INTEGRIN-BETA CYTOPLASMIC DOMAINS DIFFERENTIALLY CYTOSKELETAL PROTEINS

Integrin cytoplasmic domains connect these receptors to the cytoskelet on, Furthermore, integrin-cytoskeletal interactions involve ligand bin ding (occupancy) to the integrin extracellular domain and clustering o f the integrin, To construct mimics of the cytoplasmic face of an occu pied and clustered integrin, we fused the cytoplasmic domains of integ rin beta subunits to an N terminal sequence containing four heptad rep eat sequences. The heptad repeats form coiled coil dimers in which the cytoplasmic domains are parallel dimerized and held in an appropriate vertical stagger. In these mimics we found 1) that both conformation and protein binding properties are altered by insertion of Gly spacers C-terminal to the heptad repeat sequences; 2) that the cytoskeletal p roteins talin and filamin are among the polypeptides that bind to the integrin beta 1A tail, Filamin, but not talin binding, is enhanced by the insertion of Gly spacers; 3) binding of both cytoskeletal proteins to beta 1A is direct and specific, since it occurs with purified tali n and filamin and is inhibited in a point mutant (beta 1A(Y788A)) or i n splice variants (beta 1B, beta 1C) known to disrupt cytoskeletal ass ociations of beta 1 integrins; 4) that the muscle-specific splice vari ant, beta 1D, binds talin more tightly than beta 1A and is therefore p redicted to form more stable cytoskeletal associations; and 5) that th e beta 7 cyto plasmic domain binds filamin better than beta 1A, The st ructural specificity of these associations suggests that these mimics offer a useful approach for the analysis of the interactions and struc ture of the integrin cytoplasmic face.