Agonist and Antagonist Radioligands and Photoaffinity Labels for the Adenosine A₁ Receptor

Summary

Experiments illustrating the use of recently developed ¹²⁵I-labeled agonist and antagonist radioligands and photoaffinity labels have been described. Adenosine A₁ receptors of rat cerebral cortex were characterized with arylamine-containing radioligands: an agonist, ¹²⁵I-aminobenzyladenosine (¹²⁵I-ABA), and an antagonist, 1-propyl-8-phenylxanthine derivative ¹²⁵I-BW A827. In optimal binding conditions, both radioligands bind with K_D of < 1 nM to receptors in membranes or to receptors solubilized in detergent. Photoaffinity labels have been synthesized from the radioligands by converting the arylamines to the corresponding arylazides. The resulting antagonist compound photoincorporates into a 34 000-dalton polypeptide with greater specificity and 10 times greater efficiency than the agonist.

High-affinity agonist binding to membranes pretreated with chelator is MgCl₂-dependent and is inhibited by NaCl. After solubilization in detergent, high-affinity agonist binding to adenosine receptors displays less MgCl₂-dependence than agonist binding to membranes. Guanine nucleotides and N-ethylmaleimide abolish high-affinity agonist binding and slightly enhance antagonist binding. In addition, high concentrations of salts (>0.5 N markedly enhance antagonist radioligand binding. The data suggest that agonists and antagonists bind preferentially to different conformations of the same population of adenosine receptors and that the 34 000-dalton photolabeled polypeptide is a component of the adenosine A₁ receptor and not an adjacent protein. Since agonists bind with high affinity to only a subpopulation of receptors, antagonist radioligands may be preferable to agonists for identifying solubilized receptors during their purification.