MULTIPLE MECHANISMS INVOLVING PROTEIN-PHOSPHORYLATION ARE LINKED TO DESENSITIZATION OF MUSCARINIC RECEPTORS

Agonists induce phosphorylation of m2 muscarinic receptors (mAChR) in several cell types. This phosphorylation correlates with desensitizati on. The mechanisms underlying mAChR phosphorylation have been investig ated using several in vitro approaches. Protein kinase C phosphorylate d the purified and reconstituted m2 mAChR to a stoichiometry of approx imately 5 mols P/mol receptor; this phosphorylation resulted in the de creased ability of receptors to activate G-proteins. Although the phos phorylation by PKC was not modulated by agonist binding to the mAChR, heterotrimeric G-proteins were able to completely block the PKC-mediat ed effects. If significant receptor/G-protein coupling occurs in viva, agonists would be required to promote dissociation of the G-proteins from the receptors and reveal the phosphorylation sites for PKC. Membe rs of the G-protein coupled receptor kinase (GRK) family also phosphor ylated the purified and reconstituted m2 mAChR. In contrast to PKC, th e GRKs phosphorylated the m2 mAChR strictly in an agonist-dependent ma nner. GRK mediated phosphorylation perturbed receptor/G-protein coupli ng. In addition, phosphorylation allowed for arrestin binding to the m 2 mAChR which should further contribute to desensitization. Using a ne w strategy that does not require purification and reconstitution of re ceptors for GRK studies, the m3 mAChR were revealed as substrates for the GRKs. For both the m2 and m3 receptor subtypes, the most effective kinases were GRK 2 and 3. Phosphorylation of the receptors by these e nzymes was stimulated by low concentrations of G-proteins and by membr ane phospholipids. Thus, multiple mechanisms involving protein phospho rylation appear to contribute to the overall process of mAChR desensit ization.