Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast.
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 alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(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.
Erlenbach
I
I
Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
Kostenis
E
E
Schmidt
C
C
Hamdan
F F
FF
Pausch
M H
MH
Wess
J
J
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1; D043587:Receptor, Muscarinic M3; D043642:Receptor, Muscarinic M5; D011976:Receptors, Muscarinic / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D008024:Ligands; D043643:Receptor, Muscarinic M1; D043587:Receptor, Muscarinic M3; D043642:Receptor, Muscarinic M5; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins