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 3HN6-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 3HN6-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.
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