Receptor mediated activation of heterotrimeric G-proteins

Abstract

Receptor mediated activation of heterotrimeric G-proteins is visualized in living cells by monitoring fluorescence resonance energy transfer (FRET) between subunits of a G protein fused to cyan and yellow fluorescent proteins. The G-protein heterotrimer rapidly dissociates and reassociates upon addition and removal of cognate ligand. Energy transfer pairs of G-proteins enables direct in situ detection and have applications for drug screening and GPCR de-orphaning.

1 . A functional a α subunit of a heterotrimeric G protein comprising an amino acid sequence encoding a fluorescent or luminescent protein.  
     
     
         2 . The protein of  claim 1  wherein the amino acid sequence encoding a fluorescent or luminescent protein is cyan fluorescent protein.  
     
     
         3 . The protein of  claim 1  wherein the amino acid sequence encoding a fluorescent or luminescent protein is yellow fluorescent protein.  
     
     
         4 . The protein of  claim 1  wherein the heterotrimeric G protein is Gα2 of  D. discoideum.    
     
     
         5 . The protein of  claim 1  wherein said subunit has a helical domain and said amino acid sequence is within the helical domain of said subunit.  
     
     
         6 . A functional β subunit of a heterotrimeric G protein comprising an amino acid sequence encoding a fluorescent or luminescent protein.  
     
     
         7 . The protein of  claim 6  wherein the amino acid sequence encoding a fluorescent or luminescent protein is cyan fluorescent protein.  
     
     
         8 . The protein of  claim 6  wherein the amino acid sequence encoding a fluorescent or luminescent protein is yellow fluorescent protein.  
     
     
         9 . The protein of  claim 6  wherein the heterotrimeric G protein is Gβ of  D. discoideum.    
     
     
         10 . The protein of  claim 6  wherein said amino acid sequence is at the β subunit's N-terminus.  
     
     
         11 . A functional heterotrimeric G protein comprising an a subunit comprising a first amino acid sequence encoding a first fluorescent or luminescent protein and a α or γ subunit comprising a second amino acid sequence encoding a second fluorescent or luminescent protein, wherein said first and second fluorescent or luminescent proteins are capable of fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET).  
     
     
         12 . The functional heterotrimeric G protein of  claim 11  wherein a β subunit comprises the second amino acid sequence.  
     
     
         13 . The functional heterotrimeric G protein of  claim 11  wherein said first and said second amino acid sequences are within 100 angstroms of each other.  
     
     
         14 . The functional heterotrimeric G protein of  claim 11  wherein the first fluorescent or luminescent protein is cyan fluorescent protein.  
     
     
         15 . The functional heterotrimeric G protein of  claim 11  wherein the first fluorescent or luminescent protein is yellow fluorescent protein.  
     
     
         16 . The functional heterotrimeric G protein of  claim 11  wherein the second fluorescent or luminescent protein is cyan fluorescent protein.  
     
     
         17 . The functional heterotrimeric G protein of  claim 11  wherein the second fluorescent or luminescent protein is yellow fluorescent protein.  
     
     
         18 . The functional heterotrimeric G protein of  claim 11  wherein the first fluorescent or luminescent protein is cyan fluorescent protein and the second fluorescent or luminescent protein is yellow fluorescent protein.  
     
     
         19 . The functional heterotrimeric G protein of  claim 11  wherein the first fluorescent or luminescent protein is yellow fluorescent protein and the second fluorescent or luminescent protein is cyan fluorescent protein.  
     
     
         20 . The functional heterotrimeric G protein of  claim 11  wherein said first amino acid sequence is within a helical domain of said a α subunit.  
     
     
         21 . The functional heterotrimeric G protein of  claim 11  wherein said second amino acid sequence is at the N-terminus of said β subunit.  
     
     
         22 . The functional heterotrimeric G protein of  claim 11  wherein the α and β subunits are  D. discoideum  G protein subunits .  
     
     
         23 . The functional heterotrimeric G protein of  claim 13  wherein said first amino acid sequence is within a helical domain of said a α subunit and said second amino acid sequence is at the N-terminus of said β subunit.  
     
