Constitutively translocating cell line

Abstract

The present invention relates to agonist-independent methods of screening for compounds that alter GPCR desensitization. Included in the present invention are cell lines containing GRKs, in which GPCRs are desensitized in the absence of agonist; the GRKs may be modified. The present invention relates to methods to determine if a GPCR is expressed at the plasma membrane, and if the GPCR has an affinity for arrestin. Modified GPCRs which have increased arrestin affinity are included in the present invention. These modified GPCRs are useful in methods to screen for compounds that alter desensitization, including both the agonist-independent methods and agonist-dependent methods described herein.

1 - 13 . (canceled) 
     
     
         14 . A method of identifying a compound that alters GPCR phosphorylation, comprising:
 (a) providing a cell comprising a GPCR and a GRK;   (b) exposing said cell to the compound(s); and   (c) determining whether GRK phosphorylation of the GPCR is altered in the presence of the compound(s).   
     
     
         15 . The method of  claim 14 , wherein the cellular distribution of the GPCR or GRK is determined. 
     
     
         16 . The method of  claim 14 , wherein a difference between (1) the distribution of the GPCR or GRK in the cell in the presence of the compound and (2) the distribution of the GPCR or GRK in the cell in the absence of the compound(s) is monitored. 
     
     
         17 . The method of  claim 16 , wherein a difference is correlated between (1) and (2) to the phosphorylation of the GPCR. 
     
     
         18 . The method of  claim 14 , wherein the GRK is not located in the plasma membrane, indicating that GRK phosphorylation of the GPCR is altered. 
     
     
         19 . The method of  claim 14 , wherein the phosphorylation state of the GPCR is determined. 
     
     
         20 . The method of  claim 14 , wherein the activity of the GRK is determined. 
     
     
         21 . The method of  claim 17 , wherein the ability of the GPCR to be internalized is determined. 
     
     
         22 . A method of determining if a GPCR of interest is expressed at the plasma membrane, comprising:
 (a) providing a cell comprising a GPCR, an arrestin, and a GRK, wherein the arrestin is detectably labeled;   (b) determining the cellular distribution of the arrestin; and   (c) correlating the cellular distribution of the arrestin to the ability of the GPCR to be expressed at the plasma membrane.   
     
     
         23 . The method of  claim 22 , wherein the arrestin is localized in vesicles, pits, endosomes, or elsewhere in the desensitization pathway. 
     
     
         24 . A method of determining if a GPCR of interest is expressed at the plasma membrane, comprising:
 (a) providing a cell comprising a GPCR and a GRK, wherein the GRK is detectably labeled;   (b) determining the cellular distribution of the GRK; and   (c) correlating the cellular distribution of the GRK to the ability of the GPCR to be expressed at the plasma membrane.   
     
     
         25 . The method of  claim 24 , wherein the GRK is localized at the plasma membrane. 
     
     
         26 . A method of analyzing the ability of a GPCR to bind arrestin, comprising:
 (a) providing a cell comprising a GPCR, an arrestin, and a GRK, wherein the arrestin is detectably labeled;   (b) determining the cellular distribution of the arrestin; and   (c) correlating the cellular distribution of the arrestin to the ability of the GPCR to bind arrestin.   
     
     
         27 . The method of  claim 26 , wherein the arrestin or the GPCR is localized in vesicles, pits, or endosomes. 
     
     
         28 . A compound identified by claim  1 . 
     
     
         29 - 36 . (canceled) 
     
     
         37 . A modified GPCR comprising a NPXXY motif and a carboxyl terminal tail,
 wherein said carboxyl terminal tail comprises a putative site of palmitoylation and one or more clusters of phosphorylation,   wherein the carboxyl terminal tail comprises a retained portion of a carboxyl-terminus region of a first GPCR portion fused to a portion of a carboxyl-terminus from a second GPCR, and   wherein the second GPCR comprises the one or more clusters of phosphorylation and further comprises a second putative site of palmitoylation approximately 10 to 25 amino acid residues downstream of a second NPXXY motif.   
     
     
         38 . The modified GPCR of  claim 37 , wherein the first GPCR is a Class A receptor. 
     
     
         39 . The modified GPCR of  claim 37 , wherein the first GPCR is hGPR3, hGPR6, hGPR12, hSREB2, hSREB3, hGPR8, or hGPR22. 
     
     
         40 . The modified GPCR of  claim 37 , wherein the second GPCR is a Class B receptor. 
     
     
         41 . The modified GPCR of  claim 37 , wherein the Class B receptor is selected from the group consisting of a vasopressin V2 receptor, a neurotensin-1 receptor, a substance P receptor and an oxytocin receptor. 
     
     
         42 . A nucleic acid encoding a modified GPCR of  claim 37 . 
     
     
         43 . A nucleic acid selected from the group consisting of SEQ ID Nos: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90. 
     
     
         44 . An expression vector comprising the nucleic acid of  claim 42 . 
     
     
         45 . A host cell comprising the expression vector of  claim 44 . 
     
     
         46 . A host cell comprising the nucleic acid of  claim 42 . 
     
     
         47 . A method of screening compounds for GPCR activity comprising the steps of:
 (a) providing a cell that expresses at least one modified GPCR according to  claim 37 , wherein said cell further comprises arrestin conjugated to a detectable molecule;   (b) exposing the cell to the compound;   (c) detecting location of the arrestin within the cell;   (d) comparing the location of the arrestin within the cell in the presence of the compound to the location of the arrestin within the cell in the absence of the compound; and   (e) correlating a difference between (1) the location of the arrestin within the cell in the presence of the compound and (2) the location of the arrestin within the cell in the absence of the compound.   
     
     
         48 . The method of  claim 47 , wherein the arrestin is detected in endosomes, endocytic vesicles, or pits. 
     
     
         49 . A kit for identifying a molecule that modulates the activity of a GPCR, comprising a cell that expresses at least one modified GPCR according to  claim 37 , wherein said cell further comprises a molecule involved in desensitization conjugated to a detectable molecule. 
     
     
         50 . A method of inhibiting arrestin translocation to the AT1AR receptor by administering an effective amount of losartan to a patient in need thereof. 
     
     
         51 . (canceled) 
     
     
         52 . The method of claim  29 , wherein the GPCR is AT1AR. 
     
     
         53 . The method of claim  29 , wherein the disease is renal disease, diabetes and nephropathy, diabetes mellitus, type 2 diabetes, nephropathy, hypertension, congestive heart failure, endothelial dysfunction, vascular inflammation, or heart disease.