MAPPING IN-VIVO ASSOCIATIONS OF CYTOPLASMIC PROTEINS WITH INTEGRIN BETA-1 CYTOPLASMIC DOMAIN MUTANTS

Integrins promote formation of focal adhesions and trigger intracellul ar signaling pathways through cytoplasmic proteins such as talin, alph a-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subuni t has been shown to bind talin and alpha-actinin in in vitro assays, a nd these proteins may link integrin to the actin cytoskeleton either d irectly or through linkages to other proteins such as vinculin. Howeve r, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vi vo assay to address these questions. Microbeads were coated with anti- chicken beta 1 antibodies to selectively cluster chicken beta 1 integr ins expressed in cultured mouse fibroblasts. The ability of cytoplasmi c domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-typ e integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha -actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin t o co-localize talin and FAK was found to require a sequence near the C -terminus of beta 1. The region of beta 1 required to co-localize alph a-actinin was found to reside in a different sequence, several amino a cids further from the C-terminus of beta 1. Deletion of 13 residues fr om the C-terminus blocked co-localization of talin, FAK, and actin, bu t not alpha-actinin. Association of alpha-actinin with clustered integ rin is therefore not sufficient to induce the co-localization of F-act in.