ADP-RIBOSYLATION CYCLES

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Integrin alpha7 is a major substrate for a cell-surface ADP- ribosyltransferase in intact, differentiated mouse myoblasts (myotubes). To address the question of the role of post-translational modification of integrin alpha7, we studied interactions of the dimer of integrin alpha7 and beta1 with the extracellular matrix protein laminin in solution and in intact cells. It was observed that integrin alpha7beta1 bound to EHS laminin (laminin-1, composed of alpha1, beta1 and gamma1 chains), but did not bind to endogenous laminin expressed in C2C12 myotubes. Northern blot analysis demonstrated that C2C12 myotubes synthesized mRNA's for all laminin-1 subunits. C2C12 laminin was however, immunologically distinct from EHS laminin; it was not recognized by 5D3 anti-laminin-1 monoclonal antibody, whereas 5A2 and LT3 antibodies reacted equally well with C2C12 and EHS laminins. Following deglycosylation of EHS laminin, separation of the subunits by SDS-PAGE, Western blotting, and partial amino acid sequencing of the protein bands, the epitope recognized by 5D3 antibody was localized to the gamma1 laminin chain. It is postulated that integrin alpha7beta1 binding site is contained within the same laminin subunit. Incubation of intact C2C12 myotubes with EHS laminin or plating of C2C12 cells on EHS laminin-coated dishes resulted in the binding of EHS laminin to the cell surface and subsequent co-immunoprecipitation of laminin and integrin alpha7. The dose response analysis and the divalent cation dependence of the binding suggested that the interaction of EHS laminin and integrin alpha7beta1 occurred after lysis of cells rather than at the cell surface. EHS laminin and integrin alpha7beta1 were chemically cross-linked after they formed a complex in solution. The two proteins, however, did not undergo cross-linking at the cell surface, suggesting that in intact, resting myotubes integrin alpha7beta1 interacted poorly with EHS laminin, which may reflect a limited accesibility of integrin alpha7beta1 for laminin in the membrane to or an inactive state of the integrin.