A chimeric rat brain P2Y1 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 P2Y1 receptor. Therefore, we generated stably transfected human embryonic kidney 293 cells expressing the rat P2Y1 wild-type receptor (rP2Y1-wt) or the receptor tagged at the C-terminus with the enhanced GFP (rP2Y1-eGFP). The chimeric rP2Y1-eGFP receptor is localized mainly to the plasma membrane as revealed by Western blotting of subcellular fractions. Both receptors were analyzed by measuring Ca2+ responses to short pulses of the agonists in single cells by continuous superfusion with medium. The rP2Y1-eGFP receptor was coupled to Ca2+ release as was the rP2Y1-wt receptor. 2-Methylthio adenosine 5'-diphosphate and -triphosphate (2-MeSATP and 2-MeSADP) were the most potent agonists at the heterologously expressed receptors, with EC50 values of 50 to 70 nM for rP2Y1-eGFP and 0.06 to 0.4 nM for rP2Y1-wt. These potencies of the two P2Y-selective agonists at rP2Y1-wt receptor-expressing cells are the highest values reported so far. This increase is probably due to a receptor reserve. In both rP2Y1-wt- and in rP2Y1-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-thiotriphosphate) (ATPalphaS) are full agonists at the rP2Y1 receptor at both transfected cell lines. The rP2Y1-eGFP receptor has the same ligand selectivity as the rP2Y1-wt receptor (2-MeSADP = 2-MeSATP > ADP > ATPalphaS, ATP >> UTP). Thus, the GFP-tagged P2Y1 receptor is fully active and shows regular signal transduction coupling. It provides the means for biochemical characterization, since it can be solubilized and is a tool for further physiological analysis.
Vöhringer
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Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke-Universität, Magdeburg, Germany.
Schäfer
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Reiser
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D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D008024:Ligands; D008164:Luminescent Proteins; C546382:P2RY1 protein, human; C546384:P2ry1 protein, rat; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002118:Calcium