Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

Journal Article · Fri Sep 01 04:00:00 EDT 1989 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.86.17.6572· OSTI ID:5364458

Barrington, W W; Jacobson, K A; Hutchison, A J; Williams, M; Stiles, G L ^[1]

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1. Duke Univ. Medical Center, Durham, NC (USA)

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 and is decreased in the presence of 10(-4) M guanosine 5'-(beta, gamma-imido)triphosphate.

• OSTI ID: 5364458
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 86:17; ISSN PNASA; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADENOSINE
AFFINITY
AMP
ANIMALS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BLOOD PLATELETS
BODY FLUIDS
CATTLE
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CROSS-LINKING
CYCLASES
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
DOSE-RESPONSE RELATIONSHIPS
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KINETICS
LIGANDS
LYASES
MAMMALS
MAN
MATERIALS
MEMBRANE PROTEINS
MEMBRANES
MOLECULAR STRUCTURE
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
POLYMERIZATION
PRIMATES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
RIBOSIDES
RUMINANTS
VERTEBRATES