Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells: REGULATION OF RECEPTOR DOWN-REGULATION BY HETERODIMERIZATION (original) (raw)

2005, Journal of Biological Chemistry

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 M 1 , M 2 , and M 3 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 M 1 , M 2 , or M 3 mAChR is predominantly composed of high affinity homodimers. Saturation curve analysis of cells expressing two receptor subtypes demonstrates the existence of high affinity M 1 /M 2 , M 2 /M 3 , and M 1 /M 3 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 M 2 receptor exhibits much higher susceptibility than the M 3 receptor to agonistinduced down-regulation. Coexpression of M 3 mAChR with increasing amounts of the M 2 subtype in JEG-3 cells resulted in an increased agonist-induced down-regulation of M 3 , suggesting a novel role of heterodimerization in the mechanism of mAChR long term regulation. . 3 The abbreviations used are: GPCR, G protein-coupled receptor; mAChR, muscarinic acetylcholine receptor; TMD, transmembrane domain; BRET, bioluminescence resonance energy transfer; RLuc, Renilla luciferase; YFP, yellow fluorescence protein, GFP, green fluorescence protein; Smo, smoothened; QNB, quinuclidinyl benzilate; i3, third intracellular loop; PBS, phosphate-buffered saline; HA, hemagglutinin.