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 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.
Akiho
H
H
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Tokumitsu
Y
Y
Noda
M
M
Nomura
Y
Y
D016475:3T3 Cells; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002472:Cell Transformation, Viral; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016391:Genes, src; D066298:In Vitro Techniques; D020120:Iodocyanopindolol; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D010869:Pindolol / Q000031:analogs & derivatives / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D008565:Membrane Proteins; D010750:Phosphoproteins; D011943:Receptors, Adrenergic, beta; D019000:Phosphotyrosine; D014443:Tyrosine; D020120:Iodocyanopindolol; D010869:Pindolol; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases