American Cyanamid Company

Basf Aktiengesellschaft

Bradley Alton Ozenberger

Mark Henry Pausch

John Richard Hadcock

Donald R Kirsch

Deborah Tardy Chaleff

Eileen Marie Kajkowski

Kathleen H Young

Laura Price

Jeffrey David Hulmes

Cecil Mark Eppler

Laura A Price

C Mark Eppler

Expression vectors and yeast cells that contain a heterologous G protein-coupled receptor gene and a gene mutation that causes increased sensitivity to receptor activation or a gene mutation that permits transcriptional activation of pheromone-responsive genes without cell cycle arrest. Methods of making the yeast cells.

Heterologous G protein coupled receptors expressed in yeast, their fusion with G proteins and use thereof in bioassay

This invention relates to modified host cells which express heterologous fused proteins and methods of screening for test samples having peptide binding activity; wherein the modified host cell comprises: (a) a gene sequence encoding a heterologous fusion protein; the fusion protein comprising a first peptide of a peptide binding pair, or segment of the first peptide, which is joined to either a DNA binding domain or its corresponding transcriptional activation domain of a transcriptional activation protein; (b) a gene sequence encoding a heterologous fusion protein, the fusion protein comprising a second peptide of the peptide binding pair in (a), or a segment thereof, fused to either a DNA binding domain or its corresponding transcriptional activation domain, whichever one is not employed in (a); (c) a reporter gene operatively associated with the transcriptional activation protein, or a portion thereof; (d) optionally, a deletion or mutation in the chromosomal DNA of the host cell for the transcriptional activation protein if present in the selected host cell.

Cell systems having specific interaction of peptide binding pairs

A first aspect of the present invention is directed to a transformed yeast cell containing a first heterologous DNA sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second heterologous DNA sequence which codes for a G protein a subunit or portions thereof fused to DNA sequences from the yeast G protein in a subunit. A second aspect of the present invention is a transformed yeast cell containing a heterologous DNA sequence which codes for a G protein coupled receptor. A third aspect of the present invention is a method of assaying compounds to determine effects on cell growth.

The present invention is directed to 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 &agr;&bgr;&ggr; 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 a cytoplasmic side of the cell membrane. In some embodiments, a nucleotide sequence encoding a heterologous or chimeric G&agr; protein is expressed in conjunction with nucleotide sequences from the yeast G protein &bgr;&ggr; subunits. A second aspect of the present invention provides expression vectors and yeast cells transformed therewith encoding chimeric yeast/heterologous G protein coupled receptors. A third aspect of the present invention is directed to methods of assaying compounds using such expression constructs and yeast cell expression systems to determine the effects of ligand binding to the heterologous receptors expressed in the systems.

Method for detecting ligand binding to G protein coupled receptors

This invention relates to novel modified host cells which express heterologous fused proteins and methods of screening for test samples having peptide-binding activity; wherein the modified host cell comprises: (a) a gene sequence encoding a heterologous fusion protein; said fusion protein comprising a first peptide of a peptide binding pair, or segment of said first peptide, which is joined to either a DNA binding domain or its corresponding transcriptional activation domain of a transcriptional activation protein; (b) a gene sequence encoding a heterologous fusion protein, said fusion protein comprising a second peptide of the peptide binding pair in (a), or a segment thereof, fused to either a DNA binding domain or its corresponding transcriptional activation domain, whichever one is not employed in (a); (c) a reporter gene operatively associated with the transcriptional activation protein, or a portion thereof; (d) optionally, a deletion or mutation in the chromosomal DNA of the host cell for the transcriptional activation protein if present in the selected host cell.

The present invention relates to a method for isolating and cloning receptor DNA sequences. The invention also provides novel DNA sequences encoding a novel somatostatin receptor subtype.

Receptor identification method

cDNAs encoding proteins closely related to opioid receptors

Isolated DNAs encoding opiorph receptor polypeptides are provided. Recombinant cloning vectors which include these DNA sequences and cells which include these vectors are also provided. Methods for detecting the expression, in a tissue, of mRNA encoding a polypeptide encoded by this DNA are encompassed as well as methods for producing these polypeptides. These isolated polypeptides and antibodies to these polypeptides are also contemplated.

Proteins closely related to opioid receptors

Identification of compounds affecting specific interaction of peptide

Somatostatin receptor protein

Method of detecting expression of proteins closely related to opioid

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.

Recombinant expression vectors for expression of heterologous proteins

A first aspect of the present invention is directed to a transformed yeast cell containing a first heterologous DNA sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second heterologous DNA sequence which codes for a G protein .alpha. subunit or portions thereof fused to DNA sequences from the yeast G protein a subunit. A second aspect of the present invention is a transformed yeast cell containing a heterologous DNA sequence which codes for a G protein coupled receptor. A third aspect of the present invention is a method of assaying compounds to determine effects on cell growth.

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Yardley, PA, United States of America

Bradley Alton Ozenberger