SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.

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...

We discovered a constitutively activating mutation (CAM) V308E for the neurotensin NT1 receptor. Molecular dynamics (MD) performed for the CAM NT1-V308E exhibiting a high spontaneous activity, and for the wild-type NT1 without basal activity, show dramatic conformational changes for the CAM. To test if the two MD models could be valuable active and inactive templates for building molecular models for other class-A GPCR, supposed active and inactive models were built by homology for the cholecystokinin CCK1 receptor. Virtual screening of a corporate library with 250 000 compounds was performed with the two CCK1 models, and a differential virtual screening analysis (DVS), led us to isolate 250 predicted agonists and 250 predicted antagonists. The two sets were merged and the compounds were tested in CCK1 agonist and antagonist cellular assays. An excellent correlation was obtained between predictions and biological results. The effective profiling provided by DVS with active and inactive molecular models, opens new perspectives for finding agonists and antagonists for other class-A GPCR, notably for orphan GPCRs for which no ligands are known.