Single-cell biosensor for the measurement of GPCR ligands in a test sample

Abstract

The present invention is related to the detection of GPCR ligands in a test sample by using a single cell biosensor expressing a GPCR. Preferably, the test sample is derived from a biological or environmental sample. This invention may be used to detect the presence of a disease or to detect the presence of a harmful agent in the environment. Included in the present invention is an array of biosensors that detect ligands of various GPCRs.

1 . A method of detecting the presence or absence of acetylcholinesterase in a test sample, the method comprising the steps of:
 (a) providing a cell comprising a G protein-coupled receptor (GPCR) and a detectably labeled arrestin, wherein the cellular distribution of the detectably labeled arrestin changes in response to activation of the GPCR, and wherein the GPCR is activated by acetylcholine;   (b) applying acetylcholine to the cell and determining the cellular distribution of the detectably labeled arrestin in response to the acetylcholine and in the absence of a test sample;   (c) exposing the cell to the test sample;   (d) applying acetylcholine to the cell and determining the cellular distribution of the detectably labeled arrestin in response to the acetylcholine and in the presence of the test sample; and   (e) comparing the cellular distribution of the detectably labeled arrestin in the presence of the test sample to the cellular distribution of the detectably labeled arrestin in the absence of the test sample,   
       wherein a change in the cellular distribution of the detectably labeled arrestin in the presence of the test sample in response to acetylcholine as compared to the cellular distribution of the detectably labeled arrestin in the absence of the test sample indicates the presence of an acetylcholinesterase in the test sample. 
     
     
         2 . A method of detecting the presence or absence of an acetylcholinesterase inhibitor in a test sample, the method comprising the steps of:
 (a) providing a cell comprising a G protein-coupled receptor (GPCR) and a detectably labeled arrestin, wherein the cellular distribution of the detectably labeled arrestin changes in response to activation of the GPCR by acetylcholine;   (b) combining the test sample with acetylcholinesterase and acetylcholine;   (c) exposing the cell to the test sample combined with acetylcholinesterase and acetylcholine;   (d) determining the cellular distribution of the detectably labeled arrestin in response to the test sample combined with acetylcholinesterase and acetylcholine,   
       wherein translocation of the detectably labeled arrestin to the plasma membrane or endosomes of the cell indicates that an acetylcholinesterase inhibitor is present in the test sample. 
     
     
         3 . The method of  claim 1  or  2  wherein the test sample is a member selected from serum, tissue, blood or urine. 
     
     
         4 . The method of  claim 1  or  2 , wherein the GPCR is a muscarinic receptor. 
     
     
         5 . The method of  claim 4 , wherein the muscarinic receptor is a chimera comprising a high affinity tail from a vasopressin receptor. 
     
     
         6 . The method of  claim 1  or  2 , wherein the arrestin is β-arrestin. 
     
     
         7 . The method of  claim 1  or  2 , wherein the detectable label is a radioisotope, an epitope tag, an affinity label, an enzyme, a fluorescent group, or a chemiluminescent group. 
     
     
         8 . The method of  claim 1 , wherein an increase in a local concentration of the detectably labeled arrestin in plasma membrane, clathrin-coated pits, endocytic vesicles, or endosomes as compared to cytosol results in an increase in local signal intensity. 
     
     
         9 . The method of  claim 1 , wherein a signal intensity of the labeled arrestin in the plasma membrane, clathrin-coated pits, endocytic vesicles or endosomes is increased as compare do a level of signal instensity in the cytosol. 
     
     
         10 . The method of  claim 1 , wherein the cellular distribution of the detectably labeled arrestin is quantified.