Essential Role of Dynamin in Internalization of M2Muscarinic Acetylcholine and Angiotensin AT1AReceptors

doi:10.1074/jbc.M001736200

Essential Role of Dynamin in Internalization of M₂Muscarinic Acetylcholine and Angiotensin AT_1AReceptors*

From the Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany

Abstract

Most G protein-coupled receptors (GPCRs), including the M₁ muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M₂ mAChR and the angiotensin AT_1A receptor (AT_1AR) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M₂mAChR and AT_1AR in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4,5-bisphosphate, strongly inhibits internalization of M₁ and M₂ mAChRs and AT_1ARs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M₁ mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M₁ mAChR internalization. In contrast, M₂ mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M₁ mAChR but also for M₂ mAChR and AT_1AR internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.

Footnotes

↵* This work was supported by a grant from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Supported by an IFORES predoctoral fellowship from the Universitätsklinikum Essen.
↵§ These authors contributed equally to this work.
↵¶ To whom correspondence should be addressed: Institut für Pharmakologie, Universitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany. Tel.: 49-201-723-3462; Fax: 49-201-723-5968; E-mail: van_koppen@uni-essen.de.
Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M001736200
↵2 Y. Werbonat, N. Kleutges, K. H. Jakobs, and C. J. van Koppen, unpublished observations.
Abbreviations:

GPCR

G protein-coupled receptor

AT_1AR

angiotensin AT_1A receptor

BSA

bovine serum albumin

mAChR

muscarinic acetylcholine receptor

NMS

N-methylscopolamine

PIP₂

phosphatidylinositol 4,5-bisphosphate

HBS

HEPES-buffered saline

DMEM

Dulbecco's modified Eagle's medium
- Received February 28, 2000.
- Revision received April 26, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.