Vinculin activates inside-out signaling of integrin αIIbβ3 in Chinese hamster ovary cells.
Although vinculin is used frequently as a marker for integrin-mediated focal adhesion complexes, how it regulates the activation of integrin is mostly unknown. In this study, we examined whether vinculin would activate integrin in Chinese hamster ovary (CHO) cells expressing human integrin αIIbβ3. Silencing of vinculin by lentiviral transduction with a short hairpin RNA sequence affected the binding of PAC-1 (an antibody recognizing activated human αIIbβ3) to a constitutively active form of αIIbβ3 (α6Bβ3) expressed on CHO cells, while its inhibitory effects were much weaker than those of talin-1. Overexpression of an active form of vinculin without intramolecular interactions, but not the full length one, induced PAC-1 binding to native αIIbβ3 expressed on CHO cells in a manner dependent on talin-1. On the other hand, silencing of talin-1, but not vinculin, failed to induce cell spreading of α6Bβ3-CHO cells on fibrinogen, even in the presence of PT 25-2, a monoclonal antibody that activates αIIbβ3. Thus, an active form of vinculin could induce αIIbβ3 inside-out signaling through the actions of talin-1, while vinculin was dispensable for outside-in signaling.
Ohmori
Tsukasa
T
Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan. tohmori@jichi.ac.jp
Kashiwakura
Yuji
Y
Ishiwata
Akira
A
Madoiwa
Seiji
S
Mimuro
Jun
J
Honda
Shigenori
S
Miyata
Toshiyuki
T
Sakata
Yoichi
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D020868:Gene Silencing; D006801:Humans; D039001:Integrin alpha Chains / Q000096:biosynthesis* / Q000276:immunology; D039661:Integrin beta3 / Q000096:biosynthesis* / Q000276:immunology; D051379:Mice; D016608:Talin / Q000235:genetics / Q000378:metabolism; D016596:Vinculin / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D039001:Integrin alpha Chains; D039661:Integrin beta3; D016608:Talin; D016596:Vinculin