title: Photoaffinity labeling of A1-adenosine receptors.
abstract: The ligand-binding subunit of the A1-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R-N6-phenylisopropyladenosine, R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific ligand for A1-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R-AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A1-subtype. It competes for [3H]N6-phenylisopropyladenosine binding to A1-receptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of [3H]N6-phenylisopropyladenosine binding after extensive washing; the Ki value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity label of high specific radioactivity (125I-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for 125I-AHPIA binding to rat brain membranes with an order of potency characteristic for A1-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of Mr = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A1-subtype. The results indicate that 125I-AHPIA identifies the ligand-binding subunit of the A1-adenosine receptor, which is a peptide with Mr = 35,000.
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism; D000273:Adipose Tissue / Q000378:metabolism; D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D001792:Blood Platelets / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D010660:Phenylisopropyladenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051381:Rats; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D011983:Receptors, Purinergic
chemical_list: D000345:Affinity Labels; D001386:Azides; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D011983:Receptors, Purinergic; D010660:Phenylisopropyladenosine; C049136:2-azido-N(6)-4-hydroxyphenylisopropyladenosine; D000262:Adenylyl Cyclases; D000241:Adenosine