Chemical modification of α2-adrenoceptors : Possible role for tyrosine in the ligand binding site

Abstract

Tetranitromethane (TNM) is a reagent which reacts with the tyrosine and cysteine residues of proteins. Chemical modification of partially purified human platelet α 2-adrenoceptors with TNM resulted in an irreversible loss of binding activity. Typically, an 80–90% decrease in binding activity occurred with a 60-min exposure to 320 μM TNM. The loss of α 2-adrenoceptor activity caused by TNM could be prevented if α 2-adrenergic ligands were present during exposure of the receptor to TNM. The protection afforded by α 2-adrenergic ligands was does-dependent and showed a positive correlation with the affinity of the ligand for the α 2-adrenoceptor. Prazosin, an α 1-specific antagonist, and propranolol, a β-adrenergic antagonist, did not protect α 2-adrenoceptors against the inactivation caused by TNM. Saturation curve analysis revealed that the decrease in α 2-adrenoceptor activity caused by TNM was due to a decrease in B max with no change in K d. α 2-Adrenoceptors were also inactivated with the sulfhydryl-specific reagent phenylmercuric chloride (PMC). The receptor inactivation caused by PMC could be reversed completely by subsequent treatment with dithiothreitol. Treatment of α 2-adrenoceptors with combinations of TNM and PMC showed that the receptor inactivation caused by TNM was most likely due to an interaction with tyrosine residues. These results indicate that tyrosine residues have a function in the conformational stability of α 2-adrenoceptors and may be directly involved with ligand binding to the receptor.