Characterization of a novel adenosine binding protein sensitive to cyclic AMP in rat brain cytosolic and particulate fractions

A novel binding site for the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), which was enriched in rat forebrain, was characterized in cytosolic and particulate preparations. The site showed a pharmacological profile different from other [H-3]NECPA binding proteins and was named adenotin 2. [H-3]NECA was bound in the presence of 100 mu M 2-chloroadenosine with a K-d of 45.4 nM and a B-max of 4711 fmol/mg in the cytosol and a K-d of 72.4 nM and a B-max of 4844 fmol/mg in the crude membrane fraction. The presence of two different binding sites on adenotin 2 for [H-3]NECA was shown in kinetic experiments. This protein showed identical pharmacological profiles in both subcellular preparations. [H-3]NECA was displaced by purine analogues with a rank order of potency of NECA > 3'5' cyclic AMP (cAMP) > 5'-deoxy-5'-chloroadenosine > S-adenosylhomocysteine approximate to 5'-deoxy-5'-methylthioadenosine (MeSA) > adenosine approximate to adereine. cAMP inhibited [H-3]NECA binding allosterically, whereas adenine and MeSA acted competitively. Inhibitors and activators of protein kinases such as N-(2-aminoethyl)-5-isoquinolinesulfonamide, Sp-adenosine cyclic monophophothioate and (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxy-carbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H, 8H, 11H-2, 7b, 11a-triazadibenzo (a,g)cycloocta(cde)-trinden-1-one (K 252a) interacted with [H-3]NECA binding to adenotin 2 in nanomolar concentrations. Adenosine-5'-O-(3-thiotriphosphate) (100 mu M) increased the affinity of [H-3]NECA to a K-d of 9 nM and diminished the affinity of cAMP. The pharmacological characteristics of this novel binding site for [H-3]NECA resemble those of the inhibition of phosphorylation processes by adenosine and its derivatives in heart and smooth muscle but are distinct from known adenosine receptors, adenosine binding proteins and protein kinases. Copyright (C) 1996 Elsevier Science Inc.