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RECEPTORS AND SIGNAL TRANSDUCTION

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

¹Department of Preclinical and Clinical Pharmacology, University of Florence, Florence; ²Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa; and ³Department of Pharmacological Sciences, University of Milan, Milan, Italy

Submitted 23 December 2008 ; accepted in final form 16 July 2009

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 NH₂ 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]GTPS 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]GTPS 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 ATPS also acted as an antagonist. GPR17 coupled to G_i and, to a lesser extent, G_q proteins. UDP-glucose and LTD₄ 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.

uracil nucleotides; cysteinyl leukotrienes; G protein-coupled receptors; big-conductance K⁺ channels; neuronal damage; guanosine 5'-O-(3-thiotriphosphate)

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