title: Alteration of receptor/G-protein interaction by putative endogenous protein kinase activity in Dictyostelium discoideum membranes.
abstract: Membranes of Dictyostelium discoideum cells were incubated under phosphorylation conditions and washed, and the effects on cAMP binding to chemotactic receptors in the absence and presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) were investigated. Most experiments were done with adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), which is a good substrate for many kinases, but the product, protein phosphorothioate, is not easily hydrolyzed by phosphatases. Pretreatment of membranes under phosphorylating conditions with MgATP gamma S alters the site heterogeneity of the cAMP-binding forms, without a significant effect on the total number of binding sites. A similar effect was induced by GTP gamma S under nonphosphorylation conditions. The effects of MgATP gamma S were rapid (t1/2 = 1 min), irreversible, and not induced by Mg2+ or ATP gamma S alone or by magnesium adenylyl imidodiphosphate and magnesium adenylyl (beta, gamma-methylene)diphosphate. MgATP induced a smaller inhibition than MgATP gamma S, which was potentiated by the addition of exogenous cAMP-dependent protein kinase. The effect of MgATP was rapidly reversible; reversibility was reduced by the phosphatase inhibitor NaF. These results suggest that the effects of MgATP gamma S are mediated by an endogenous protein kinase. The major 35S-thiophosphorylated band detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a protein with Mr = 36,000. The phosphorylation of a protein with the molecular weight of the cAMP receptor (Mr = 40,000-45,000) was not observed.
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002633:Chemotaxis; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D004023:Dictyostelium / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D011953:Receptors, Cyclic AMP; D013873:Thionucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D019204:GTP-Binding Proteins; D008274:Magnesium