Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.
Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, 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 vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type 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 to 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 alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.
Lewis
J M
JM
Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Schwartz
M A
MA
D016475:3T3 Cells; D000185:Actinin / Q000032:analysis / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000378:metabolism; D002645:Chickens; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019012:Integrin beta1; D016023:Integrins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D016608:Talin / Q000378:metabolism; D016596:Vinculin / Q000032:analysis / Q000378:metabolism
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D019012:Integrin beta1; D016023:Integrins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D016608:Talin; D000185:Actinin; D016596:Vinculin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse