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 t

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• 作者：Vö ; hringer ; Christian ; Schä ; fer ; Rainer ; Reiser ; Georg
• 关键词：purinergic receptor ; receptor expression ; GFP tag ; human embryonic kidney cells (HEK 293)
• 刊名：Biochemical Pharmacology
• 出版年：2000
• 出版时间：April 1, 2000
• 年：2000
• 卷：59
• 期：7
• 页码：791-800
• 全文大小：445 K

文摘

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) (ATPαS) 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 > ATPαS, 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.