Purification and characterization of the human platelet α₂-adrenergic receptor

Human platelet α₂-adrenergic receptors have been purified ~80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall yield starting from the membranes is ~2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of M(r) 64,000. The specific binding activity of the α₂-adrenergic receptor after four chromatographic steps is 14.5 nmol/mg protein. This value is consistent with the expected theoretical specific activity (15.6 nmol/mg) for a protein with a molecular mass of 64,000 daltons if it is assumed that there is one ligand-bind site/receptor molecule. The purified protein can be covalently labeled with the alkylating α-adrenergic ligand, [³H]phenoxybenzamine. This labeling is specific, and it shows that the M(r) 64,000 protein contains the ligand binding site of the α₂-adrenergic receptor. In addition, the competitive binding of ligands to the purified receptor protein shows the proper α₂-adrenergic specificity. The α₂-adrenergic receptor contains an essential sulfhydryl residue. Thus, exposure of the purified receptor to the sulfhydryl-specific reagent, phenylmercuric chloride, resulted in an 80% loss of binding activity. This loss of binding activity was prevented when exposure to phenylmercuric chloride was done in the presence of α₂-adrenergic ligand, and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified α₂-adrenergic receptors was obtained with Staphylococcus aureus V-8 protease, α-chymotrypsin, and papain. In a comparison with purified β₂-adrenergic receptors, no common partial proteolytic products were found.