Assay methods and compositions for detecting and evaluating the intracellular transduction of an extracellular signal
First Claim
1. A method for identifying extracellular signals that modulate cell surface protein-mediated activity, comprising:
- comparing the difference in the amount of transcription of a reporter gens in a recombinant cell in the presence of the signal with the amount of transcription in the absence of said signal, or with the amount of transcription in the absence of the cell surface protein, wherein;
the recombinant cell contains a reporter gens construct and expresses the cell surface protein; and
transcription of the reporter gens is under the control of at least one transcriptional control element whose activity is regulated by the cell surface protein.
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Abstract
Transcription based assays that identify extracellular signals that modulate the activity of cell surface proteins are provided. Extracellular signals are identified by measuring the amount of transcription of a reporter gene in a recombinant cell that expresses the cell surface protein and contains DNA encoding the reporter gene under the transcriptional control of a promoter that is regulated, directly or indirectly, by the cell surface protein. The assays, provide a means for identifying potential pharmaceutical compounds that can be used to treat disease by virtue of their agonistic or antagonistic effects on the cell surface protein. Recombinant cells that express cell surface receptors and that contain reporter gene constructs that include transcriptional regulatory elements that are responsive to the activity of the cell surface receptors are also provided.
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Citations
48 Claims
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1. A method for identifying extracellular signals that modulate cell surface protein-mediated activity, comprising:
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comparing the difference in the amount of transcription of a reporter gens in a recombinant cell in the presence of the signal with the amount of transcription in the absence of said signal, or with the amount of transcription in the absence of the cell surface protein, wherein; the recombinant cell contains a reporter gens construct and expresses the cell surface protein; and transcription of the reporter gens is under the control of at least one transcriptional control element whose activity is regulated by the cell surface protein. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 35, 36, 43, 44, 45, 46)
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24. A recombinant cell, comprising:
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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. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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37. A method for identifying compounds that modulate cell surface protein-mediated activity by detecting intracellular transduction of a signal generated upon interaction of the compound with a cell surface protein, comprising:
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comparing the amount of transcription of a reporter gene or the amount of reporter gene product expressed in a first recombinant cell in the presence of the compound with the amount of transcription or product in a second recombinant cell; and selecting compounds that change the amount of transcription of a reporter gene or the amount of reporter gene product expressed in the first recombinant cell in the presence of the compound compared to the amount of transcription or product in the absence of the compound, or compared to the amount of transcription or product in the second recombinant cell, wherein; the first recombinant cell contains a reporter 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 reporter 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) a reporter gene that encodes a detectable transcriptional or translational product and that is in operative association with the transcriptional control element. Transcription based assays that identify extracellular signals that modulate the activity of cell surface proteins are provided. Extracellular signals are identified by measuring the amount of transcription of a reporter gene in a recombinant cell that expresses the cell surface protein and contains DNA encoding the reporter gene under the transcriptional control of a promoter that is regulated, directly or indirectly, by the cell surface protein. The assays, provide a means for identifying potential pharmaceutical compounds that can be used to treat disease by virtue of their agonistic or antagonistic effects on the cell surface protein. Recombinant cells that express cell surface receptors and that contain reporter gene constructs that include transcriptional regulatory elements that are responsive to the activity of the cell surface receptors are also provided. - View Dependent Claims (38, 39, 40, 41, 42, 47, 48)
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Specification