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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 ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). 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 [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX 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 [3H]PIA and [3H]DPCPX 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.
Highlights
From the Pharmakologisches Institut der Universität Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, Federal Repubüc of Germany
From the selective protection of [3 8] PIA and [3 H]DPCPX binding from inactivation, it is concluded that agonists and antagonists oocupy different domains at the binding site
In order to obtain further information about the molecular structure of the receptor protein, we used the method of chemical modification with reagents, which are specific for amino acids or functional groups as a useful tool to probe the recognition site of receptors and other proteins
Summary
From the Pharmakologisches Institut der Universität Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, Federal Repubüc of Germany. Chemical modification of amino acid residues was used to probe the Iigand recognition site of A1 adenosine receptors from rat brain membranes. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding This effect is explained by modification of tbe inhibitory guanine nucleotide binding protein. A1 adenosine receptors from different tissues and species have been well characterized by pharmacological and biochemical methods (for review, see Ref. 1) They are coupled to adenylate cyclase via the inhibitory guanine nucleotide binding protein Gh while A2 receptors are coupled in a stimulatory manner via Ga (2, 3). In order to obtain further information about the molecular structure of the receptor protein, we used the method of chemical modification with reagents, which are specific for amino acids or functional groups as a useful tool to probe the recognition site of receptors and other proteins. We propose a possible explanation for the Iack of hydrophilic ligands with high affinity for this receptor subtype
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