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AssigneeAmerican Cyanamid Company
InventorsMark H. Pausch, Bradley A. Ozenberger, John R. Hadcock, Laura A. Price, Eileen M. Kajkowski, Donald R. Kirsch, Deborah T. Chaleff
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The present invention is directed to a expression vectors and yeast cells transformed therewith containing a first heterologous nucleotide sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second nucleotide sequence which codes for all or a portion of a G protein .alpha..beta..gamma. complex. Said heterologous protein is physically expressed in a host cell membrane in proper orientation for both stereoselective binding of ligands, as well as functional interaction with G proteins on the cytoplasmic side of the cell membrane. In some embodiments, a nucleotide sequence encoding a heterologous or chimeric G.alpha. protein is expressed in conjunction with nucleotide sequences from the yeast G protein .beta..gamma. subunits. A second aspect of the present invention provides expression vectors and transformed yeast cells encoding chimeric yeast/heterologous G protein coupled receptors. A third aspect of the present invention is directed to methods of assaying compounds using these expression constructs and yeast cell expression systems to determine the effects of ligand binding to the heterologous receptors expressed in the systems.
A nucleotide expression construct capable of expressing a functional G protein coupled receptor heterologous to yeast cells, comprising a nucleotide sequence encoding said heterologous receptor in the absence of fusion of said heterologous nucleotide sequence to a segment of a nucleotide sequence encoding a G protein coupled receptor endogenous to yeast cells, and having regulatory elements capable of functioning in yeast, comprising a promoter positioned upstream to a yeast specific translational initiation site in correct reading frame with the heterologous nucleotide sequence.
A nucleotide expression construct comprising a nucleotide sequence encoding a functional heterologous G protein-coupled receptor in the absence of fusion of said heterologous nucleotide sequence to a nucleotide sequence encoding an endogenous yeast G protein-coupled receptor and further comprising a promoter capable of functioning in yeast, said promoter is positioned upstream to a translational initiation site in correct reading frame with said heterologous sequence, wherein said heterologous receptor activates a pheromone induced mating signal transduction pathway in a yeast host cell having a gene mutation selected from the group consisting of sst2, STE50, sgv1, ste2, ste3, pik1, and afr1.
A nucleotide expression construct comprising a reporter gene under the control of a pheromone responsive promoter and a heterologous G protein-coupled receptor gene under the control of a promoter capable of functioning in yeast, wherein said heterologous nucleotide sequence is not fused to a nucleotide sequence encoding an endogenous yeast G protein-coupled receptor, and wherein said heterologous receptor activates a pheromone induced mating signal transduction pathway in a yeast host cell having a mutation at a SCG1/GPA1 gene and expressing a hybrid G.alpha. subunit.
A nucleotide expression construct comprising a heterologous G protein-coupled receptor gene under the control of a promoter capable of functioning in yeast wherein said heterologous nucleotide sequence is not fused to a nucleotide sequence enconding an endogenous yeast G protein-coupled receptor, and wherein the heterologous receptor activates a pheromone induced mating signal transduction pathway in a yeast cell having a gene mutation effective to disconnect a pheromone responsive signal transduction pathway of said yeast cell from said yeast cell's natural cell cycle arrest pathway.
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