Agonist Regulation of Muscarinic M2/M3 Receptor Heteromer and M2 Homomer Stability, but not of the Corresponding M3 Homomer

D Aslanoglou, E Alvarez Curto, G Milligan. University of Glasgow, Glasgow, UK

Muscarinic receptors (M1-M5) belong to class A of the G protein coupled receptor (GPCR) family. There is growing evidence that many GPCRs exist as dimers or higher-order oligomers (1) and muscarinic receptors are no exception (2). Herein, as for the co-existence of homomers and heteromers of the dopamine D2 and D3 receptors (3) we demonstrate such combinations of co-expressed human M2 (hM2WT) and a RASSL (Receptor Activated Solely by Synthetic Ligand) form of the human M3 receptor (hM3RASSL) using N-terminal SNAP and CLIP tags in combination with homogeneous time resolved FRET (HTRF) (3). Stable Flp-In™ T-REx™ 293 cell lines able to inducibly express each of these receptor forms upon addition of doxycycline, and a cell line able to express both hM3RASSL constitutively and hM2WT in a doxycycline inducible manner were generated.

In these cells both hM3RASSL and hM2WT were detected after treatment with different concentrations of doxycycline via Western Blots using tag-specific antibodies. Radioligand binding using [3H]-QNB indicated that similar amounts of hM2WT and hM3RASSL were expressed following induction with 5 ng.ml-1 doxycycline; Bmax (no dox) = 2603 ± 200 fmol.mg protein-1; Bmax (+ dox) = 5465 ± 244 fmol.mg protein-1). Following induction with doxycycline each of hM2WT and hM3RASSL homo-oligomers and hM2WT-hM3RASSL heteromers were identified. Unlike the corresponding homo-oligomers in cells expressing either receptor alone, occupancy of hM2WT-hM3RASSL heteromers with the hM2WT agonist carbachol resulted in a marked, time and concentration-dependent (pIC50= 5.2 ± 0.25) decrease in detected heteromers and a concomitant, concentration-dependent (pEC50 = 5.5 ± 0.2) increase in hM2WT homomers. The formation of hM2WT-hM3RASSL heteromers was significantly decreased (P=0.007) by 1.2 fold in the presence of 1 mM carbachol, and by 1.3 fold when 1 mM carbachol was added in the presence of 100 µM CNO (P=0.037). There was a 2.3 fold increase detected in the hM2WT homomers, in the presence of 1mM carbachol (P=0.0002) or 1 mM carbachol and 100 µM CNO together (P=0.001).

Despite the presence of hM2WT-hM3RASSL heteromers the functional pharmacology of hM2WT and hM3RASSL receptor specific agonists (carbachol and clozapine N-oxide respectively) were largely unaltered.

1. Milligan G, Mol Pharmacol 84:158, 2013

2. Alvarez-Curto E et al, J Biol Chem 285:23318, 2010

3. Pou C et al, JBC 287:8864, 2012