Rapid agonist-mediated phosphorylation of m3-muscarinic receptors revealed by immunoprecipitation.
A specific antiserum against the human m3-muscarinic receptor subtype was made by subcloning a variant region of the third intracellular loop of the m3-receptor (Ser345-Leu463) into a bacterial expression plasmid that produced a fusion protein with glutathione S-transferase. In immunoblot studies this anti-serum identified the human m3-receptor expressed in transfected Chinese hamster ovary (CHO) cells (CHO-m3 cells, 1343 fmol/mg protein) as a diffuse band at approximately 97-110 kDa. In vivo labeling of the ATP pool in CHO-m3 cells with [32P]orthophosphate followed by immunoprecipitation of solubilized m3-receptors revealed that the unstimulated receptor existed in a phosphorylated form. Incubation of CHO-m3 cells with the cholinergic agonist carbachol (1 mM) increased the phosphorylated state of the receptor dramatically, primarily at serine. The time course for agonist-dependent phosphorylation was very rapid occurring within seconds of agonist addition and was maintained for at least 30 min. The muscarinic antagonist atropine (10 microM) inhibited agonist-stimulated phosphorylation. Neither forskolin (10 microM) nor the calcium ionophore, ionomycin (1 microM), had any effect on the state of phosphorylation of the m3-receptor, eliminating a role for cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase in the agonist-dependent phosphorylation of m3-receptors. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (100 nM) did increase m3-receptor phosphorylation, an effect that was inhibited by the selective protein kinase C inhibitor RO-318220 (10 microM). However, agonist-stimulated m3-receptor phosphorylation was not inhibited by RO-318220 indicating that protein kinase C was not involved in agonist-induced m3-receptor phosphorylation. In conclusion the phosphorylation of m3-receptors, in vivo, was increased following the application of muscarinic agonist or PMA. The response to agonist was mediated via a kinase distinct from protein kinase C, protein kinase A and Ca2+/calmodulin dependent protein kinase, whereas the effect of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate was mediated by protein kinase C.
Tobin
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AB
Department of Pharmacology and Therapeutics, Leicester University, United Kingdom.
Nahorski
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SR
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001483:Base Sequence; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010277:Parasympathomimetics / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011494:Protein Kinases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D007106:Immune Sera; D009838:Oligodeoxyribonucleotides; D010277:Parasympathomimetics; D010710:Phosphates; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D015759:Ionomycin; D001285:Atropine; D000255:Adenosine Triphosphate; D002217:Carbachol; D005982:Glutathione Transferase; D011494:Protein Kinases; D013755:Tetradecanoylphorbol Acetate