title: Chemical modification and irreversible inhibition of striatal A2a adenosine receptors.
abstract: The ligand recognition site of A2a-adenosine receptors in rabbit striatal membranes was probed using non-site-directed labeling reagents and specific affinity labels. Exposure of membranes to diethylpyrocarbonate at a concentration of 2.5 mM, followed by washing, was found to inhibit the binding of [3H]CGS 21680 and [3H]xanthine amine congener to A2a receptors, by 86 and 30%, respectively. Protection from diethylpyrocarbonate inactivation by an adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine, and an antagonist, theophylline, suggested the presence of two histidyl residues on the receptor, one associated with agonist binding and the other with antagonist binding. Binding of [3H]CGS 21680 or [3H]xanthine amine congener was partially restored after incubation with 250 mM hydroxylamine, further supporting histidine as the modification site. Preincubation with disulfide-reactive reagents, dithiothreitol or sodium dithionite, at greater than 5 mM inhibited radioligand binding, indicating the presence of essential disulfide bridges in A2a receptors, whereas the concentration of mercaptoethanol required to inhibit binding was greater than 50 mM. A number of isothiocyanate-bearing affinity labels derived from the A2a-selective agonist 2-[(2-aminoethylamino) carbonylethylphenylethylamino]-5'-N- ethylcarboxamidoadenosine (APEC) were synthesized and found to inhibit A2a receptor binding in rabbit and bovine striatal membranes. Binding to rabbit A1 receptors was not inhibited. Preincubation with the affinity label 4-isothiocyanatophenylaminothiocarbonyl-APEC (100 nM) diminished the Bmax for [3H]CGS 21680 binding by 71%, and the Kd was unaffected, suggesting a direct modification of the ligand binding site. Reversal of 4-isothiocyanatophenylaminothiocarbonyl-APEC inhibition of [3H]CGS 21680 binding with hydroxylamine suggested that the site of modification by the isothiocyanate is a cysteine residue. A bromoacetyl derivative of APEC was ineffective as an affinity label at submicromolar concentrations.
authors.affiliation: Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: K A
authors.initials: KA
authors.lastname: Jacobson
authors.forename: G L
authors.initials: GL
authors.lastname: Stiles
authors.forename: X D
authors.initials: XD
authors.lastname: Ji
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide); D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004336:Drug Antagonism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008969:Molecular Sequence Data; D010627:Phenethylamines / Q000378:metabolism; D058914:Purinergic Antagonists; D011817:Rabbits; D011869:Radioligand Assay; D011983:Receptors, Purinergic / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013806:Theophylline / Q000494:pharmacology; D014970:Xanthines / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D006898:Hydroxylamines; D010627:Phenethylamines; D058914:Purinergic Antagonists; D011983:Receptors, Purinergic; D014970:Xanthines; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D019811:Hydroxylamine; D019830:Adenosine-5'-(N-ethylcarboxamide); C051869:8-(4-((2-aminoethyl)aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine; D013806:Theophylline; D000241:Adenosine; D004047:Diethyl Pyrocarbonate