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Functional expression of M 1 , M 3 and M 5 muscarinic acetylcholine receptors in yeast
Author(s) -
Erlenbach Isolde,
Kostenis Evi,
Schmidt Clarice,
Hamdan Fadi F.,
Pausch Mark H.,
Wess Jürgen
Publication year - 2001
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2001.00344.x
Subject(s) - muscarinic acetylcholine receptor , receptor , biology , g protein , g protein coupled receptor , protein subunit , yeast , biochemistry , muscarinic acetylcholine receptor m2 , muscarinic acetylcholine receptor m5 , saccharomyces cerevisiae , muscarinic acetylcholine receptor m3 , microbiology and biotechnology , gene
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 α‐subunit, Gpa1p, or a Gpa1 mutant subunit that contained C‐terminal mammalian Gα s sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Gα q hybrid subunit containing C‐terminal mammalian Gα q sequence, indicating that the M 1 , M 3 and M 5 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.