     
         24 . The functional heterotrimeric G protein of  claim 23  wherein the first fluorescent or luminescent protein is cyan fluorescent protein and the second fluorescent or luminescent protein is yellow fluorescent protein.  
     
     
         25 . The functional heterotrimeric G protein of  claim 24  wherein the α and β subunits are  D. discoideum  G protein subunits.  
     
     
         26 . A nucleic acid encoding a functional a α subunit of a heterotrimeric G protein which comprises an amino acid sequence encoding a fluorescent or luminescent protein.  
     
     
         27 . The nucleic acid of  claim 26  wherein the amino acid sequence encoding a fluorescent or luminescent protein is cyan fluorescent protein.  
     
     
         28 . The nucleic acid of  claim 26  wherein the amino acid sequence encoding a fluorescent or luminescent protein is yellow fluorescent protein.  
     
     
         29 . The nucleic acid of  claim 26  wherein the heterotrimeric G protein is Gα2 of  D. discoideum.    
     
     
         30 . The nucleic acid of  claim 26  wherein said subunit has a helical domain and the amino acid sequence is within the helical domain of said subunit.  
     
     
         31 . A nucleic acid encoding a functional β subunit of a heterotrimeric G protein which comprises an amino acid sequence encoding a fluorescent or luminescent protein.  
     
     
         32 . The nucleic acid of  claim 31  wherein the amino acid sequence encoding a fluorescent or luminescent protein is cyan fluorescent protein.  
     
     
         33 . The nucleic acid of  claim 31  wherein the amino acid sequence encoding a fluorescent or luminescent protein is yellow fluorescent protein.  
     
     
         34 . The nucleic acid of  claim 31  wherein the heterotrimeric G protein is Gβ of  D. discoideum.    
     
     
         35 . The nucleic acid of  claim 31  wherein the amino acid sequence is at the β subunit's N-terminus.  
     
     
         36 . The nucleic acid of  claim 26  wherein the nucleic acid comprises a vector for replication and expression of said subunit.  
     
     
         37 . The nucleic acid of  claim 31  wherein the nucleic acid comprises a vector for replication and expression of said subunit.  
     
     
         38 . A eukaryotic cell comprising a nucleic acid according to any of  claims 26  to  37   
     
     
         39 . A eukaryotic cell which comprises: 
 (a) a nucleic acid encoding a functional a α subunit or a heterotrimeric G protein which comprises an amino acid sequence encoding a first fluorescent or luminescent protein; and    (b) a nucleic acid encoding a functional β of γ subunit of a heterotrimeric G protein which comprises an amino acid sequence encoding a second fluorescent or luminescent protein, wherein the cell expresses a functional heterotrimeric G protein which is capable of FRET or BRET.    
     
     
         40 . The eukaryotic cell of  claim 39  wherein the second fluorescent or luminescent protein is encoded in the β subunit.  
     
     
         41 . The eukaryotic cell of  claim 39  which further expresses a G protein coupled receptor.  
     
     
         42 . The eukaryotic cell of  claim 38  wherein the cell is a 3T3 cell.  
     
     
         43 . The eukaryotic cell of  claim 39  wherein the cell is a 3T3 cell.  
     
     
         44 . The eukaryotic cell of  claim 41  wherein the cell is a 3T3 cell.  
     
     
         45 . The eukaryotic cell of  claim 38  wherein the cell is a  D. discoideum  cell.  
     
     
         46 . The eukaryotic cell of  claim 39  wherein the cell is a  D. discoideum  cell.  
     
     
         47 . The eukaryotic cell of  claim 41  wherein the cell is a  D. discoideum  cell.  
     
     
         48 . The eukaryotic cell of  claim 41  wherein the receptor is for a ligand selected from the group consisting of: a chemoattractant, an odorant, a hormone, and a neurotransmitter.  
     
     
         49 . A method for detecting conformational changes in a protein, comprising: 
 detecting changes in fluorescent resonance energy transfer (FRET) of an artificial protein subjected to a change in environmental conditions, wherein the artificial protein comprises at least two subunits, wherein a first subunit comprises a cyan fluorescent protein and a second subunit comprises a yellow fluorescent protein, wherein the cyan fluorescent protein and the yellow fluorescent protein are within  100  angstroms of each other, wherein a change in FRET indicates that the change in environmental conditions caused the artificial protein to change its conformation.    
     
