Regulation of M3 RASSL by phosphorylation

Rudi Prihandoko1, Elisa Alvarez-Curto2, Graeme Milligan2, Andrew B. Tobin1. 1Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, LE1 9HN, United Kingdom, 2Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, United Kingdom.

To investigate the role of a specific GPCR signalling in vivo, mutant receptors which no longer respond to their endogenous ligand(s) but can still be fully activated by synthetic molecules have been generated. These receptors are known as RASSLs (receptors activated solely by synthetic ligands). Recently, a RASSL based on the M3 mAChR has been developed. This receptor contains two mutations within the ligand binding pocket (Y149C, A239G) and was shown to be unresponsive to acetylcholine but responded fully to clozapine-N-oxide (CNO). Subsequent functional studies have shown that this receptor couples normally to the phosphoinositide hydrolysis and ERK1/2 phosphorylation pathways in response to CNO. This receptor also undergoes agonist dependent internalization. However the phosphorylation profile of this receptor has not been described. Using a number of biochemical techniques including in vivo 32P-labeling, phospho-peptide mapping and western blotting with phospho-specific antibodies, we show that the M3 RASSL was phosphorylated in a similar manner as the wild-type M3 mAChR. Hence this reverse engineered receptor fully recapitulated the signalling and regulatory properties of the wild-type M3 mAChR and may provide an avenue for studying phosphorylation/arrestin dependent signalling in vivo in addition to G-protein mediated signalling.