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

Abstract

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 hNT 1 -R revealed multiple c-myc cross-reacting polypeptides of high molecular mass, suggesting that hNT 1 -R was glycosylated in yeast. High-affinity binding sites for 125 Competition binding studies of neurotensin with SR142948 and SR48692, two nonpeptidic antagonists of hNT 1 -R, indicated that the yeast-produced recombinant receptor displayed the same pharmacological properties as hNT 1 -R expressed in mammalian cells. Interestingly, neurotensin activated the pheromone pathway in hNT 1 -R-expressing cells in a dose-dependent fashion, as revealed by a b-galactosidase activity assay with a pheromone-responsive Fus1::lacZ construct. Mutational inactivation of the SST2 and STE2 genes increased the level of b-galactosidase activity in response to neurotensin by twofold. Recombinant hNT 1 -Rproducing cells, which lacked the endogenous G-proteincoupled receptor for the alpha pheromone, mated with wild-type MATa haploid cells in response to neurotensin, leading to bona fide diploid zygote formation. This is the first report of a mammalian receptor that can replace the endogenous pheromone receptor when produced in yeast, by signaling a fully effective, agonist-induced, mating process.Keywords: neurotensin; G-protein coupled receptor; heterologous expression; signal transduction; yeast.The tridecapeptide neurotensin (NT) originally discovered in bovine hypothalamus displays a wide range of biological activities, including roles in nociception, hypothermia, control of pituitary hormone secretion and muscle relaxation [1,2]. In the brain NT acts as a neurotransmitter/neuromodulator [3,4]. In particular, it modulates dopamine transmission in the nigrostrial and mesolimbic pathways [5]. Two neurotensin receptors termed NT 1 -R and NT 2 -R have been cloned to date. The high affinity receptor NT 1 -R has been cloned from rat brain and from the human cell line HT29 [6,7]. The type 2 neurotensin receptor, which exhibits a lower affinity for NT, has been cloned from various sources: rat brain, mouse brain and recently from a human cortex cDNA library [8][9][10]. These two receptors belong to the family of GTP-binding-protein coupled receptors (GPCR) with seven transmembrane domains (TMs). The biochemical and pharmacological properties of hNT 1 -R have been extensively studied (reviewed in [2]). The interaction of neurotensin with the NT 1 -R receptor modulates the intracellular levels of cGMP, cAMP and inositol phosphates [11,12].Recently, various mammalian G-protein-coupled receptors, including beta(2)adrenergic, alpha(2)-C2 adrenergic, M1 and M5 muscarinic, D 2S dopamine somatostatin subtype 2, A (2a) adenosine, opsin, growth hormone releasing hormone, Edg-2/Vzg-1, C5a, VPAC1 and m opioid, have successfully been expressed in the yeast S. cerevisiae [13][14][15][16][17][18][19][20][21][22][23][24][25]. Product...

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