Expression and pharmacological characterization of the human M1 muscarinic receptor in Saccharomyces cerevisiae.
The yeast S. cerevisiae has been examined as a heterologous host for the expression of mammalian neurotransmitter receptors which couple to guanine nucleotide regulatory (G) proteins. A cloned gene encoding the M1 subtype of human muscarinic receptor (HM1) was transformed into S. cerevisiae on a high copy plasmid under the control of the promoter for the yeast alcohol dehydrogenase (ADH) gene. Northern blotting demonstrated the presence of HM1 transcripts in transformants, and crude membranes prepared from these cells showed saturable binding of the muscarinic antagonist [3H]N-methyl scopolamine with a Kd of 179 pM and Bmax of 20 fmol/mg protein. Competition binding studies revealed pharmacological properties for these sites which were comparable to those reported for the M1 site in mammalian tissues. Yeast expressing HM1 did not exhibit high affinity agonist binding or cell-cycle arrest in the presence of muscarinic agonists, indicating that the mammalian receptor did not couple to the endogenous yeast G protein.
Payette
P
P
National Research Council of Canada, Biotechnology Research Institute, Montréal, Qué.
Gossard
F
F
Whiteway
M
M
Dennis
M
M
D001483:Base Sequence; D015152:Blotting, Northern; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D019832:N-Methylscopolamine; D010276:Parasympatholytics / Q000378:metabolism; D010277:Parasympathomimetics / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000502:physiology; D012602:Scopolamine Derivatives / Q000378:metabolism
D010276:Parasympatholytics; D010277:Parasympathomimetics; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D012602:Scopolamine Derivatives; D019832:N-Methylscopolamine