Regulation of the M1 RASSL by Phosphorylation
G-protein coupled receptors or seven-transmembrane receptors have been shown to undergo phosphorylation in response to agonist occupation that mediates downstream signalling in the cell. To investigate these receptors, in vivo, our laboratory is currently employing a chemical genetics approach based on a mutant receptor called a RASSL (receptors activated solely by synthetic ligands). RASSL receptors are engineered so that the receptor no longer responds to its endogenous ligand(s) but instead can be activated by a previously inert pharmacological ligand. A RASSL based on the acetylcholine M1-muscarinic receptor has been developed which contains modifications in the third- (T106C) and fifth- (A196G) trans-membrane domains. The M1-RASSL receptor mutant has been shown to be unresponsive to acetylcholine (Ach) but responds to the chemical ligand, clozapine-N-oxide (CNO). This was determined in assays that measure the accumulation of inositol phosphate in the presence of a lithium block of inositol 1-phosphatase. Basal levels of phosphate as measured by the Inositol Phosphate accumulation assay (IPx) = 6067.664 ± 1414.394 cpm/mg. Stimulation with 10mM CNO resulted in levels to increase to 49456.65 ± 12166.24 cpm/mg. Initial studies from other laboratories have shown that the M1-RASSL receptor couples normally to phosphoinositide hydrolysis and ERK1/2 phosphorylation (Roth et al., 2007). However, the phosphorylation profile of this receptor mutant has up until now not been described. Here we show using Western blotting with phospho-specific antibodies raised against residues which have been shown to be phosphorylated, that the M1-RASSL receptor is phosphorylated in response to CNO in a similar manner to the wild type M1-receptor following stimulation with Ach. In the wild-type receptor phosphorylation at residue 228 was seen to increase by approximately 7-fold in response to Ach but no response was seen to CNO. In contrast, the M1-RASSL receptor did not respond to Ach but CNO stimulation resulted in an approximate 7-fold increase in receptor phosphorylation at residue 228. We also show that the M1-RASSL receptor when stimulated with CNO behaves in a similar manner when the wild-type M1-muscarinic receptor is stimulated with Ach with regards to its coupling to phosphoinositide hydrolysis. We anticipate these results to be the starting point for more sophisticated studies using the M1-RASSL receptor, for example, studying-protein mediated signalling and phosphorylation/arrestin dependent signalling in vivo and in vitro.