Journal
JOURNAL OF NEUROCHEMISTRY
Volume 77, Issue 5, Pages 1327-1337Publisher
WILEY
DOI: 10.1046/j.1471-4159.2001.00344.x
Keywords
G protein-coupled receptors; G proteins; receptor/G protein coupling; muscarinic receptors; Saccharomyces cerevisiae
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The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M-1, M-3 and M-5, 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 (B-max <5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M-1, M-3 and M-5 muscarinic receptors resulted in dramatic increases in B-max 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 M-1, M-3 and M-5 receptors were unable to productively interact with the endogenous yeast G protein a-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Ga, sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/G alpha (q), hybrid subunit containing C-terminal mammalian G alpha (q), sequence, indicating that the M,, M, and Mg 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 cc-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.
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