Functional expression of rat M5 muscarinic acetylcholine receptor in yeast.
We have produced the rat M5 muscarinic acetylcholine receptor, an integral membrane protein, in the yeast Saccharomyces cerevisiae. This was achieved by placing an M5 gene in the yeast vector under the control of the yeast alpha-factor promoter and leader sequence. Northern blotting revealed the presence of M5 transcripts in yeast transformed with the M5 plasmid constructs. Crude extract prepared from the transformant yeasts showed saturable binding of the muscarinic antagonist [3H]-N-methyl scopolamine ([3H]NMS) with a kd of 22.77 nM and Bmax of 134.76 fmole per mg protein. Results deduced from saturation binding assay of intact cell demonstrated clearly that the M5 receptor was translocated across the membrane of the endoplasmic reticulum using the secretion signal on alpha-leader sequence and its binding site was still functional. Yeast expressing M5 receptor did not exhibit cell-cycle arrest in the presence of carbachol, a acetylcholine agonist, indicating that the recombinant M5 receptor could not couple directly to the endogenous yeast pheromone signaling G-protein.
Huang
H J
HJ
Graduate Institute of Botany, National Taiwan University, Taipei, Republic of China.
Liao
C F
CF
Yang
B C
BC
Kuo
T T
TT
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D019832:N-Methylscopolamine; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D051381:Rats; D011976:Receptors, Muscarinic / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012602:Scopolamine Derivatives / Q000378:metabolism; D014158:Transcription, Genetic
D021382:Protein Sorting Signals; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D012602:Scopolamine Derivatives; D019832:N-Methylscopolamine