Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway.
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.
Price
L A
LA
Cyanamid Agricultural Research Center, Princeton, New Jersey 08543-0400, USA.
Kajkowski
E M
EM
Hadcock
J R
JR
Ozenberger
B A
BA
Pausch
M H
MH
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D010675:Pheromones / Q000502:physiology; D051381:Rats; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000502:physiology; D017481:Receptors, Somatostatin / Q000502:physiology*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000502:physiology*; D015398:Signal Transduction; D013004:Somatostatin / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors
D017931:DNA Primers; D010675:Pheromones; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D017481:Receptors, Somatostatin; D011994:Recombinant Proteins; D014157:Transcription Factors; D013004:Somatostatin; D019204:GTP-Binding Proteins