Neurotensin induces mating in Saccharomyces cerevisiae cells that express human neurotensin receptor type 1 in place of the endogenous pheromone receptor

Heterologous expression of the human neurotensin receptor type I (hNT(1)-R) has been achieved in the yeast Saccharomyces cerevisiae. Immunoanalysis of membranes prepared from cells expressing a c-myc-tagged version of hNT1-R revealed multiple c-myc cross-reacting polypeptides of high molecular mass, suggesting that hNT1-R was glycosylated in yeast. High-affinity binding si tes for I-125-labeled-[monoiodo-Tyr3]neurotensin ([I-125-Tyr3]NT) were dete cted on hNT(1)-R-expressing cells with K-d and B-max values of 3.2 nM and o f 500 receptors per cell, respectively. Competition binding studies of neur otensin with SR142948 and SR48692, two nonpeptidic antagonists of hNT(1)-R, indicated that the yeast-produced recombinant receptor displayed the same pharmacological properties as hNT1-R expressed in mammalian cells. Interest ingly, neurotensin activated the pheromone pathway in hNT(1)-R-expressing c ells in a dose-dependent fashion, as revealed by a beta -galactosidase acti vity assay with a pheromone-responsive Fus1::lacZ construct. Mutational ina ctivation of the SST2 and STE2 genes increased the level of beta -galactosi dase activity in response to neurotensin by twofold. Recombinant hNT(1)-R-p roducing cells, which lacked the endogenous G-protein-coupled receptor for the alpha pheromone, mated with wild-type MAT alpha haploid cells in respon se to neurotensin, leading to bona fide diploid zygote formation. This is t he first report of a mammalian receptor that can replace the endogenous phe romone receptor when produced in yeast, by signaling a fully effective, ago nist-induced, mating process.