MOLECULAR-CLONING AND CHARACTERIZATION OF THE HUMAN-A(3) ADENOSINE RECEPTOR

The human A3 adenosine receptor was cloned from a striatal cDNA librar y using a probe derived from the homologous rat sequence. The cDNA enc odes a protein of 318 amino acids and exhibits 72% and 85% overall ide ntity with the rat and sheep A3 adenosine receptor sequences, respecti vely. Specific and saturable binding of the adenosine receptor agonist N6-(4-amino-3-[I-125]iodobenzyl)adenosine [I-125]ABA was measured on the human A3 receptor stably expressed in Chinese hamster ovary cells with a K(d) = 10 nM. The potency order for adenosine receptor agonists was N-ethylcarboxamidoadenosine (NECA) greater-than-or-equal-to (R)-N 6-phenyl-2-propyladenosine [(R)-PIA] > N6-cyclopentyladenosine (CPA) > (S)-N6-phenyl-2-propyladenosine [(S)-PIA]. The human receptor was blo cked by xanthine antagonists, most potently by inobenzyl)-8-(4-oxyacet ate)phenyl-1-propylxanthine (I-ABOPX) with a potency order of I-ABOPX > 1,3-dipropyl-8-(4-acrylate)phenylxanthine greater-than-or-equal-to x anthine amino congener >> 1,3-dipropyl-8-cyclopentylxanthine. Adenosin e, NECA, (R)- and (S)-PIA, and CPA inhibited forskolin-stimulated cAMP accumulation by 30-40% in stably transfected cells; I-ABA is a partia l agonist. When measured in the presence of antagonists, the dose-resp onse curves of NECA-induced inhibition of forskolin-stimulated cAMP ac cumulation were right-shifted. Antagonist potencies determined by Schi ld analyses correlated well with those established by competition for radioligand binding. The A3 adenosine receptor transcript is widesprea d and, in contrast to the A1, A2a, and A2b transcripts, the most abund ant expression is found in the lung and liver. The tissue distribution of A3 mRNA is more similar to the widespread profile found in sheep t han to the restricted profile found in the rat. This raises the possib ility that numerous physiological effects of adenosine may be mediated by A3 adenosine receptors.