Functional expression of M1, M3 and M5 muscarinic acetylcholine receptors in yeast

The goal of this study was to functionally express the three Gq‐coupled muscarinic receptor subtypes, M1, M3 and M5, in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full‐length receptors resulted in very low numbers of detectable muscarinic binding sites (Bmax < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M1, M3 and M5 muscarinic receptors resulted in dramatic increases in Bmax values (53–214 fmol/mg). To monitor productive receptor/G‐protein coupling, we used specifically engineered yeast strains that required agonist‐stimulated receptor/G‐protein coupling for cell growth. These studies showed that the shortened versions of the M1, M3 and M5 receptors were unable to productively interact with the endogenous yeast G protein α‐subunit, Gpa1p, or a Gpa1 mutant subunit that contained C‐terminal mammalian Gαs sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Gαq hybrid subunit containing C‐terminal mammalian Gαq sequence, indicating that the M1, M3 and M5 muscarinic receptors retained proper G‐protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling‐competent form in yeast. The strategy described here, which involves structural modification of both receptors and co‐expressed G proteins, should facilitate the functional expression of other classes of G protein‐coupled receptors in yeast.

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