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Decrease in coupling of Gs in v-src-transformed NIH-3T3 fibroblasts: possible involvement of tyrosine phosphorylation
1Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Abstract:
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 beta 2-adrenoceptors. When the membranes were phosphorylated with ATP, however, the binding affinity of isoproterenol to beta 2-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. GTP gamma S- 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 GTP gamma S 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 [gamma-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 alpha-subunit of Gs and consequently causes a decrease in the coupling of beta 2-receptors to Gs and in the coupling of Gs to adenylyl cyclase.
Insights
Rous sarcoma virus (RSV) infection reduces beta 2-adrenoceptor function in NIH-3T3 cells by phosphorylating Gs proteins. This impairs cAMP signaling, affecting cell communication.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Rous sarcoma virus (RSV) transformation of NIH-3T3 fibroblasts alters cellular signaling pathways.
- Tyrosine protein kinase pp60v-src plays a key role in RSV-induced transformation.
- Isoproterenol-stimulated cAMP accumulation is a critical indicator of beta-adrenergic receptor function.
Purpose of the Study:
- To investigate the impact of pp60v-src on beta 2-adrenoceptor function and cAMP signaling in NIH-3T3 cells.
- To elucidate the molecular mechanisms underlying the observed alterations in signaling pathways.
- To determine the role of pp60v-src in the phosphorylation of key signaling proteins.
Main Methods:
- Measurement of isoproterenol-stimulated cAMP accumulation.
- Analysis of beta 2-adrenoceptor binding affinity.
- Assessment of adenylyl cyclase activity stimulated by GTP gamma S and forskolin.
- Phosphorylation studies using [gamma-32P]ATP and identification of phosphoproteins via GTP-agarose binding and immunoprecipitation.
Main Results:
- RSV-transformed cells exhibited significantly lower cAMP accumulation compared to normal cells.
- A decrease in beta 2-adrenoceptor number and binding affinity was observed in transformed cells.
- pp60v-src mediated phosphorylation of a 46-kDa protein, identified as the alpha-subunit of Gs, was detected.
- Treatment with anti-pp60v-src antibody or staurosporine restored normal signaling function.
Conclusions:
- pp60v-src directly phosphorylates the alpha-subunit of the stimulatory GTP-binding protein (Gs).
- This phosphorylation event leads to reduced coupling between beta 2-adrenoceptors and Gs, and between Gs and adenylyl cyclase.
- The findings reveal a novel mechanism by which viral tyrosine kinases disrupt cellular signal transduction pathways.