Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-10-05
2004-11-30
Landsman, Robert (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C530S300000, C530S350000, C530S402000
Reexamination Certificate
active
06824990
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of detecting oligomerization of G protein coupled receptors (GPCRs) as well as to the identification of agonists and antagonists of G protein coupled receptors utilizing fluorescence resonance energy transfer (FRET).
BACKGROUND OF THE INVENTION
Hormones, sensory stimuli, neurotransmitters, chemokines and other molecules signal by activating G protein coupled receptors. These receptors were thought to function as monomers, however, the present invention provides the surprising result that G protein coupled receptors are oligomeric in intact cells and membranes. Therefore, oligomerization of G protein coupled receptors is important for G protein coupled receptor signaling and regulation. By utilizing the methods of the present invention, oligomerization of homologous as well as heterologous G protein coupled receptors can be detected by FRET. Additionally, screening assays are provided for identification of agonists and antagonists of G protein coupled receptors.
SUMMARY OF THE INVENTION
The present invention provides a method of detecting oligomerization of G protein coupled receptors comprising: a) obtaining a first G protein coupled receptor fusion protein containing a fluorescence donor; b) obtaining a second G protein coupled receptor fusion protein containing a fluorescence acceptor; c) transfecting a cell with the G protein coupled receptor fusion proteins of a) and b); d) exciting the fluorescence donor at a particular wavelength; e) detecting fluorescence emission of the acceptor (FRET), such that if this emission is greater than the emission detected in control cells expressing only the acceptor, oligomerization of the G protein coupled receptors has been detected.
Also provided by the present invention is a method of determining whether a receptor agonist activates G protein coupled receptors by enhancing oligomerization or activates G protein coupled receptors by disrupting oligomerization comprising: a) obtaining a first G protein coupled receptor fusion protein containing a fluorescence donor; b) obtaining a second G protein coupled receptor fusion protein containing a fluorescence acceptor; c) transfecting a cell with the G protein coupled receptor fusion proteins of a) and b); d) contacting the cell with an agonist; e) exciting the fluorescence donor at a particular wavelength; f) detecting fluorescence resonance energy transfer (FRET), such that if the efficiency of FRET detected is greater in the cells contacted with the agonist than the efficiency of FRET detected in cells prior to the addition of the agonist, receptor activation has occurred by enhancing oligomerization and if the efficiency of FRET detected is less in the cells contacted with the agonist than the efficiency of FRET detected in cells prior to the addition of the agonist, receptor activation has occurred by disrupting oligomerization.
The present invention further provides a method of screening for an agonist that activates G protein coupled receptors by enhancing oligomerization comprising: a) obtaining a first G protein coupled receptor fusion protein containing a fluorescence donor; b) obtaining a second G protein coupled receptor fusion protein containing a fluorescence acceptor; c) transfecting a cell with the G protein coupled receptor fusion proteins of a) and b); d) contacting the cell with an agonist; e) exciting the fluorescence donor at a particular wavelength; f) detecting fluorescence resonance energy transfer (FRET), such that if the efficiency of FRET detected is greater in the cells contacted with the agonist than the efficiency of FRET detected in cells prior to the addition of the agonist, receptor activation has occurred by enhancing oligomerization.
Further provided by the present invention is a method of screening for an agonist that activates G protein coupled receptors by disrupting oligomerization comprising: a)obtaining a first G protein coupled receptor fusion protein containing a fluorescence donor; b) obtaining a second G protein coupled receptor fusion protein containing a fluorescence acceptor; c) transfecting a cell with the G protein coupled receptor fusion proteins of a) and b); d) contacting the cell with an agonist; e) exciting the fluorescence donor at a particular wavelength; f) detecting fluorescence resonance energy transfer (FRET), such that if the efficiency of FRET detected is less in the cells contacted with the agonist than the efficiency of FRET detected in cells prior to the addition of the agonist, receptor activation has occurred by disrupting oligomerization.
The present invention also provides a method of screening for an agonist of the interaction between G protein coupled receptors comprising: a) obtaining a first G protein coupled receptor fusion protein containing a fluorescence donor; b) obtaining a second G protein coupled receptor fusion protein containing a fluorescence acceptor; c) transfecting a cell with the G protein coupled receptor fusion proteins of a) and b); c) contacting the cell with a test compound; d) exciting the fluorescence donor at a particular wavelength; e) detecting fluorescence resonance energy transfer (FRET), such that if the efficiency of FRET detected is greater in cells contacted with the compound than the efficiency of FRET detected in cells prior to the addition of the test compound, the test compound is an agonist of the interaction between G protein coupled receptors.
The present invention also provides a method of screening for an antagonist of the interaction between G protein coupled receptors comprising: a) obtaining a first G protein coupled receptor fusion protein containing a fluorescence donor; b) obtaining a second G protein coupled receptor fusion protein containing a fluorescence acceptor; c) transfecting a cell with the G protein coupled receptor fusion proteins of a) and b); d) contacting the cell with a test compound; e) exciting the fluorescence donor at a particular wavelength; e) detecting fluorescence resonance energy transfer FRET), such that if the efficiency of FRET detected is less than the efficiency of FRET detected in cells prior to the addition of the test compound, the test compound is an antagonist of the interaction between G protein coupled receptors.
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Landsman Robert
Needle & Rosenberg P.C.
Washington University
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