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J. Biol. Chem., Vol. 281, Issue 9, 5416-5425, March 3, 2006

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Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells

REGULATION OF RECEPTOR DOWN-REGULATION BY HETERODIMERIZATION^*

Juan C. Goin¹ and Neil M. Nathanson²

From the Centro de Estudios Farmacológicos y Botánicos, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires 1414, Argentina and the Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195

Although previous pharmacological and biochemical data support the notion that muscarinic acetylcholine receptors (mAChR) form homo- and heterodimers, the existence of mAChR oligomers in live cells is still a matter of controversy. Here we used bioluminescence resonance energy transfer to demonstrate that M₁, M₂, and M₃ mAChR can form constitutive homo- and heterodimers in living HEK 293 cells. Quantitative bioluminescence resonance energy transfer analysis has revealed that the cell receptor population in cells expressing a single subtype of M₁, M₂, or M₃ mAChR is predominantly composed of high affinity homodimers. Saturation curve analysis of cells expressing two receptor subtypes demonstrates the existence of high affinity M₁/M₂, M₂/M₃, and M₁/M₃ mAChR heterodimers, although the relative affinity values were slightly lower than those for mAChR homodimers. Short term agonist treatment did not modify the oligomeric status of homo- and heterodimers. When expressed in JEG-3 cells, the M₂ receptor exhibits much higher susceptibility than the M₃ receptor to agonist-induced down-regulation. Coexpression of M₃ mAChR with increasing amounts of the M₂ subtype in JEG-3 cells resulted in an increased agonist-induced down-regulation of M₃, suggesting a novel role of heterodimerization in the mechanism of mAChR long term regulation.

Received for publication, July 11, 2005 , and in revised form, November 22, 2005.

^* This work was supported by National Institutes of Health Grants NS26920 and HL44948 and by grants from the Pew Latin American Program for the Biomedical Sciences and Fundación Antorchas. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–4.

¹ Researcher for Consejo Nacional de Investigaciones Científicas y Técnicas.

² To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington, Box 357750, Seattle, WA 98195-7750. Tel.: 206-543-9457; Fax: 206-616-4230; E-mail: nathanso{at}u.washington.edu.

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