Structural Basis of M3 Muscarinic Receptor Dimer/Oligomer Formation

Abstract

Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M 3 muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.The superfamily of G protein-coupled receptors (GPCRs) 3 represents the largest group of cell surface receptors found in nature (1, 2). Following activation by extracellular ligands such as neurotransmitters, hormones, or sensory stimuli, GPCRs regulate an extraordinarily large number of important physiological responses, reflecting the fact that nearly 50% of drugs in current clinical use target specific GPCRs (3). A characteristic structural feature of all GPCRs is a transmembrane (TM) core consisting of a bundle of seven TM helices (TM1-7). The TM receptor core plays a critical role in propagating the ligandinduced conformational changes to the cytoplasmic receptor surface, enabling the receptor to interact with specific classes of heterotrimeric G proteins (2, 4).Accumulating evidence suggests that GPCRs are able to form dimers and/or higher order oligomeric complexes (5-10). Most convincingly, studies with class C GPCRs, exemplified by the ␥-aminobutyric acid, type B, and metabotropic glutamate receptors, have demonstrated that dimer formation is required for agonist-induced G protein activation, at least in this subclass of GPCRs (for recent reviews see Refs. 7,8). Class C GPCRs form stable dimeric interactions through well character...

Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

“…The specific region that is involved in the dimer interface varies, but there are common themes that emerge. TM4 is a common interface for GPCRs to interact with one another (15,35,47,48). Our recent studies have mapped the homodimer interface for PAR4 to a region on TM4 (15 , and Leu-202…”

Oligomerization of GPCRs involved in endocrine regulation

“…Several GPCR-GPCR protomer interfaces have been reported under involvement of TMH4 (Carrillo et al 2004, Hernanz-Falcon et al 2004, Guo et al 2005, Mancia et al 2008), TMH1, and TMH5-6 (Hebert et al 1996, McMillin et al 2011, Yanagawa et al 2011 or the extracellular N-terminal region (Uddin et al 2012). The intermolecular interactions are constituted between single amino acids or between multitudes of side chains.…”

Design and Functional Characterization of a Novel, Arrestin-Biased Designer G Protein-Coupled Receptor

“…Approximately 48 h after transfection, cells were incubated with increasing concentrations of CNO, and changes in intracellular calcium levels were determined using FLIPR technology (Molecular Devices, Sunnyvale, CA). All measurements were performed in 96-well plates, as described previously McMillin et al, 2011). CNO concentration-response curves were analyzed using Prism 4.0 software.…”

“…For bioluminescence resonance energy transfer (BRET) studies, we generated plasmids [vector backbone, pcDNA3.1(ϩ)] coding for receptors in which the C terminus of Rq or Rq(R165L) was fused to the coding sequence of Renilla reniformis luciferase 8 (Luc), yielding Rq-Luc and Rq(R165L)-Luc, respectively. These plasmids were obtained by using a strategy similar to the one we used to generate M 3 -Luc (McMillin et al, 2011). The correctness of all coding regions was verified by sequencing.…”