Methods of testing for agonists and antagonists of cell surface receptors

Abstract

Disclosed 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 α ). 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 α-subunit (yeast G α ). The cells are useful for screening compounds which affect the rate of dissociation of G α from G βτ in a cell. Also disclosed is a novel DNA expression vector useful for making cells as described above. 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.

That which is claimed is:  
     
         1 . 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 α ), wherein said first and second heterologous DNA sequences are capable of expression in said cell, and wherein said cell is incapable of expressing an endogenous G protein α-subunit (yeast G α ).  
     
     
         2 . A transformed yeast cell according to  claim 1 , wherein said first heterologous DNA sequence is carried by a plasmid.  
     
     
         3 . A transformed yeast cell according to  claim 1 , wherein said second heterologous DNA sequence is carried by a plasmid.  
     
     
         4 . A transformed yeast cell according to  claim 1 , wherein said mammalian G protein α subunit is selected from the group consisting of G s  α subunits, G i  α subunits, G o  α subunits, G z  α subunits, and transducin α subunits.  
     
     
         5 . A transformed yeast cell according to  claim 1  which expresses a complex of the G protein β subunit and the G protein τ subunit (G βτ ).  
     
     
         6 . A transformed yeast cell according to  claim 5  which expresses endogenous G βτ .  
     
     
         7 . A transformed yeast cell according to  claim 1 , wherein said first heterologous DNA sequence codes for a mammalian G protein-coupled receptor selected from the group consisting of dopamine receptors, muscarinic cholinergic receptors, α-adrenergic receptors, β-adrenergic receptors, opiate receptors, cannabinoid receptors, and serotonin receptors.  
     
     
         8 . A transformed yeast cell according to  claim 1  further comprising a third heterologous DNA sequence, wherein said third heterologous DNA sequence comprises a pheromone-responsive promotor and an indicator gene positioned downstream from said pheromone-responsive promoter and operatively associated therewith.  
     
     
         9 . A transformed yeast cell according to  claim 8 , wherein said pheromone responsive promoter is selected from the group consisting of the BAR1 gene promoter and the FUS1 gene promoter, and wherein said indicator gene is selected from the group consisting of the HIS3 gene and the LacZ gene.  
     
     
         10 . A method of testing a compound for the ability to affect the rate of dissociation of G α  from G βτ  in a cell, comprising: 
 providing 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 α , wherein said first and second heterologous DNA sequences are capable of expression in said cell, wherein said cell is incapable of expressing endogenous G α , and wherein said cell expresses G βτ ;  
 contacting said compound to said cell; and  
 detecting the rate of dissociation of G a  from G βτ  in said cell.  
 
     
     
         11 . A method according to  claim 10 , wherein said yeast cells are provided in an aqueous solution and said contacting step is carried out by adding said compound to said aqueous solution.  
     
     
         12 . A method according to  claim 10 , wherein said mammalian G protein a subunit is selected from the group consisting of G s  α subunits, G i  α subunits, G o  α subunits, G z  α subunits, and transducin α subunits.  
     
     
         13 . A method according to  claim 10 , wherein said yeast cell expresses endogenous G βτ .  
     
     
         14 . A method according to  claim 10 , wherein said first heterologous DNA sequence codes for a mammalian G protein-coupled receptor selected from the group consisting of dopamine receptors, muscarinic cholinergic receptors, α-adrenergic receptors, β-adrenergic receptors, opiate receptors, cannabinoid receptors, and serotonin receptors.  
     
     
         15 . A method according to  claim 10 , said yeast cell further comprising a third heterologous DNA sequence, wherein said third heterologous DNA sequence comprises a pheromone-responsive promotor and an indicator gene positioned downstream from said pheromone-responsive promoter and operatively associated therewith; 
 and wherein said detecting step is carried out by monitoring the expression of said indicator gene in said cell.    
     
     
         16 . A DNA expression vector capable of expressing a transmembrane protein into the cell membrane of yeast cells, comprising: 
 a first segment comprising at least a fragment of the extreme amino-terminal coding sequence of a yeast G protein coupled receptor; and    a second segment downstream from said first segment and in correct reading frame therewith, said second segment comprising a DNA sequence encoding a heterologous G protein coupled receptor.    
     
     
         17 . A DNA expression vector according to  claim 16 , wherein a fragment of the extreme amino-terminal coding sequence of said heterologous G protein coupled receptor is absent.  
     
     
         18 . A DNA expression vector according to  claim 16 , wherein said first and second segments are operatively associated with a promoter operative in a yeast cell.  
     
