System for screening agonists/antagonists of cellular signaling pathways
AssigneePresident and Fellows of Harvard College
InventorsYu Wang, Andrew P. McMahon
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Abstract
The present invention identifies a method for investigating the response of a cell membrane-associated protein in a living cell to a drug by labeling the protein with a visual marker, and also selectively labeling the membrane portion of the protein with another visual marker, such that upon exposure of the cell to a stimulus, the translocation of the cell membrane-associated protein may be observed directly.
Claims15
A method for screening a drug affect on the Hedgehog (Hh) signaling pathway comprising the steps of:
- culturing a first population and second population of cells, wherein both populations express a A::Smoothened::Fluorescent Protein construct (A::Smo::FP), and wherein A is a fragment of an acyl carrier protein that suffices for post-translational phosphopantetheinylation of A::Smo::FP protein;
- labeling both the first and second cell populations with a fluorophore using AcpS;
- contacting the first cell population with a Hh ligand;
- contacting the second cell population with a Hh ligand and a drug;
- observing the cilial translocation of said A::Smo::FP in the first cell population and in the second cell population by visualizing the FP and fluorophore;
- comparing the cilial translocation of said A::Smo::FP in the first cell population with the second cell population;
- wherein a difference in cilial translocation of said A::Smo::FP in the first cell population and the second cell population provides an indication of whether said drug affects the Hh signaling pathway.
A method for investigating the response of a cell membrane protein in a living cell to a drug comprising:
- (a) identifying a cell membrane protein for investigation, wherein said membrane protein responds to a stimulus by translocation;
- (b) obtaining a gene that expresses a recombinant version of said membrane protein;
- (c) creating a genetic construct that adds (i) at least one acyl carrier protein peptide tag to the membrane-associated portion of said membrane protein and (ii) at least one visual marker to said recombinant protein, wherein said construct is expressed in a cell as a chimeric membrane protein;
- (d) introducing said construct into a cell such that said chimeric membrane protein is expressed;
- (e) labeling the membrane portion of said chimeric membrane protein with a fluorophore via AcpS;
- (f) contacting said cell with a drug;
- (g) observing translocation activity of said chimeric membrane protein within said cell by observing the fluorophore and the visual marker of the chimeric membrane protein;
- wherein an effect of the drug on translocation activity of the chimeric membrane protein indicates whether the drug is an antagonist, agonist, or does not affect translocation of said cell membrane protein.
A cell culture system comprising a population of cells that expresses a reporter construct comprising A::Smo::FP, and wherein A is a fragment of an acyl carrier protein that suffices for post-translational phosphopantetheinylation in a living cell, Smo is Smoothened protein, and FP is a fluorescent protein.
Dependencies are inferred from the claim text.