Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin alphaIIbbeta3.
Platelet aggregation requires agonist-induced alphaIIbbeta3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered alphaIIbbeta3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates alphaIIbbeta3, 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 alphaIIbbeta3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or beta3 that disrupt their mutual interaction block both talin recruitment and alphaIIbbeta3 activation. However, one talin mutant (L325R) is recruited to alphaIIbbeta3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated alphaIIbbeta3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and alphaIIbbeta3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to alphaIIbbeta3 by RIAM mediates agonist-induced alphaIIbbeta3 activation, with implications for hemostasis and thrombosis.
Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Naohide
N
Watanabe
Laurent
L
Bodin
Manjula
M
Pandey
Matthias
M
Krause
Shaun
S
Coughlin
Vassiliki A
VA
Boussiotis
Mark H
MH
Ginsberg
Sanford J
SJ
Shattil
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D005453:Fluorescence; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D044463:Receptor, PAR-1 / Q000378:metabolism; D016608:Talin / Q000819:agonists* / Q000378:metabolism*; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C521189:RIAM protein, mouse; D044463:Receptor, PAR-1; D016608:Talin; D020827:rap1 GTP-Binding Proteins