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

Tetranitromethane (TNM) is a reagent which reacts with the tyrosine and cysteine residues of proteins. Chemical modification of partially purified human platelet α₂-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 α₂-adrenoceptor activity caused by TNM could be prevented if α₂-adrenergic ligands were present during exposure of the receptor to TNM. The protection afforded by α₂-adrenergic ligands was does-dependent and showed a positive correlation with the affinity of the ligand for the α₂-adrenoceptor. Prazosin, an α₁-specific antagonist, and propranolol, a β-adrenergic antagonist, did not protect α₂-adrenoceptors against the inactivation caused by TNM. Saturation curve analysis revealed that the decrease in α₂-adrenoceptor activity caused by TNM was due to a decrease in B_max with no change in K_d. α₂-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 α₂-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 α₂-adrenoceptors and may be directly involved with ligand binding to the receptor.