The A1 adenosine receptor. Identification of the binding subunit by photoaffinity cross-linking

Adenosine modifies the catalytic activity of adenylate cyclase through both inhibitory (A1 or Ri) as well as stimulatory (A2 or Ra) cell surface receptors. We developed 125I-labeled N6-2-(4-aminophenyl)ethyladenosine as a selective ligand to probe the structure of A1 receptors. The binding of this radioligand to rat cerebral cortex or adipocyte membranes is saturable, reversible, and of high affinity (KD approximately 2 nM). A1 receptor agonists antagonize binding stereoselectivity and with a potency order appropriate for A1 receptors. The heterobifunctional cross-linking reagent N-succinimidyl-6-(4-azido-2-nitrophenylamino)hexanoate covalently couples the radioligand to a protein of Mr = 38,000 in both tissues as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Inhibition of covalent labeling by adenosine analogs exhibited the stereoselectivity and potency order typical of A1 receptor ligands. Guanine nucleotides reduced both specific binding and covalent incorporation of the radioligand, evidence that the radioligand is an A1 receptor agonist. These results suggest that the A1 receptor binding subunit of both brain and adipocytes resides on a protein of Mr = 38,000. The new radioligand should prove useful in studying the structure and regulation of A1 receptors.