A NOVEL PHOSPHOGLUCOMUTASE-RELATED PROTEIN IS CONCENTRATED IN ADHERENS JUNCTIONS OF MUSCLE AND NONMUSCLE CELLS

Using five monoclonal antibodies raised against a human uterine smooth muscle extract, we have identified a novel antigen which runs as a cl osely spaced doublet in SDS-gels. The proteins (60/63 M)a) co-purify, are present in a 1:1 ratio as judged by Coomassie Blue staining, and a re immunologically closely related, if not identical. No N-terminal se quence could be obtained from a mixture of the 60/63 kDa proteins, but the sequence of four polypeptides liberated by V8 protease or cyanoge n bromide cleavage showed that the proteins are closely related to the glycolytic enzyme phosphoglucomutase type 1. Affinity-purified polycl onal antibodies and three different monoclonal antibodies to the 60/63 kDa proteins cross-reacted with rabbit skeletal muscle phosphoglucomu tase type 1, whilst two additional monoclonal antibodies were specific for the 60/63 M)a proteins. Peptide maps of the 60/63 kDa proteins an d phosphoglucomutase 1 are markedly different, and the purified protei ns have no detectable phosphoglucomutase activity. Staining of culture d smooth muscle cells and fibroblasts with antibodies to 60/63 kDa pro teins showed that the antigen is concentrated in focal contacts at the ends of actin bundles and is also associated with actin filaments. Ab out 60% of the cellular 60/63 kDa proteins were found in the detergent -insoluble fraction, suggesting a physical association with the cytosk eleton. The highest levels of protein immunoreactivity were found in m uscles. The antigen is concentrated in muscle adherens junctions, incl uding smooth muscle dense plaques, cardiomyocyte intercalated disks, a nd striated muscle myotendinous junctions. Among epithelial cells, the 63 kDa isoform of the protein was found only in cultured keratinocyte s where immunofluorescent staining was localized in cell-to-cell adher ens junctions. Expression of the 60/63 M)a proteins in vascular smooth muscle cells is developmentally regulated and correlates with the dif ferentiated contractile phenotype of these cells.