DESENSITIZATION AND INTERNALIZATION OF THE M2 MUSCARINIC ACETYLCHOLINE-RECEPTOR ARE DIRECTED BY INDEPENDENT MECHANISMS

The phenomenon of acute desensitization of G-protein-coupled receptors has been associated with several events, including receptor phosphory lation, loss of high affinity agonist binding, receptor:G-protein unco upling, and receptor internalization. However, the biochemical events underlying these processes are not fully understood, and their contrib utions to the loss of signaling remain correlative, In addition, the n ature of the kinases and the receptor domains which are involved in mo dulation of activity have only begun to be investigated, In order to d irectly measure the role of G-protein-coupled receptor kinases (GRKs) in the desensitization of the m2 muscarinic acetylcholine receptor (m2 mAChR), a dominant-negative allele of GRK2 was used to inhibit recept or phosphorylation by endogenous GRK activity in a human embryonic kid ney cell line. The dominant-negative GRK2K(220R) reduced agonist depen dent phosphorylation of the m2 mAChR by similar to 50% and prevented a cute desensitization of the receptor as measured by the ability of the m2 mAChR to attenuate adenylyl cyclase activity, In contrast, the ago nist-induced internalization of the m2 mAChR was unaffected by the GRK 2(K220R) construct. Further evidence linking receptor phosphorylation to acute receptor desensitization was obtained when two deletions of t he third intracellular loop were made which created m2 mAChRs that did not become phosphorylated in an agonist dependent manner and did not desensitize. However, the mutant mAChRs retained the ability to intern alize. These data provide the first direct evidence that GRK-mediated receptor phosphorylation is necessary for m2 mAChR desensitization; th e likely sites of in vivo phosphorylation are in the central portion o f the third intracellular loop (amino acids 282-323). These results al so indicate that internalization of the m2 receptor is not a key event in desensitization and is mediated by mechanisms distinct from GRK ph osphorylation of the receptor.