Agonist-Induced Desensitization/Resensitization of Human G Protein-Coupled Receptor 17: A Functional Cross-Talk between Purinergic and Cysteinyl-Leukotriene Ligands

Abstract

G protein-coupled receptor (GPR) 17 is a P2Y-like receptor that responds to both uracil nucleotides (as UDP-glucose) and cysteinyl-leukotrienes (cysLTs, as LTD 4 ). By bioinformatic analysis, two distinct binding sites have been hypothesized to be present on GPR17, but little is known on their putative cross-regulation and on GPR17 desensitization/resensitization upon agonist exposure. In this study, we investigated in GPR17-expressing 1321N1 cells the cross-regulation between purinergic-and cysLT-mediated responses and analyzed GPR17 regulation after prolonged agonist exposure. Because GPR17 receptors couple to G i proteins and adenylyl cyclase inhibition, both guanosine 5Ј-O-(3-[35 S]thio)triphosphate ([ 35 S]GTP␥S) binding and the cAMP assay have been used to investigate receptor functional activity. UDP-glucose was found to enhance LTD 4 potency in mediating activation of G proteins and vice versa, possibly through an allosteric mechanism. Both UDP-glucose and LTD 4 induced a time-and concentration-dependent GPR17 loss of response (homologous desensitization) with similar kinetics. GPR17 homologous desensitization was accompanied by internalization of receptors inside cells, which occurred in a time-dependent manner with similar kinetics for both agonists. Upon agonist removal, receptor resensitization occurred with the typical kinetics of G protein-coupled receptors. Finally, activation of GPR17 by UDP-glucose (but not vice versa) induced a partial heterologous desensitization of LTD 4 -mediated responses, suggesting that nucleotides have a hierarchy in producing desensitizing signals. These findings suggest a functional cross-talk between purinergic and cysLT ligands at GPR17. Because of the recently suggested key role of GPR17 in brain oligodendrogliogenesis and myelination, this cross-talk may have profound implications in fine-tuning cell responses to demyelinating and inflammatory conditions when these ligands accumulate at lesion sites.

Is GPR17 a P2Y/Leukotriene Receptor? Examination of Uracil Nucleotides, Nucleotide Sugars, and Cysteinyl Leukotrienes as Agonists of GPR17

“…Although multiple follow-up studies consistent with this original report have been published by the same group (Parravicini et al, 2008;Pugliese et al, 2009;Buccioni et al, 2011;Daniele et al, 2011;Coppi et al, 2013), Benned-Jensen and Rosenkilde (2010) reported that UDP and UDP-glucose, but not cysteinyl leukotrienes, promoted GPR17-dependent guanosine 59-O-(3-[…”

“…Early characterization of GPR17 mostly focused on (Daniele et al, 2011). However, using the same conditions (cAMP levels elevated by 10 mM forskolin) and the same cells (1321N1), we failed to observe inhibition of cAMP accumulation in over a dozen independent experiments, even though the receptor was expressed at the cell surface based on an intact cell RIA.…”

“…However, the authors were unable to demonstrate inhibition of cAMP accumulation by two different immunocompetitive cAMP assays and required the increased sensitivity of the GloSensor assay to observe inhibition of adenylyl cyclase. In contrast, Daniele et al (2011) reported robust inhibition of cAMP accumulation in 1321N1 cell using a competitive protein-binding assay. Thus, there are important discrepancies even among laboratories that observe activation of GPR17 with nucleotides and nucleotide sugars.…”

“…Activation of GPR17 also was reported to inhibit accumulation of cAMP when expressed in 1321N1 cells (Ciana et al, 2006;Daniele et al, 2011). Therefore, we also examined the capacity of uracil nucleotides, UDP-sugars, and cysteinyl leukotrienes to inhibit adenylyl cyclase in 1321N1 cells expressing GPR17.…”

The Regulated Expression, Intracellular Trafficking, and Membrane Recycling of the P2Y-like Receptor GPR17 in Oli-neu Oligodendroglial Cells

“…Recent work has led to the "deorphanization" of the G protein-coupled receptor (GPCR) 5 referred to as GPR17, which is located at an intermediate phylogenetic position between the purinergic P2Y receptors and the CysLT 1 and CysLT 2 receptors for cysteinyl-leukotrienes (cysLTs; [1][2][3][4][5][6]. Both recombinant and native GPR17 receptors respond to uracil nucleotides (e.g.…”