsberg1, and Sanford J. Shattil1

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 {alpha}IIbβ3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered {alpha}IIbβ3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates {alpha}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 {alpha}IIbβ3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or β3 that disrupt their mutual interaction block both talin recruitment and {alpha}IIbβ3 activation. However, one talin mutant (L325R) is recruited to {alpha}IIbβ3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated {alpha}IIbβ3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and {alpha}IIbβ3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to {alpha}IIbβ3 by RIAM mediates agonist-induced {alpha}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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