Characterization of major phosphoproteins in the cGMP-mediated protein phosphorylation system of vascular smooth muscle membranes

Go (215-250 kD) and G(1) (120-140 kD), the unidentified major phosphoprotei ns in the cGMP-mediated protein phosphorylation system of vascular smooth m uscle membranes, were compared for biochemical and immunological properties with the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R, 240 kD) a nd the myosin-binding subunit (MBS, 138 kD) of myosin phosphatase, both of them substrates for cGMP-dependent protein kinase. Two microsomal proteins that were immunoreactive with antibodies to InsP3R and MBS were detected, a nd comigrated with G(0) and G(1), respectively, on SDS-PAGE. When thiophosp horylated G(0) and G(1) were subjected to immunoprecipitation, MBS antibody induced the precipitation of a 138-kD phosphoprotein, but did not signific antly affect the amount of G(1) remaining in the supernatant, while InsP(3) R antibody precipitated G(0) almost completely. Unexpectedly, InsP(3)R anti body coprecipitated a large portion of G(1), which did not cross-react with either antibody to MBS or InsP(3)R. Just like InsP(3)R, G(0) bound to the calmodulin column in a Ca2+-dependent manner, and, again, a large portion o f G(1) was copurified with G(0). These results suggest that G(0) is identic al to InsP(3)R, while G1 consists of several phosphoproteins, including the 138-kD protein associated with InsP(3)R as a major component. MBS is not G (1) or may represent only a minor component of it.