chrvoehringer.pdf [2.63 mb] |Charakterisierung des heterolog exprimierten metabotropen purinergen P2Y1-Rezeptors aus dem Hirn der Ratte (rP2Y1) in HEK293-Zellen|
|GPCR Purinergic receptor green fluorescent protein human embryonic kidney (HEK293) cells receptor regulation desensitization ca2+ signalling heterologous expression|
Extracellular nucleoside di- and triphosphates modulate a wide variety of physiological responses through activation of a family of G protein-coupled receptors, referred to as P2Y receptors. This study presents the successfull stable expression of a chimera of the rat brain P2Y1 receptor (rP2Y1) and green fluorescent protein in HEK293 cells, characterizing the heterologously expressed receptor proteins as high affinity receptors for specific P2Y1-ligands, coupled to intracellular Ca2+ release. Data concerning the characterization of receptor mediated Ca2+ signalling, posttranslational receptor glycosylation and agonist-induced receptor trafficking were generated in experiments with rP2Y1-GFP expressing HEK293 cells and were compared to such data generated with stably transfected HEK 293 cells expressing the rat brain P2Y1 wild type receptor (rP2Y1-wt). Both receptors were analyzed by measuring Ca2+ responses in single cells. The rP2Y1-eGFP receptor is coupled to Ca2+ release like the rP2Y1-wt receptor is and it could be demonstrated that P2Y1-induced Ca2+ release is mediated via PLC/InsP3 activation and causes capacitative Ca2+ entry. Experiments using a PLC inhibitor, U73122, confirm the functional activation of PLCb through rP2Y1-eGFP activation. The P2Y1 selective agonists, 2-MeSADP and 2-MeSATP, were most potent at the heterologously expressed receptors. We found a ligand selectivity typical for P2Y1 receptors (2-MeSADP = 2-MeSATP > ADP > ATPaS, ATP >> UTP). Western blotting experiments demonstrate the plasma membrane localization of rP2Y1-eGFP and lectin staining with Concanavalin A indicates glycosylation of the recombinant rP2Y1 receptor. Investigation of transfected cells with fluorescence microscopy revealed a intense plasma membrane staining with GFPFluorescence. Fluorescence microscopy and Ca2+ measurements confirm that the rP2Y1-eGFP receptor is subjected to processes leading to agonist-induced internalization and homologous desensitization. Analysis of agonist-induced [Ca2+]i signals revealed that homologous receptor desensitization is dependent of agonist concentration and duration of the stimulus. The desensitization effect was decreased after preincubation with the CaMKinase inhibitor KN-62 and increased after preincubation with the phosphatase inhibitor okadaic acid or the protein kinase C activator TPA, indicating involvement of PPA2 in kinase regulation. The used rP2Y1-eGFP expressing cell line was therfore shown to be a suitable tool to determine the physiological role of the rP2Y1 receptor and it磗 signaling pathways.