Photoaffinity labeling of the porcine brain α₂-adrenergic receptor using a radioiodinated arylazide derivative of rauwolscine: Identification of the hormone-binding subunit

A functionalized derivative of the α₂-selective antagonist rauwolscine formed the basis for a photoaffinity adduct that has allowed identification of the hormone-binding subunit of the brain α₂-adrenergic receptor protein. Rauwolscine carboxylate underwent reaction with 4-N-t-butyloxycarbonylaminoaniline, leading to the synthesis of rauwolscine 4-aminophenyl carboxamide (Rau-AmPC). Rau-AmPC was radioiodinated and converted to the arylazide derivative, 17α-hydroxy-20α-yohimban-16β-[N-(4-azido-3-[¹²⁵I]iodo) phenyl] carboxamide (¹²⁵I-Rau-AzPC), via a diazonium salt intermediate. The characterization of ¹²⁵I-Rau-AzPC as a photolabile probe employed α₂-adrenergic receptors, which were first solubilized from porcine brain membranes and partially purified by affinity chromatography utilizing a yohimbine-agarose affinity matrix. In the partially purified receptor preparation incubated with ¹²⁵I-Rau-AzPC, photolysis resulted in covalent labeling of a major (M(r), 62,000) peptide as determined by NaDodSO₄/PAGE and autogradiography. Labeling of this peptide was inhibited by the α₂-selective antagonist, yohimbine, and the non-subtype-selective α-antagonist, phentolamine, but not by the α₁-antagonist, prazosin, or the β-receptor antagonist, (-)-alprenolol. The α-adrenergic agonist epinephrine also inhibited labeling in a stereoselective manner. These data indicate that the photolabeled M(r) 62,000 peptide is the hormone-binding subunit of the α₂-adrenergic receptor protein. The availability of this radioiodinated photoaffinity probe for the α₂-adrenergic receptor should facilitate further structural and biophysical characterization of the receptor protein.