ISSN:
1471-4159
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Medicine
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1046/j.1471-4159.2001.00344.x
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