Functional characterization of two isoforms of the P2Y-like receptor GPR17: [35S]GTPgammaS binding and electrophysiological studies in 1321N1 cells

Am J Physiol Cell Physiol. 2009 Oct;297(4):C1028-40. doi: 10.1152/ajpcell.00658.2008. Epub 2009 Jul 22.

Abstract

The previously "orphan" G protein-coupled receptor GPR17 is structurally related to both P2Y nucleotide receptors and to receptors for cysteinyl leukotrienes. Genomic analysis revealed two putative open reading frames encoding for a "short" and a "long" receptor isoform of 339- and 367-amino acids, respectively, with the latter displaying a 28-amino acid longer NH(2) terminus. The short isoform has been recently "deorphanized," revealing dual responses to uracil nucleotides and cysteinyl leukotrienes. No information regarding the ligand specificity, tissue distribution, or pathophysiological roles of the long receptor isoform is available. In the present study, we cloned human long-GPR17, determined its tissue distribution, and characterized its pharmacological specificity in 1321N1 cells by [35S]GTPgammaS binding (which measures the ability of G protein-coupled receptor agonists to increase GTP binding to G proteins) and whole cell patch-clamp recording measuring receptor coupling to K+ channels. [35S]GTPgammaS binding in long-GPR17-expressing 1321N1 cells revealed concentration-dependent responses to uracil nucleotides (UDP-galactose = UDP > UDP-glucose) and cysteinyl leukotrienes (LTC4 > LTD4), which were counteracted by a purinergic (cangrelor) and a cysteinyl leukotriene antagonist (montelukast), respectively. The nonhydrolyzable ATP analog ATPgammaS also acted as an antagonist. GPR17 coupled to Gi and, to a lesser extent, Gq proteins. UDP-glucose and LTD(4) also induced increases in overall outward K+ currents, which were antagonized by the purinergic antagonists MRS2179 and cangrelor and by montelukast. We conclude that the previously uncharacterized long-GPR17 isoform is a functional receptor that is stimulated by both uracil nucleotides and cysteinyl leukotrienes. We also show that the signaling pathway of GPR17 involves the generation of outward K+ currents, an important protective mechanism that, in brain, is specifically aimed at reducing neuronal hyperexcitability and resultant neuronal injury.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Cloning, Molecular
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism*
  • Humans
  • Leukotriene D4 / metabolism
  • Leukotrienes / metabolism*
  • Patch-Clamp Techniques
  • Protein Isoforms / genetics
  • Protein Isoforms / physiology
  • Radioligand Assay
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / physiology*
  • Sulfur Radioisotopes
  • Uridine Diphosphate / metabolism
  • Uridine Diphosphate Glucose / metabolism

Substances

  • GPR17 protein, human
  • Leukotrienes
  • Protein Isoforms
  • Receptors, G-Protein-Coupled
  • Sulfur Radioisotopes
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Uridine Diphosphate
  • Leukotriene D4
  • Uridine Diphosphate Glucose