Muscarinic acetylcholine receptors (M1-M5) belong to the class A family of transmembrane G protein coupled receptors (GPCRs) and mediate various signalling processes. M1, M3 and M5 predominantly couple to Gq and promote intracellular calcium ion release from the endoplasmic reticulum. M2 and M4 preferentially couple Gi inhibiting adenylyl cyclase activity to thus decrease cAMP production and acting to regulate various ion channels. There is growing evidence that many GPCRs can exist as dimers or higher-order oligomers (Milligan, 2013) and muscarinic receptors are no exception (Alvarez-Curto et al., 2010). Herein, combinations of homomers and heteromers of co-expressed human M2 (hM2WT) and a RASSL (Receptor Activated Solely by Synthetic Ligand) form of the human M3 receptor (hM3RASSL) (Alvarez-Curto et al., 2011) were demonstrated to occur using N-terminal SNAP and CLIP tags in combination with homogeneous time resolved FRET (htrFRET). 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 the 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 in the cells co-expressing the two receptors. Expression of the receptors was observed at the surface of live cells following labelling of the expressed receptors with SNAP and CLIP-specific cell impermeant substrates. Following induction with doxycycline each of hM2WT and hM3RASSL homomers and hM2WT-hM3RASSL heteromers were identified. Detection of oligomers was achieved following co-labelling with htrFRET-compatible substrates. Occupancy of hM2WT-hM3RASSL heteromers with the hM2WT agonist carbachol resulted in a marked, time and concentration-dependent decrease in detected heteromers and a concomitant, concentration-dependent increase in hM2WT homomers. The dynamics of interchange between heteromers and homomers was investigated by using a multiplex labelling approach and htrFRET. This method involved labelling with one energy donor and two energy acceptors capable of emitting at distinct wavelengths. Results confirmed the hM2WT-hM3RASSL heteromer to hM2WT homomer transition upon selective carbachol-mediated activation of hM2WT. A small increase in the hM3RASSL homomer was detected upon activation of the hM3RASSL with the selective agonist clozapine-N-oxide, but this was only observed in the absence of heteromers. Despite the presence of hM2WT-hM3RASSL heteromers the functional pharmacology of hM2WT and hM3RASSL receptor specific agonists was largely unaltered.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:679579 |
Date | January 2016 |
Creators | Aslanoglou, Despoina |
Publisher | University of Glasgow |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
Source | http://theses.gla.ac.uk/7048/ |
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