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US 2003/0148390 A1Published application
Dose response-based methods for identifying receptors having alterations in signaling
OwnerAlan S. Kopin, Martin Beinborninventors
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Abstract
The invention provides methods of identifying receptors having altered signaling. In particular, the invention provides a sensitive dose response assay for the identification of receptors having alterations in ligand dependent or ligand independent signaling.
Claims22
A method of screening for a ligand that alters a receptor response, said method comprising:
- (a) co-transfecting a first host cell with:
- (i) an expression vector, said expression vector comprising a promoter operably linked to a receptor, and
- (ii) a reporter construct, said reporter construct comprising a response element and a promoter operably linked to a reporter gene, said response element being sensitive to a signal induced by said receptor;
- (b) co-transfecting a second host cell with said reporter construct and a negative control vector;
- (c) measuring the level of expression of said reporter construct in said first host cell and in said second host cell, at varying concentrations of said reporter construct or at varying concentrations of said expression vector or said negative control vector, whereby dose-response curves are generated for said expression of said reporter construct in said first and said second host cells;
- (d) identifying said receptor as a receptor with altered signaling by its ability to increase or decrease said level of expression in the first host cell compared to said level of expression in the second host cell over a range of at least two different concentrations of said reporter construct, said negative control vector, or said expression vector;
- and
- (e) contacting said receptor with altered signaling identified in step (d) with a candidate ligand;
- and
- (f) measuring the activity of said receptor in the presence and in the absence of said candidate ligand, whereby an alteration in the activity of said receptor in the presence of said ligand relative to the activity of said receptor in the absence of said ligand indicates that said candidate ligand is a ligand that alters a receptor response.
7 more elements
A method of screening for a ligand that alters a receptor response, said method comprising:
- (a) co-transfecting a first host cell with:
- (i) a reporter construct, said reporter construct comprising a G protein response element and a promoter operably linked to a reporter gene,
- (ii) a first expression vector, said first expression vector comprising a promoter operably linked to a G protein-coupled receptor, and
- (iii) a second expression vector, said second expression vector comprising a promoter operably linked to a chimeric G protein, wherein said chimeric G protein is capable of receiving a signal from said G protein-coupled receptor and increasing the expression of said reporter construct;
- (b) co-transfecting a second host cell with said reporter construct, said second expression vector, and a negative control vector;
- (c) measuring the level of expression of said reporter construct in said first host cell and said second host cell, wherein an increased or decreased level of expression in the first host cell compared to the second host cell identifies said receptor as a G protein-coupled receptor with altered signaling;
- (d) contacting said receptor with altered signaling identified in step (c) with a candidate ligand;
- and
- (e) measuring the activity of said receptor in the presence and in the absence of said candidate ligand, whereby an alteration in the activity of said receptor in the presence of said ligand relative to the activity of said receptor in the absence of said ligand indicates that said candidate ligand is a ligand that alters a receptor response.
6 more elements
Dependencies are inferred from the claim text.