A new protein of adhesion plaques and ruffling membranes

Abstract

A protein with a molecular weight on SDS polyacrylamide gels of 215,000 (referred to here as 215K) was purified from chicken gizzard smooth muscle. Antibodies against this protein localized it in fibroblasts to adhesion plaques (focal contacts), to regions underlying cell surface fibronectin, and to ruffling membranes. In the first two distributions it was similar to vinculin in cellular location, and this was confirmed by double-label immunofluorescence microscopy, but the concentration of 215K in membrane ruffles distinguished it from vinculin. There was no cross-reaction of the antibody against 215K with vinculin, and immunoprecipitation and antibody staining of SDS gels of whole cells revealed a single cross-reactive component with a molecular weight of 215,000. Immunoprecipitation from cultures labeled with [32P]phosphate revealed 215K to be a phosphoprotein. Transformation of rat or chicken fibroblasts by Rous sarcoma virus resulted in a reorganization of 215K, in some cases into complex intracellular structures. The localization of 215K where microfilament bundles terminate as well as in close relation to cell surface fibronectin and in membrane ruffles suggests that the protein has some function in the organization of actin filaments at or close to regions of actin-membrane attachment.