An orphan
G protein-coupled receptor from rat has recently been discovered to be activated by the nucleobase
adenine (Proc Natl Acad Sci USA 99:8573-8578, 2002). In the present study, we show for the first time that the
adenine receptor is expressed in membrane preparations of native tissues and cell lines in high density, including rat brain cortex, rat brain striatum, and the mouse
neuroblastoma x rat
glioma hybrid cell line NG108-15. Saturation analysis with [3H]
adenine at rat brain cortical membranes exhibited a single high-affinity binding site with a KD value of 27.2 nM, and a binding capacity of 2.28 pmol/mg of
protein. Kinetic studies revealed unusual binding kinetics of [3H]
adenine with rapid association and slow dissociation. A series of compounds were investigated in [3H]
adenine competition experiments at rat brain cortex. Only minor substitution of the
adenine structure was tolerated, the most potent compounds of the present series being
2-fluoroadenine (Ki value of 620 nM), 8-thioadenine (Ki value of 2.77 microM), N6-methyladenine (Ki value of 3.64 microM), and
7-methyladenine (Ki value of 4.13 microM), all of which were partial agonists (40-60% intrinsic activity).
Adenine dose dependently inhibited
forskolin-stimulated
adenylate cyclase in membrane preparations of NG108-15 cells as well as in intact cells, showing that the receptor is functional in NG108-15 cells.
Reverse transcriptase-polymerase chain reaction experiments followed by sequencing indicate that the NG108-15 cells express the murine ortholog of the
adenine receptor. Moreover, preliminary radioligand binding studies with [3H]
adenine at membranes of human
astrocytoma 1321N1 cells suggest that a human ortholog of the
rat adenine receptor exists.