title: Kindlins, integrin activation and the regulation of talin recruitment to αIIbβ3.
abstract: Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment.
authors.affiliation: Department of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Bryan N
authors.initials: BN
authors.lastname: Kahner
authors.forename: Hisashi
authors.initials: H
authors.lastname: Kato
authors.forename: Asoka
authors.initials: A
authors.lastname: Banno
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ginsberg
authors.forename: Sanford J
authors.initials: SJ
authors.lastname: Shattil
authors.forename: Feng
authors.initials: F
authors.lastname: Ye
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D039661:Integrin beta3 / Q000378:metabolism*; D008565:Membrane Proteins / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology*; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism*
chemical_list: C475301:FERMT1 protein, human; D039661:Integrin beta3; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D040201:Platelet Membrane Glycoprotein IIb