A Peptide Derived from a β2-Adrenergic Receptor Transmembrane Domain Inhibits Both Receptor Dimerization and Activation

doi:10.1074/jbc.271.27.16384

A Peptide Derived from a β₂-Adrenergic Receptor Transmembrane Domain Inhibits Both Receptor Dimerization and Activation*

From the ^‡ Département de biochimie and Le Groupe de Recherche sur le Systéme Nerveux Autonome, Université de Montréal, Montréal, Quebec, Canada H3C 3J7 and the
^∥ Centre de Recherche, Hôpital du Sacre-Coeur de Montréal et Département de Médecine, Université de Montréal, Montréal, Quebec, Canada H4J 1C5

^∥ Medical Research Council of Canada Scientist. To whom correspondence should be addressed. Tel.: 514-343-6319; Fax: 514-343-2210; E-mail: bouvier{at}bch.umontreal.ca

Abstract

One of the assumptions of the mobile receptor hypothesis as it relates to G protein-coupled receptors is that the stoichiometry of receptor, G protein, and effector is 1:1:1 (Bourne, H. R., Sanders, D. A., and McCormick, F. (1990) Nature 348, 125–132). Many studies on the cooperativity of agonist binding are incompatible with this notion and have suggested that both G proteins and their associated receptors can be oligomeric. However, a clear physical demonstration that G protein-coupled receptors can indeed interact as dimers and that such interactions may have functional consequences was lacking. Here, using differential epitope tagging we demonstrate that β₂-adrenergic receptors do form SDS-resistant homodimers and that transmembrane domain VI of the receptor may represent part of an interface for receptor dimerization. The functional importance of dimerization is supported by the observation that a peptide derived from this domain that inhibits dimerization also inhibits β-adrenergic agonist-promoted stimulation of adenylyl cyclase activity. Moreover, agonist stimulation was found to stabilize the dimeric state of the receptor, while inverse agonists favored the monomeric species, which suggests that interconversion between monomeric and dimeric forms may be important for biological activity.

Footnotes

↵^§ Supported by fellowships from the Heart and Stroke Foundation of Canada and the Fonds de la Recherche en Santé du Québec. Present address: Centre de recherche, Institut de cardiologie de Montréal et Dépt. de médecine, Université de Montréal, 5000 rue Bélanger est, Montréal, Quebec, Canada H1T 1C8.
↵^¶ Both supported by scholarships from the Heart and Stroke Foundation of Canada.
↵^” Supported by a studentship from the Medical Research Council of Canada.
↵^‴ Career scientist of the Medical Research Council of Canada.
↵* This work was supported by grants from the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada (to M. B.). 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.
↵¹ The abbreviations used are:

β₂AR

β₂-adrenergic receptor

BASED

bis[β-(4-azidosalicylamindo)ethyl]disulfide

M2-R

M2 muscarinic acetylcholine receptor

D1-R

D1 dopamine receptor

GPCR

G protein-coupled receptor

GpA

glycophorin A

HA

influenza hemagglutinin

TM VI

transmembrane domain 6

V2-R

V2 vasopressin receptor

CYP

cyanopindolol

PAGE

polyacrylamide gel electrophoresis

mAb

monoclonal antibody.
↵² G. Y. K. Ng, B. F. O'Dowd, H. T., Chung, S. P. Lee, M. R. Brann, and S. R. George, personal communication.
- Received March 1, 1996.
- Revision received April 3, 1996.