Desensitization and Internalization of the m2 Muscarinic Acetylcholine Receptor Are Directed by Independent Mechanisms

Abstract

The phenomenon of acute desensitization of G-protein-coupled receptors has been associated with several events, including receptor phosphorylation, loss of high affinity agonist binding, receptor:G-protein uncoupling, and receptor internalization. However, the biochemical events underlying these processes are not fully understood, and their contributions to the loss of signaling remain correlative. In addition, the nature of the kinases and the receptor domains which are involved in modulation of activity have only begun to be investigated. In order to directly measure the role of G-proteincoupled receptor kinases (GRKs) in the desensitization of the m2 muscarinic acetylcholine receptor (m2 mAChR), a dominant-negative allele of GRK2 was used to inhibit receptor phosphorylation by endogenous GRK activity in a human embryonic kidney cell line. The dominant-negative GRK2 K220R reduced agonist-dependent phosphorylation of the m2 mAChR by ϳ50% and prevented acute desensitization of the receptor as measured by the ability of the m2 mAChR to attenuate adenylyl cyclase activity. In contrast, the agonist-induced internalization of the m2 mAChR was unaffected by the GRK2 K220R construct. Further evidence linking receptor phosphorylation to acute receptor desensitization was obtained when two deletions of the 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 internalize. These data provide the first direct evidence that GRK-mediated receptor phosphorylation is necessary for m2 mAChR desensitization; the likely sites of in vivo phosphorylation are in the central portion of the third intracellular loop (amino acids 282-323). These results also indicate that internalization of the m2 receptor is not a key event in desensitization and is mediated by mechanisms distinct from GRK phosphorylation of the receptor.

Phosphorylation and Desensitization of Human Endothelin A and B Receptors

“…This K220R mutant of GRK2 cannot perform a phosphotransferase reaction (35); therefore, for any receptor substrate of GRK2, the K220R mutant can act as a competitive inhibitor of any GRK that could bind to and subsequently phosphorylate the receptor (18,21,(35)(36)(37). Characteristically, however, the GRK2 K220R mutant inhibits GRK action incompletely; even when present in 15-fold molar excess over GRK2, the K220R mutant inhibits only 60% of agonist-stimulated ␤ 2 -adrenergic receptor phosphorylation in vitro (35).…”

Involvement of Receptor Cycling and Receptor Reserve in Resensitization of Muscarinic Responses in SH‐SY5Y Human Neuroblastoma Cells

“…Many G protein-coupled receptors become phosphorylated in response to agonist stimulation (Palmer et al, 1995;Pals-Rylaarsdam et al, 1995;Pippig et al, 1995;Heurich et al, 1996), and this modification has been linked to the functional desensitization of the receptor (Pippig et al, 1995;Garland et al, 1996). Furthermore, subsequent dephosphorylation of the receptor by protein phosphatases may play a role in receptor resensitization (Krueger et al, 1997).…”

“…However, loss of receptors from the surface is often too slow to account for the rapid onset of desensitization (Roth et al, 1991;Tobin et al, 1992;Fisher et al, 1994), and the effects of site-directed mutations on receptor internalization and desensitization are often disparate (Barak et al, 1994;Pals-Rylaarsdam et al, 1995), suggesting that the major functional role of internalization is not simply to cause desensitization. An alternative hypothesis is that receptor cycling is involved in receptor resensitization (Yu et al, 1993).…”

Dynamic Targeting of the Agonist-stimulated m2 Muscarinic Acetylcholine Receptor to Caveolae in Cardiac Myocytes

“…40), the data in this manuscript support the hypothesis that, following stimulation by agonist, cardiac m2 mAchR translocation to caveolae may be necessary to initiate specific downstream signaling cascades. Interestingly, several recent studies have shown that internalization of the m2 and m4 mAchR is mediated by mechanisms distinct from the phosphorylation by the G protein-coupled receptor kinase (GRK) family known to lead to receptor desensitization (41,42). The translocation of muscarinic receptors within caveolae should allow their interaction with the heterotrimeric G protein complexes known to be concentrated within these plasmalemmal microdomains (12,26,28) and lead, after recruitment of co-factors and intermediate effector proteins, to the activation of eNOS, a resident caveolar protein in cardiac myocytes.…”