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- Title
Structural aspects of M<sub>3</sub> muscarinic acetylcholine receptor dimer formation and activation.
- Authors
Jianxin Hu; Thor, Doreen; Yaru Zhou; Tong Liu; Yan Wang; McMillin, Sara M.; Mistry, Rajendra; Challiss, R. A. John; Costanzi, Stefano; Wess, Jürgen
- Abstract
To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M3 muscarinic acetyl-choline receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. [35S]GTPγS binding/Gαq/11 immunoprecipitation assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (~50% reduction in receptor-G-protein coupling, as compared to control M3R). These data support the novel concept that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Given the high degree of structural homology among family A GPCRs, these findings should be of broad significance.
- Subjects
DIMERS; MUSCARINIC receptors; CHOLINERGIC receptors; G proteins; MEMBRANE proteins
- Publication
FASEB Journal, 2012, Vol 26, Issue 2, p604
- ISSN
0892-6638
- Publication type
Article
- DOI
10.1096/fj.11-191510