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 IC(50) concentrations of 8 and 15 nM, respectively. The
adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (
DPCPX, 1 muM) as well as the P2 receptor antagonist
suramin (300 muM) 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 [(3)H]
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 >> N(6)-
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.