Binding of Gq protein stabilizes the activated state of the muscarinic receptor type 1

Activation of G protein coupled receptors (GPCRs) induces various cellular responses through interactions with G proteins. The key trigger of GPCR activation is agonist binding. It is reportedly known that the agonist-bound active conformation of the GPCRs, such as the muscarinic acetylcholine receptor type 1 (M1R), can be affected by the coupling of G proteins and by depolarization of the membrane potential. Here we aimed at investigating their effects on the structural rearrangements of the M(1)Rs between the active and quiescent states, using the fluorescence resonance energy transfer (FRET) technique. For this purpose, fluorescent M1R constructs that maintained intact activation of the Gq pathway and interaction with Gq were used. We captured the agonist-induced conformational changes of the M1R as the FRET decreases and found that the FRET decreases were enhanced by co-expression of the Gq subunits. In addition, co-expression of the Gq subunits decelerated the recovery of the declined FRET upon removal of the agonists, which was slower than the dissociation of the Gq subunits from the receptor. These results suggested that Gq binding stabilizes the agonist-induced activated conformation of the M1R. We also found that depolarization of the membrane potential slightly but significantly enhanced the agonist-induced FRET decrease, by accelerating the agonist-induced conformational changes. Thus, structural rearrangement analyses by FRET revealed that Gq coupling stabilizes the active conformation of the M1R and also suggested that depolarization accelerates the transition from quiescent to activation conformation. (C) 2012 Elsevier Ltd. All rights reserved.