Abstract
Each subtype of the muscarinic receptor family of G protein-coupled receptors is activated by similar concentrations of the neurotransmitter acetylcholine or closely related synthetic analogs such as carbachol. However, pharmacological selectivity can be generated by the introduction of a pair of mutations to produce Receptor Activated Solely by Synthetic Ligand (RASSL) forms of muscarinic receptors. These display loss of potency for acetylcholine/carbachol alongside a concurrent gain in potency for the ligand clozapine N-oxide. Co-expression of a form of wild type human M2 and a RASSL variant of the human M3 receptor resulted in concurrent detection of each of M2-M2 and M3-M3 homomers alongside M2-M3 heteromers at the surface of stably transfected Flp-In(TM) T-REx(TM) 293 cells. In this setting occupancy of the receptors with a muscarinic antagonist was without detectable effect on any of the muscarinic oligomers. However, selective agonist occupancy of the M2 receptor resulted in enhanced M2-M2 homomer interactions but decreased M2-M3 heteromer interactions. By contrast, selective activation of the M3 RASSL receptor did not significantly alter either M3-M3 homomer or M2-M3 heteromer interactions. Selectively targeting closely related receptor oligomers may provide novel therapeutic opportunities.
Highlights
Muscarinic receptors can form both homo- and hetero-oligomers
Cells were co-incubated with forskolin (5 M) and ligands for 30 min in a white Proxiplate-384 Plus. This step was followed by lysis of cells using a mixture of detection reagents prepared in lysis buffer according to manufacturer’s instructions and incubation for 1 h at room temperature. Homogeneous Time-resolved FRET (htrFRET) was measured on a PheraStar FS and the reduction of cAMP levels was calculated as ratio of 665/620 nm
In cells induced with doxycycline to allow co-expression of VSVSNAP-hM2WT and HA-CLIP-hM3RASSL, as noted above, coaddition of a combination of SNAP-Lumi4Tb and CLIP-Red resulted in detection of htrFRET signal, consistent with interactions between the two receptors (Fig. 8A)
Summary
Muscarinic receptors can form both homo- and hetero-oligomers. Results: Co-expression of M2 and M3 receptors resulted in concurrent detection of both homomer and heteromer interactions and regulation of M2-containing forms by agonist. Pharmacological selectivity can be generated by the introduction of a pair of mutations to produce Receptor Activated Solely by Synthetic Ligand (RASSL) forms of muscarinic receptors These display loss of potency for acetylcholine/carbachol alongside a concurrent gain in potency for the ligand clozapine N-oxide. Co-expression of a form of wild type human M2 and a RASSL variant of the human M3 receptor resulted in concurrent detection of each of M2-M2 and M3-M3 homomers alongside M2-M3 heteromers at the surface of stably transfected Flp-InTM T-RExTM 293 cells. In this setting occupancy of the receptors with a muscarinic antagonist was without detectable effect on any of the muscarinic oligomers. We demonstrate concurrent detection of M2-M2, M3-M3, and M2-M3 interactions at the surface of cells and distinct agonist regulation of these interactions
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