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Cloning and Functional Expression of a Novel G_i Protein-Coupled Receptor for Adenine from Mouse Brain

Department of Pharmacology and Toxicology (I.v.K., K.H.) and Pharmaceutical Institute, Pharmaceutical Chemistry I (A.C.S., B.B.A.A., A.A., C.E.M.), University of Bonn, Bonn, Germany

An orphan G protein-coupled receptor from the rat has recently been demonstrated to act as a transmembrane receptor for the nucleobase adenine. The receptor is possibly involved in nociception. Here we report the cloning and functional expression of an additional G_i-coupled receptor for adenine (Genbank accession code DQ386867). mRNA for this receptor was obtained from mouse brain and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. The new mouse protein sequence shares only 76% identity with that of the rat adenine receptor, suggesting that the receptors are not species homologs but distinct receptor subtypes. In human 1321N1 astrocytoma cells stably expressing the new mouse receptor, adenine and 2-fluoroadenine inhibited the isoproterenol-induced cAMP formation with IC₅₀ concentrations of 8 and 15 nM, respectively. The adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 µM) as well as the P2 receptor antagonist suramin (300 µM) failed to change the responses to adenine. In contrast, pretreatment of cells with pertussis toxin abolished the effect of adenine. When the novel adenine receptor was expressed in Sf21 insect cells, a specific binding site for [³H]adenine was detected. In competition assays, the rank order of potency of selected ligands was identical to that obtained in membranes from NG108-15 cells and rat brain cortex (adenine > 2-fluoroadenine > 7-methyladenine > 1-methyladenine >> N⁶-dimethyladenine). In summary, our data show that a second mammalian DNA sequence encodes for a G_i-coupled GPCR activated by low, nanomolar concentrations of adenine.

Address correspondence to: Ivar von Kügelgen, Department of Pharmacology and Toxicology, University of Bonn, D-53113 Bonn, Germany. E-mail: kugelgen{at}uni-bonn.de