Molecular Characterization of A(1) and A(2a) Adenosine Receptors.

Detailed amino acid sequence analyses of A(1) and A(2a) adenosine receptors were assembled by analogy to other G-protein-coupled receptors and correlated with pharmacological observations. Sites for phosphorylation, palmitoylation, and sodium binding have been proposed. Striatal A(2a) receptors from human and other species were photoaffinity-labeled using the selective, radioiodinated agonist PAPA-APEC. Selective chemical affinity labels for A(1) and A(2a) receptors have been introduced. For example, an isothiocyanate, p-DITC-APEC (100 nM), irreversibly diminished the B(max) for [(3)H]CGS 21680 (2-[4-[(2-carboxyethyl) phenyl] ethylamino]-5'-N-ethylcarboxamidoadenosine) binding in rabbit striatal membranes by 71% (K(d) unaffected), suggesting a direct modification of the ligand binding site. Novel trifunctional affinity labels have been designed. Rabbit and human A(2a) receptors were characterized using [(3)H]XAC binding in the presence of 50 or 25 nM CPX (8-cyclopentyl-l,3-dipropylxanthine), respectively. The inhibition of A(2) radioligand binding by the histidyl-modifying reagent diethylpyrocarbonate suggested the involvement of His residues in interactions with adenosine agonists and antagonists. Properties of transiently expressed mutants of bovine A(1) receptors in which either His(251) or His(278) residues have been substituted with Leu suggest that both histidines are important in binding.