     
         19 . A DNA expression vector according to  claim 18 , wherein said promoter is the GAL1 promoter.  
     
     
         20 . A DNA expression vector according to  claim 16 , wherein said first segment comprises at least a fragment of the extreme amino-terminal coding sequence of a yeast phereomone receptor.  
     
     
         21 . A DNA expression vector according to  claim 16 , wherein said first segment comprises at least a fragment of the extreme amino-terminal coding sequence of a yeast phereomone receptor selected from the group consisting of the STE2 gene and the STE3 gene.  
     
     
         22 . A DNA expression vector according to  claim 16 , further comprising at least a fragment of the 5′-untranslated region of a yeast G protein coupled receptor gene positioned upstream from said first segment and operatively associated therewith.  
     
     
         23 . A DNA expression vector according to  claim 16 , further comprising at least a fragment of the 5′-untranslated region of a yeast phereomone receptor gene positioned upstream from said first segment and operatively associated therewith.  
     
     
         24 . A DNA expression vector according to  claim 23 , wherein said yeast pheromone receptor gene is selected from the group consisting of the STE2 gene and the STE3 gene.  
     
     
         25 . A DNA expression vector according to  claim 16 , said vector comprising a plasmid.  
     
     
         26 . A DNA expression vector according to  claim 16 , said second segment comprising a DNA sequence encoding a mammalian G protein coupled receptor.  
     
     
         27 . A DNA expression vector according to  claim 16 , said second segment comprising a DNA sequence encoding a mammalian G protein-coupled receptor selected from the group consisting of dopamine receptors, muscarinic cholinergic receptors, α-adrenergic receptors, β-adrenergic receptors, opiate receptors, cannabinoid receptors, and serotonin receptors.  
     
     
         28 . A yeast cell carrying a DNA expression vector according to  claim 16 .  
     
     
         29 . A method for screening a plurality of compounds to identify a compound that agonizes or antagonizes a G protein-coupled receptor-mediated activity by detecting intracellular transduction of a signal generated upon interaction of the compound with the G protein-coupled receptor, comprising: 
 comparing the amount of expression of an indicator gene product in a recombinant eukaryotic cell in the presence of the compound with the amount of expression of an indicator gene product in the absence of the compound; and    selecting the compound from the plurality of compounds that changes the amount of expression of the indicator gene product in the recombinant cell in the presence of the compound compared to the amount of expression of an indicator gene product in the absence of the compound whereby the compound that modulates G protein-coupled receptor mediated activity is identified, wherein:    the recombinant cell contains an indicator gene construct and expresses the G protein-coupled receptor; and    the indicator gene construct includes: 
 (a) a transcriptional control element that is responsive to the intracellular signal that is generated by the interaction of the compound with the G protein-coupled receptor; and  
 (b) an indicator gene that encodes a detectable product and that is in operative association with the transcriptional control element.  
   
     
     
         30 . The method of  claim 29 , wherein said compound is an agonist or antagonist of said G protein-coupled receptor.  
     
     
         31 . The method of  claim 29 , wherein the amount of expression of an indicator gene product is measured by measuring the amount of indicator gene protein that is produced.  
     
     
         32 . The method of  claim 30 , wherein said compound is an antagonist.  
     
     
         33 . The method of  claim 30 , further comprising, prior to comparing the difference in the amount of expression of the indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene product is induced.  
     
     
         34 . The method of  claim 29 , wherein the G protein-coupled receptor is selected from the group consisting of muscarinic receptors, adrenergic receptors, dopamine receptors, and serotonin receptors.  
     
     
         35 . The method of  claim 29 , wherein the transcriptional control element includes a promoter sequence which is responsive to the intracellular signal.  
     
     
         36 . A method for screening a plurality of compounds to identify a compound that agonizes or antagonizes a G protein-coupled receptor-mediated activity by detecting intracellular transduction of a signal generated upon interaction of the compound with the G protein-coupled receptor, comprising: 
 comparing the amount of expression of an indicator gene product in a first recombinant eukaryotic cell in the presence of the compound with the amount of expression of an indication gene product in the absence of the compound, or with the amount of expression in a second recombinant cell; and    selecting a compound from the plurality of compounds that changes the amount of expression of the indicator gene product in the first recombinant cell in the presence of the compound compared to the amount of expression in the absence of the compound, or compared to the amount of expression in the second recombinant cell, whereby a compound that modulates G protein-coupled receptor-mediated activity is identified wherein: 
 the first recombinant eukaryotic cell contains an indicator gene construct and expresses the G protein-coupled receptor;  
 the second recombinant cell is identical to the first recombinant cell, except that it does not express the G protein-coupled receptor; and  
 the indicator gene construct includes: 
 (a) a transcriptional control element that is responsive to the intracellular signal that is generated by the interaction of the compound with the G protein-coupled receptor; and  
 (b) an indicator gene that encodes a detectable product and that is in operative association with the transcriptional control element.  
 
