INTEGRIN CYTOPLASMIC DOMAINS MEDIATE INSIDE-OUT SIGNAL-TRANSDUCTION

We analyzed the binding of fibronectin to integrin alpha(5) beta(1) in various cells; in some cells fibronectin bound with low affinity (e.g ., K562 cells) whereas in others (e.g., CHO), it bound with high affin ity (Kd similar to 100 nM) in an energy-dependent manner. We construct ed chimeras of the extracellular and transmembrane domains of alpha(II b)beta(3) joined to the cytoplasmic domains of alpha(5) beta(1). The a ffinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha(5) beta(1) conferred an energy-dependent high affinity state on alpha(IIb )beta(3) in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha(2), alpha(6)A, alpha(6)B) conferred PAC1 binding in CH O cells, while three others (alpha(M), alpha(L), alpha(V)) did not. In the high affinity alpha chimeras, cotransfection with a truncated (be ta(3) Delta 724> or mutated (beta(3)(S-752-->P)) beta(3) subunit aboli shed high affinity binding. Thus, both cytoplasmic domains are require d for energy-dependent, cell type-specific affinity modulation. In add ition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha(IIb)beta (3). In contrast to the chimeras, the high affinity state of these mut ants was independent of cellular metabolism, cell type, and the bulk o f the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic doma ins mediate inside-out signaling. Furthermore, the highly conserved GF FKR motif of the alpha subunit cytoplasmic domain maintains the defaul t low affinity state.