A chimeric rat brain P2Y(1) receptor tagged with green-fluorescent protein: High-affinity ligand recognition of adenosine diphosphates and triphosphates and selectivity identical to that of the wild-type receptor

We tested how the green fluorescent protein (GFP) tag affects signaling of the nucleotide-activated P2Y(1) receptor. Therefore, we generated stably tr ansfected human embryonic kidney 293 cells expressing the rat P2Y(1) wild-t ype receptor (rP2Y(1)-wt) or the receptor tagged at the C-terminus with the enhanced GFP (rP2Y(1)-eGFP). The chimeric rP2Y(1)-eGFP receptor is localiz ed mainly to the plasma membrane as revealed by Western blotting of subcell ular fractions. Both receptors were analyzed by measuring Ca2+ responses to short pulses of the agonists in single cells by continuous superfusion wit h medium. The rP2Y(1)-eGFP receptor was coupled to Ca2+ release as was the rP2Y(1)-wt receptor. 2-Methylthio adenosine 5'-diphosphate and -triphosphat e (2-MeSATP and 2-MeSADP) were the most potent agonists at the heterologous ly expressed receptors, with EC50 values of 50 to 70 nM for rP2Y(1)-eGFP an d 0.06 to 0.4 nM for rP2Y(1)-wt. These potencies of the two P2Y-selective a gonists at rP2Y(1)-wt receptor-expressing cells are the highest values repo rted so far. This increase is probably due to a receptor reserve. In both r P2Y(1)-wt- and in rP2Y(1)-eGFP-expressing cells, the effect of 2-MeSATP was inhibited equally by the antagonist pyridoxal phosphate-6-azophenyl-2',4'- disulfonic acid. We established that ATP as well as adenosine 5'-O-(1-thiot riphosphate) (ATP alpha S) are full agonists at the rP2Y(1) receptor at bot h transfected cell lines. The rP2Y(1)-eGFP receptor has the same ligand sel ectivity as the rP2Y(1)-wt receptor (2-MeSADP = 2-MeSATP > ADP > ATP alpha S, ATP >> UTP). Thus, the GFP-tagged P2Y(1) receptor is fully active and sh ows regular signal transduction coupling. it provides the means for biochem ical characterization, since it can be solubilized and is a tool for furthe r physiological analysis. BIOCHEM PHARMACOL 59;7:791-800, 2000, (C) 2000 El sevier Science Inc.