   
     
     
         37 . A method for identifying extracellular signals that agonize or antagonize cell surface protein-mediated activity, comprising: 
 comparing the difference in the amount of expression of an indicator gene product in a recombinant cell in the presence of the signal with the amount of expression in the absence of said signal, or with the amount of expression in the absence of the cell surface protein, wherein: 
 the recombinant cell contains an indicator gene construct and expresses the cell surface protein; and  
 expression of the indicator gene product is under the control of at least one transcriptional control element whose activity is regulated by the cell surface protein.  
   
     
     
         38 . The method of  claim 37 , wherein said protein is a cell surface receptor.  
     
     
         39 . The method of  claim 38 , wherein said signal is an agonist or antagonist of said cell surface receptor.  
     
     
         40 . The method of  claim 39 , wherein the concentration of said agonist or antagonist is less than 100 μM.  
     
     
         41 . The method of  claim 39 , wherein the concentration of said agonist or antagonist is less than 10 μM.  
     
     
         42 . The method of  claim 39 , wherein the concentration of said agonist or antagonist is less than 1 μM.  
     
     
         43 . The method of  claim 39 , wherein the concentration of said agonist or antagonist is less than 0.1 μM.  
     
     
         44 . The method of  claim 39 , wherein the concentration of said agonist or antagonist is less than 10 nM.  
     
     
         45 . The method of  claim 37 , wherein the amount of expression is determined by measuring the amount of indicator gene product that is produced.  
     
     
         46 . The method of  claim 39 , wherein said signal is an antagonist.  
     
     
         47 . The method of  claim 46 , further comprising, prior to or simultaneously with comparing the difference in the amount of expression of the indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene is induced.  
     
     
         48 . The method of  claim 37 , wherein the cell surface protein is a cell surface receptor selected from: muscarinic receptors, adrenergic receptors, dopamine receptors, serotonin receptors.  
     
     
         49 . The method of  claim 37 , wherein the transcriptional control element includes a promoter sequence which is responsive to intracellular signals.  
     
     
         50 . A recombinant cell, comprising: 
 DNA that encodes a heterologous cell surface protein whose activity is modulated by extracellular signals; and    a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional control element that is regulated by said cell surface protein; wherein 
 said cell surface protein is a G-protein coupled receptor.  
   
     
     
         51 . The recombinant cell of  claim 50 , wherein said cell surface protein is a cell surface receptor selected from: muscarinic receptors, adrenergic receptors, dopamine receptors, serotonin receptors.  
     
     
         52 . A method for identifying compounds that agonize or antagonize cell surface protein-mediated activity by detecting intracellular transduction of a signal generated upon interaction of the compound with a cell surface protein, comprising: 
 comparing the amount of expression of an indicator gene product in a first recombinant cell in the presence of the compound with the amount of indicator gene product in a second recombinant cell; and    selecting compounds that change the amount of expression of an indicator gene product in the first recombinant cell in the presence of the compound compared to the amount of expression in the absence of the compound, or compared to the amount of expression in the second recombinant cell, wherein: 
 the first recombinant cell contains an indicator gene construct and expresses the cell surface protein;  
 the second recombinant cell is identical to the first recombinant cell, except that it does not express the cell surface protein; and the indicator gene construct contains: 
 (a) a transcriptional control element that is responsive to the intracellular signal that is generated by the interaction of an agonist with the cell surface protein; and  
 (b) an indicator gene that encodes a detectable product and that is in operative association with the transcriptional control element.  
 
   
     
     
         53 . The method of  claim 52 , wherein the compound is an agonist or antagonist of said cell surface receptor.  
     
     
         54 . The method of  claim 52 , wherein the level of expression is determined by measuring the amount of indicator gene product that is produced.  
     
     
         55 . The method of  claim 52 , further comprising, prior to or simultaneously with comparing the difference in the amount of indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene product is induced.  
     
     
         56 . The method of  claim 55 , wherein said compound is an antagonist.  
     
     
         57 . The method of  claim 47 , wherein said compound is an antagonist.  
     