     
         50 . The method of  claim 49  wherein the environmental conditions are changed by addition of a ligand for a protein which affects conformation of said artificial protein.  
     
     
         51 . The method of  claim 49  wherein the environmental conditions are changed by the addition of a protein which affects conformation of said artificial protein.  
     
     
         52 . The method of  claim 49  wherein FRET is monitored by exciting with blue light and observing fluorescence in the yellow range.  
     
     
         53 . A method for detecting G protein coupled receptor signaling in the presence of a test sample, the method comprising: 
 contacting a cell according to  claim 41  with a test sample;    monitoring fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) in said cell; wherein a change in FRET or BRET suggests that the test sample binds to a G protein coupled receptor expressed in the cell.    
     
     
         54 . The method of  claim 53  wherein the cell is in a tissue sample.  
     
     
         55 . The method of  claim 53  wherein the cell is in a whole organ.  
     
     
         56 . A functional heterotrimeric G protein comprising an α subunit comprising a first fluorescent or luminescent moiety and a β or γ subunit comprising a second fluorescent or luminescent moiety, wherein the first and second fluorescent or luminescent moieties are capable of fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET).  
     
     
         57 . The functional heterotrimeric G protein of  claim 56  wherein a β subunit comprises the second fluorescent or luminescent moiety.  
     
     
         58 . A eukaryotic cell comprising the G protein of  claim 56 .  
     
     
         59 . The eukaryotic cell of  claim 58  wherein said first and second fluorescent or luminescent moieties is an amino acid sequence.  
     
     
         60 . The eukaryotic cell of  claim 59  wherein one of said first and second fluorescent or luminescent moieties is luciferase.  
     
     
         61 . A functional heterotrimeric G protein comprising an α subunit comprising a fluorescent or luminescent moiety and a β or γ subunit comprising a quenching moiety, wherein the quenching moiety is capable of quenching fluorescence of the fluorescent moiety or the luminescence of the luminescent moiety.  
     
     
         62 . A functional heterotrimeric G protein comprising an α subunit comprising a quenching moiety and a or γ subunit comprising a fluorescent or luminescent moiety, wherein the quenching moiety is capable of quenching fluorescence of the fluorescent moiety or the luminescence of the luminescent moiety.  
     
     
         63 . The G protein of  claim 61  or  62  wherein the fluorescent and quenching moieties are within 100 angstroms of each other.  
     
     
         64 . A functional γ subunit of a heterotrimeric G protein comprising an amino acid sequence encoding a fluorescent or luminescent protein.  
     
     
         65 . The protein of  claim 64  wherein the fluorescent or luminescent protein is cyan fluorescent protein.  
     
     
         66 . The protein of  claim 64  wherein the fluorescent or luminescent protein is yellow fluorescent protein.  
     
     
         67 . The protein of  claim 64  wherein the heterotrimeric G protein is Gγ of  D. discoideum.    
     
     
         68 . The protein of  claim 64  wherein said amino acid sequence is at the γ subunit's N-terminus.  
     
     
         69 . The protein of  claim 64  wherein the fluorescent or luminescent moiety is an amino acid sequence.  
     
     
         70 . A nucleic acid encoding a functional γ subunit of a heterotrimeric G protein which comprises an amino acid sequence encoding a fluorescent or luminescent protein.  
     
     
         71 . The nucleic acid of claim  70  wherein the fluorescent or luminescent protein is cyan fluorescent protein.  
     
     
         72 . The nucleic acid of claim  70  wherein the fluorescent or luminescent protein is yellow fluorescent protein.  
     
     
         73 . The nucleic acid of claim  70  wherein the heterotrimeric G protein is Gγ of  D. discoideum.    
     
     
         74 . The nucleic acid of claim  70  wherein the amino acid sequence is at the γ subunit's N-terminus.  
     
     
         75 . The nucleic acid of claim  70  wherein the nucleic acid comprises a vector for replication and expression of said subunit.  
     
     
         76 . A eukaryotic cell comprising a nucleic acid according to any of  claims 70  to  75 .