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

doi:10.1074/jbc.270.48.29004

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

From the (1) Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611 and
(2) Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

^¶ To whom correspondence should be addressed:
Dept. of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611.
Tel.: 312-503-2737; Fax: 312-503-0495; mhosey{at}nwu.edu.

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-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 receptor phosphorylation by endogenous GRK activity in a human embryonic kidney cell line. The dominant-negative GRK2 reduced agonist-dependent phosphorylation of the m2 mAChR by 50% 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 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.

Footnotes

↵^§ An established investigator of the American Heart Association.
↵* This work was supported in part by National Institutes of Health Grants HL 50201 (to M. M. H.) and GM 44944 (to J. L. B.), a grant-in-aid from the American Heart Association (to M. M. H.), NIEHS Grant ES00210 to Oregon State University Environment Health Sciences Center, a National Research Service Award postdoctoral fellowship (to P. W. E.), and a Howard Hughes Medical Institute Predoctoral Fellowship (to R. P. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GPR

G-protein coupled receptor

GRK

G-protein coupled receptor kinase

ACh

acetylcholine

m2 mAChR

m2 muscarinic acetylcholine receptor

AR

adrenergic receptor

βARK

β-adrenergic receptor kinase

DME

Dulbecco's modified Eagle's medium

F-12

Ham's F-12 medium

HRP

horseradish peroxidase

PCR

polymerase chain reaction

NMS

N-methyl scopolamine

QNB

1-quinuclidinyl benzilate

LH(R)

luteinizing hormone (receptor)

PBS

phosphate-buffered saline

TBS

Tris-buffered saline

PAGE

polyacrylamide gel electrophoresis

GTPS

guanosine-5′-O-(3-thio)triphosphate

HEK

human embryonic kidney.
↵²X.-L. Zhao and M. M. Hosey, unpublished observations.
- Received August 7, 1995.
- Revision received September 28, 1995.