     
         58 . The method of  claim 37 , wherein the cell is preincubated with the extracellular signal prior to comparing the difference in expression.  
     
     
         59 . The method of  claim 52 , wherein the cell is preincubated with the compound prior to comparing the difference in expression.  
     
     
         60 . The method of any one of claims  37  and  52 , wherein said cell surface protein is a G-protein coupled receptor.  
     
     
         61 . The method of  claim 36 , wherein said compound is an agonist or antagonist of said G protein-coupled receptor.  
     
     
         62 . The method of  claim 36 , wherein the amount of expression of an indicator gene product is measured by measuring the amount of indicator gene protein that is produced.  
     
     
         63 . The method of  claim 36 , wherein said compound is an antagonist.  
     
     
         64 . The method of  claim 36 , further comprising, prior to comparing the difference in the amount of expression of the indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene product is induced.  
     
     
         65 . The method of  claim 36 , wherein the G protein-coupled receptor is selected from the group consisting of muscarinic receptors, adrenergic receptors, dopamine receptors, and serotonin receptors.  
     
     
         66 . An assay for screening a plurality of compounds to determine if a compound so screened has cell surface receptor agonist or antagonist activity, which assay comprises 
 (a) challenging a recombinant eukaryotic cell with the compound, wherein the cell recombinantly expresses the G protein-coupled receptor;    (b) determining the level of indicator gene product recombinantly expressed in the cell;    (c) comparing the amount of expression of an indicator gene product in the eukaryotic cell with the amount of expression of an indicator gene product in the eukaryotic cell in the absence of the compound, and    (d) identifying a compound from the plurality of compounds that exhibits said cell surface receptor agonist or antagonist activity, wherein the indicator gene is under transcriptional control of a transcriptional control element selected to be responsive to an intracellular signal that is generated by the interaction of a compound with G protein-coupled receptor.    
     
     
         67 . The assay of  claim 66 , wherein said compound is an agonist or antagonist of said G protein-coupled receptor.  
     
     
         68 . The assay of  claim 66 , wherein the amount of expression is measured by measuring the amount of indicator gene protein that is produced.  
     
     
         69 . The assay of  claim 67 , wherein said compound is an antagonist.  
     
     
         70 . The assay of  claim 67 , further comprising, prior to comparing the difference in the amount of expression of the indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene product is induced.  
     
     
         71 . The assay of  claim 66 , wherein said cell surface protein is a G-protein coupled receptor.  
     
     
         72 . The assay of  claim 66 , wherein the cell surface receptor is selected from the group consisting of muscarinic receptors, adrenergic receptors, dopamine receptors, and serotonin receptors.  
     
     
         73 . The assay of  claim 66 , wherein the transcriptional control element includes a promoter sequence which is responsive to the intracellular signal.  
     
     
         74 . An assay for screening a plurality of compounds to determine if a compound so screened has cell surface receptor agonist or antagonist activity, which assay that comprises 
 (a) challenging a first recombinant eukaryotic cell with a compound to be screened wherein the cell recombinantly expresses the G protein-coupled receptor,    (b) determining the level of expression of an indicator gene product recombinantly expressed in the cell,    (c) comparing the amount of expression of an indicator gene product in the first recombinant eukaryotic cell with the amount of expression of an indicator gene product in a second recombinant eukaryotic cell that is identical to the first eukaryotic cell except that the second cell does not express the G protein-coupled receptor, and    (d) identifying a compound that exhibits said G protein-coupled receptor agonist or antagonist activity, wherein the indicator gene is under transcriptional control of a transcriptional control element selected to be responsive to an intracellular signal that is generated by the interaction of the compound with the G protein-coupled receptor.    
     
     
         75 . The assay of  claim 74 , wherein said compound is an agonist or antagonist of G protein-coupled receptor.  
     
     
         76 . The assay of  claim 74 , wherein the amount of expression is measured by measuring the amount of indicator gene protein that is produced.  
     
     
         77 . The assay of  claim 75 , wherein said compound is an antagonist.  
     
     
         78 . The assay of  claim 75 , further comprising, prior to comparing the difference in the amount of expression of the indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene product is induced.  
     
     
         79 . The assay of  claim 74 , wherein the G protein-coupled receptor is selected from the group consisting of muscarinic receptors, adrenergic receptors, dopamine receptors, and serotonin receptors.  
     
     
         80 . The assay of  claim 74 , wherein the transcriptional control element includes a promoter sequence which is responsive to the intracellular signal.