Abstract
Chemical modification of amino acid residues was used to probe the
ligand recognition site of A1 adenosine receptors from rat brain
membranes. The effect of treatment with group-specific reagents on
agonist and antagonist radioligand binding was investigated. The
histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss
of binding of the agonist R-N6-3H phenylisopropyladenosine (3HPIA),
which could be prevented in part by agonists, but not by antagonists.
DEP treatment induced also a loss of binding of the antagonist 3H8-cyclopentyl-1,3-dipropylxanthine
(3HDPCPX). Antagonists protected A1 receptors from this inactivation
while agonists did not. This result provided evidence for the existence
of at least 2 different histidine residues involved in ligand binding.
Consistent with a modification of the binding site, DEP did not alter
the affinity of 3HDPCPX, but reduced receptor number. From the
selective protection of 3H PIA and 3HDPCPX binding from inactivation,
it is concluded that agonists and antagonists occupy different domains
at the binding site. Sulfhydryl modifying reagents did not influence
antagonist binding, but inhibited agonist binding. This effect is
explained by modification of the inhibitory guanine nucleotide binding
protein. Pyridoxal 5-phosphate inactivated both 3HPIA and 3HDPCPX
binding, but the receptors could not be protected from inactivation
by ligands. Therefore, no amino group seems to be located at the
ligand binding site. In addition, it was shown that no further amino
acids with polar side chains are present. The absence of hydrophilic
amino acids from the recognition site of the receptor apart from
histidine suggests an explanation for the lack of hydrophilic ligands
with high affinity for A1 receptors.
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