New insights into the structural organisation of human muscarinic M3 acetylcholine receptor (M3R) dimers/higher-order oligomers

MJ Varela1, G Caltabiano2, G Milligan1. 1University of Glasgow, Glasgow, UK, 2Universitat Autonoma de Barcelona, Bellaterra, Spain

Although GPCRs have long been considered to function as monomeric units, it is now widely accepted that they can associate into dimers or higher-order oligomers. However, there still remains a lack of knowledge about the basis of these interactions (1) and identifying the structural elements governing oligomer formation, together with an understanding of how this phenomenon can modify receptor signalling is a key challenge in receptor pharmacology and drug discovery. The M3R is a member of the muscarinic acetylcholine receptor subfamily of class A GPCRs that preferentially activates G proteins of the Gq family and is associated with numerous physiological functions. In previous work we showed that, at the plasma membrane, M3R exists as a mixture of dimers that can associate reversibly to form tetramers (2). The aim of this work was to identify key structural elements involved in human (h) M3R oligomerisation interactions and models of the hM3R rhombic tetramer (2) and the atomic level, inactive state, structure of M3R (3). hM3R was modified at the N terminus to incorporate the metabotropic glutamate receptor (mGluR5) signal sequence, the epitope tag (VSV-G) and the polypeptide ‘SNAP’ tag. This construct was used as template to generate transmembrane (TM) domain mutants of the hM3R by alanine substitution mutagenesis. To examine whether the hM3R mutants were impaired in their ability to oligomerise we performed homogenous time resolved (htr)-FRET assays using the Tag-lite® system (Cisbio Bioassays). Optimal concentrations of htr-FRET donor and acceptor were established by incubating cells expressing the wild type construct with fixed donor and varying energy acceptor concentrations. The htr-FRET signal was plotted and fit to a bell shaped curve, the maximum of which described the optimal concentration ratio to identify cell surface hM3R oligomers. Htr-FRET assays were then performed in HEK293T cells transfected with increased amounts of wild type or receptor mutant plasmids, htr-FRET signal (665 nm emission after excitation at 337nm) was plotted as a function of cell surface receptor expression defined by fluorescence emission at 620 nm. A linear dependence between htr-FRET emission and receptor expression was observed for both wild type and all the mutants and data were analysed by linear regression. The resulting slope values were compared, with lower slope values indicative of reduced propensity to oligomerise (Table 1).

Residues altered to Alanine are identified by the ‘one-letter’ code and the corresponding Ballesteros and Weinstein residue location numbering system. Data are given as the mean ± S.E. of 3 independent experiments.

Overlay of identified key residues with the ‘tetramer model’ suggests both further sites of contact to explore and a potential important role for receptor-associated molecules of cholesterol

(1) Milligan G, Mol Pharmacol 84:158,2013

(2) Patowary S et al, Biochem J 452:303,2013 (3) Kruse AC et al, Nature 482:552,2012