Characterization of Major Phosphoproteins in the cGMP-Mediated Protein Phosphorylation System of Vascular Smooth Muscle Membranes

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Abstract
G₀ (215–250 kD) and G1 (120–140 kD), the unidentified major phosphoproteins in the cGMP-mediated protein phosphorylation system of vascular smooth muscle membranes, were compared for biochemical and immunological properties with the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R, 240 kD) and 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, and comigrated with G₀ and G1, respectively, on SDS-PAGE. When thiophosphorylated G₀ and G1 were subjected to immunoprecipitation, MBS antibody induced the precipitation of a 138-kD phosphoprotein, but did not significantly affect the amount of G1 remaining in the supernatant, while InsP3R antibody precipitated G₀ almost completely. Unexpectedly, InsP3R antibody coprecipitated a large portion of G1, which did not cross-react with either antibody to MBS or InsP3R. Just like InsP3R, G₀ bound to the calmodulin column in a Ca2+-dependent manner, and, again, a large portion of G1 was copurified with G₀. These results suggest that G₀ is identical to InsP3R, while G1 consists of several phosphoproteins, including the 138-kD protein associated with InsP3R as a major component. MBS is not G1 or may represent only a minor component of it.