Abstract
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.
Highlights
A specific antiserum against the human m3-musca- growth factoreceptors and G-proteiniant region of the third intracellular loop of the m3- linked receptors
Agonist-stimulated m3-receptorphosphorylationwasnotinhibitedby RO-318220 indicating that protein kinase C was not whoawsever, it would appear from thesestudiesthat receptor phosphorylation is commonly employed in the regulation of G-protein-linked receptors
Using Chinese hamster ovary (CHO) cells transfected with the human m3-muscarinic receptor cDNA we have recently identified a rapid desensitization event, in which, a 5-min pre-exposure to muscarinic agonist reduces the peak inositol 1,4,5-trisphosphate (Ins[1,4,5]P3)response seen within the first few seconds of a subsequent application of agonist [20]
Summary
Glutathione S-transferase as evident by the loss in immunoreactivity to extracts from pGEX-2T transformeEd. coli Immunoprecipitation of solubilized CHO-m3membranes from cells where the protein content had been ubiquitously radiolabeled with [35S]methionine revealed a diffuse band running at approximately 97-110 kDa (Fig. 4, lane R).This band was present in immunoprecipitates from CHO-m3 cells but not from nontransfected CHO cells. This band was exactly superimposable with the specific immunoreactive band identified in immunoblots from CHO-m3 cell membranes, thereforeidentifying this band aims munoprecipitated m3-receptor. The inclusion of the muscarinic receptor antagonist atropine (10 p ~ )0 min prior to agonist application, dramatically inhibited phosphorylation of the m3-receptor (Fig. 6). Mediated m3-receptor phosphorylation but, significantly,has no effect on agonist-mediated phosphorylation (Fig. 9)
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