Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking.
A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-[2-[(4- aminophenyl)methylcarbonylamino]ethylaminocarbonyl]- ethyl)phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-[(R)-1-methyl- 2-phenylethyl]adenosine (R-PIA) greater than (+)-N6-[(S)-1-methyl-2- phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors (see above) and is decreased in the presence of 10(-4) M guanosine 5'-[beta, gamma-imido]triphosphate. Photoaffinity crosslinking of the A1 adenosine receptor binding subunit with 125I-labeled 8-[4-[2-(4- aminophenylacetylamino)ethyl]carbonylmethyloxyphenyl]-1,3-di propylxanthine (PAPAXAC) (an A1 selective photoaffinity probe) in the same tissue reveals a 38-kDa peptide that exhibits the appropriate A1 receptor pharmacology. 125I-labeled PAPA-APEC, therefore, has identified the A2 receptor binding subunit as a 45-kDa protein that is unique and distinct from the A1 binding subunit.
Department of Medicine, Duke University Medical Center, Durham, NC 27710.
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Barrington
K A
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Jacobson
A J
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Hutchison
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Williams
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Stiles
D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000097:blood; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003342:Corpus Striatum / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007462:Iodobenzenes / Q000138:chemical synthesis / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D015394:Molecular Structure; D010777:Photochemistry; D011983:Receptors, Purinergic / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D007457:Iodine Radioisotopes; D007462:Iodobenzenes; D046911:Macromolecular Substances; D011983:Receptors, Purinergic; C060851:2-(4-(2-(2-((4-aminophenyl)methylcarbonylamino)ethylaminocarbonyl)ethyl)phenyl)ethylamino-5'-N-ethylcarboxamidoadenosine; D000262:Adenylyl Cyclases; D000241:Adenosine