Decrease in Coupling of Gs in v-src-Transformed NIH-3T3 Fibroblasts: Possible Involvement of Tyrosine Phosphorylation of Gs by pp60v-src

In Rous sarcoma virus (RSV)-transformed NIH-3T3 fibroblasts expressing pp60v-src as tyrosine protein kinase, isoproterenol-stimulated cAMP accumulation was much lower than in normal cells. The reduction in v-src-transformed cells seemed to be mainly due to a decrease in the number of β2-adrenoceptors. When the membranes were phosphorylated with ATP, however, the binding affinity of isoproterenol to β2-adrenoceptors was reduced in transformed cell membranes by 34% compared to that in normal cell membranes. The reduction in transformed cell membranes was restored to the level of normal cell membranes by treatment of membranes with anti-pp60v-src antibody. GTPγS- and cholera toxin-stimulated adenylyl cyclase activities were reduced with no change in forskolin-stimulated adenylyl cyclase activity in transformed cell membranes. The reduced effect of GTPγS was also restored by treatment with anti-pp60v-src antibody or by adding staurosporine, which inhibits a variety of protein kinases, including tyrosine protein kinase. One of the 32P-phosphoproteins phosphorylated with [γ-32P]ATP in v-src-transformed cell membranes was bound to GTP-agarose, and was a 46-kDa molecule on a sodium dodecyl sulfate-polyacrylamide gel. This 46-kDa 32P-labeled phosphoprotein was immunoprecipitated with anti-phosphotyrosine antibody or anti-stimulatory GTP-binding protein (anti-Gs) antibody. These results suggest that pp60v-src phosphorylates the α-subunit of Gs and consequently causes a decrease in the coupling of β2-receptors to Gs and in the coupling of Gs to adenylyl cyclase.