Conserved extracellular cysteine pair in the M-3 muscarinic acetylcholine receptor is essential for proper receptor cell surface localization but notfor G protein coupling

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the f irst and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and /or proper receptor folding. However, the potential importance of the two c onserved cysteine residues for proper receptor cell surface localization ha s not been investigated systematically. To address this issue, we used the rat M-3 muscarinic receptor as a model system. Most studies were carried ou t with a modified version of this receptor subtype (lacking potential N-gly cosylationsites and the central portion of the third intracellular loop) th at could be readily detected via western blot analysis. Cys-->Ala mutant re ceptors were generated, transiently expressed in COS-7 cells, and then exam ined for their subcellular distribution and functional properties. ELISA an d immunofluorescence studies showed that the presence of both conserved cys teine residues (corresponding to C140 and C220 in the rat M-3 muscarinic re ceptor sequence) is required for efficient expression of the M-3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger ass ays). These results shed new light on the functional role of the two extrac ellular cysteine residues present in most G protein-coupled receptors.