Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Utility Patent
1998-04-08
2001-01-02
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S254200
Utility Patent
active
06168927
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to yeast cells expressing heterologous G protein coupled receptors, vectors useful for making such cells, and methods of using the same.
BACKGROUND OF THE INVENTION
The actions of many extracellular signals (for example, neurotransmitters, hormones, odorants, light) are mediated by receptors with seven transmembrane domains (G protein coupled receptors) and heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins). See H. Dohlman, M. Caron, and R. Lefkowitz,
Biochemistry
26, 2657 (1987); L. Stryer and H. Bourne,
Ann. Rev. Cell Biol.
2, 391 (1988). Such G protein-mediated signaling systems have been identified in organisms as divergent as yeast and man. See H. Dohlman et al., supra; L. Stryer and H. Bourne, supra; K. Blumer and J. Thorner,
Annu.Rev. Physiol.
(in press). The &bgr;2-adrenergic receptor (&bgr;AR) is the prototype of the seven-transmembrane-segment class of ligand binding receptors in mammalian cells. In response to epinephrine or norepinephrine, &bgr;AR activates a G protein, G
s
, which in turn stimulates adenylate cyclase and cyclic adenosine monophosphate production in the cell. See H. Dohlman et al., supra; L. Stryer and H. Bourne, supra. G protein-coupled pheromone receptors in yeast control a developmental program that culminates in mating (fusion) of a and a haploid cell types to form the a/&agr; diploid. See K. Blumer and J. Thorner, supra; I. Herskowitz,
Microbiol. Rev.
52, 536 (1988).
The present invention is based on our continued research into the expression of heterologous G protein coupled receptors in yeast.
SUMMARY OF THE INVENTION
A first aspect of the present invention is a transformed yeast cell containing a first heterologous DNA sequence which codes for a mammalian G protein coupled receptor and a second heterologous DNA sequence which codes for a mammalian G protein a subunit (mammalian G
&agr;
). The first and second heterologous DNA sequences are capable of expression in the cell, but the cell is incapable of expressing an endogenous G protein &agr;-subunit (yeast G
&agr;
). The cell optionally contains a third heterologous DNA sequence, with the third heterologous DNA sequence comprising a pheromone-responsive promotor and an indicator gene positioned downstream from the pheromone-responsive promoter and operatively associated therewith.
A second aspect of the present invention is a method of testing a compound for the ability to affect the rate of dissociation of G
&agr;
from G
&bgr;&ggr;
in a cell. The method comprises: providing a transformed yeast cell as described above; contacting the compound to the cell; and then detecting the rate of dissociation of G
&agr;
from G
&bgr;&ggr;
in the cell. The cells may be provided in an aqueous solution, and the contacting step carried out by adding the compound to the aqueous solution.
A third aspect of the present invention is a DNA expression vector capable of expressing a transmembrane protein into the cell membrane of yeast cells. The vector contains a first segment comprising at least a fragment of the extreme amino-terminal coding sequence of a yeast G protein coupled receptor. A second segment is positioned downstream from the first segment (and in correct reading frame therewith), with the second segment comprising a DNA sequence encoding a heterologous G protein coupled receptor.
A fourth aspect of the present invention is a yeast cell transformed by a vector as described above.
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Caron Marc G.
Dohlman Henrik G.
King Klim
Lefkowitz Robert J.
DeConti, Jr. Giulio A.
Duke University
Lahive & Cockfield LLP
Lauro Peter C.
Ulm John
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