Functional Coupling of a Mammalian Somatostatin Receptor to the Yeast Pheromone Response Pathway

Abstract

A detailed analysis of structural and functional aspects of G-protein-coupled receptors, as well as discovery of novel pharmacophores that exert their effects on members of this class of receptors, will be facilitated by development of a yeast-based bioassay. To that end, yeast strains that functionally express the rat somatostatin receptor subtype 2 (SSTR2) were constructed. High-affinity binding sites for somatostatin ([125I-Tyr-11]S-14) comparable to those in native tissues were detected in yeast membrane extracts at levels equivalent to the alpha-mating pheromone receptor (Ste2p). Somatostatin-dependent growth of strains modified by deletion of genes encoding components of the pheromone response pathway was detected through induction of a pheromone-responsive HIS3 reporter gene, enabling cells to grow on medium lacking histidine. Dose-dependent growth responses to S-14 and related SSTR2 subtype-selective agonists that were proportional to the affinity of the ligands for SSTR2 were observed. The growth response required SSTR2, G alpha proteins, and an intact signal transduction pathway. The sensitivity of the bioassay was affected by intracellular levels of the G alpha protein. A mutation in the SST2 gene, which confers supersensitivity to pheromone, was found to significantly enhance the growth response to S-14. In sst2 delta cells, SSTR2 functionally interacted with both a chimeric yeast/mammalian G alpha protein and the yeast G alpha protein, Gpa1p; to promote growth. These yeast strains should serve as a useful in vivo reconstitution system for examination of molecular interactions of the G-protein-coupled receptors and G proteins.

Development of a Yeast Bioassay to Characterize G Protein-coupled Receptor Kinases

“…Plasmid Construction-The plasmid pMP222 was constructed by amplifying a fragment encoding the rat SSTR2 by PCR using pJH2 (13) as template and synthetic oligonucleotides (MPO249, TCTCAAGCTT-AAAAATGGAGATGAGCTCTGAG; MPO250, TCTCAGATCTTCAGAT-ACTGGTTTGGAGG) that add a 5Ј HindIII site followed by a yeast translation initiation site and a 3Ј BglII site. The fragment was cut with HindIII and BglII and cloned into corresponding sites in pPGK (14).…”

“…When FAR1 is deleted, cell growth is permitted to continue in the presence of the activated pheromone pathway. Based on this concept, a novel expression system was developed that allows yeast cells expressing the mammalian somatostatin receptor subtype 2 to couple to the pheromone pathway in the presence of the peptide agonist SST-14 (13). Activation of this pathway can then be used in a growth selection by placing a HIS3 reporter into transcriptional elements of the FUS1 gene.…”

“…His3 protein expression then permits auxotrophic growth of yeast cells on media lacking histidine. In accordance with this observation, an appropriate strain was constructed (MPY576fc) that functionally expresses the SSTR2 and exhibits agonist-dependent growth upon exposure to somatostatin (13).…”

“…As a result, transcriptional activation of additional components of the mitogen-activated protein kinase cascade occurs, leading to cell cycle arrest. Previously, it has been demonstrated that mutations of certain components of the yeast pheromone response pathway eliminates the characteristic growth arrest response (13). This in turn led to the development of a yeast-based bioassay using strains that were constructed to functionally express the rat somatostatin receptor subtype 2 (SSTR2) and consequently induce growth upon activation by somatostatin peptide.…”

Identification of Allosteric Peptide Agonists of CXCR4

“…At these positions the wild-type sequence is KPVS. The amino acid sequence for the remaining peptide (positions [5][6][7][8][9][10][11][12][13][14][15][16][17] retains the SDF-1␣ wild-type sequence. Transformation of strain YAS2 with the plasmid library yielded ϳ10 6 transformants.…”