Molecular events associated with the regulation of signaling by M-2 muscarinic receptors

Multiple events are associated with the regulation of signaling by the M-2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuati on of adenylyl cyclase by the M-2, mAChRs appears to involve agonist-depend ent phosphorylation of M-2 mAChRs by G-protein coupled receptor kinases (GR Ks) that phosphorylate the receptors in a serine/threonine rich motif in th e 3(rd) intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M-2 mAChR results in a los s of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist- induced sequestration of receptors away from their normal membrane environm ent is also regulated by agonist-induced phosphorylation of the M-2 mAChRs on the 3(rd) intracellular domain, but in HEK cells, the predominant pathwa y of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signalin g of the M-2 mAChR to G-protein regulated inwardly rectifying K channels (G IRKs) can be modified by RGS proteins. In HEK cells, expression of RGS prot eins leads to a constitutive activation of the channels through, a mechanis m that depends on G beta gamma. RGS proteins appear to increase the concent ration of flee G beta gamma in addition to acting as GAPs. Thus multiple me chanisms acting at either the level of the M-2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M-2 mAChRs.