Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin αIIbβ3
Platelet aggregation requires agonist-induced αIIbβ3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered αIIbβ3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimul...
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Published in | The Journal of cell biology Vol. 181; no. 7; pp. 1211 - 1222 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
The Rockefeller University Press
30.06.2008
Rockefeller University Press |
Subjects | |
Online Access | Get full text |
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Summary: | Platelet aggregation requires agonist-induced αIIbβ3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered αIIbβ3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates αIIbβ3, 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 αIIbβ3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or β3 that disrupt their mutual interaction block both talin recruitment and αIIbβ3 activation. However, one talin mutant (L325R) is recruited to αIIbβ3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated αIIbβ3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and αIIbβ3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to αIIbβ3 by RIAM mediates agonist-induced αIIbβ3 activation, with implications for hemostasis and thrombosis. |
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Bibliography: | 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. Correspondence to Sanford J. Shattil: sshattil@ucsd.edu |
ISSN: | 0021-9525 1540-8140 |
DOI: | 10.1083/jcb.200803094 |