Identification of the Subunit Structure of Rat Pineal Adrenergic Receptors by Photoaffinity Labeling

Abstract

The adrenergic receptors of rat pineal gland were investigated using radiolabeled ligand binding and photoaffinity labeling techniques. 125I-2-[.beta.-(4-hydroxyphenyl)ethylaminomethyl]tetralone (125I-HEAT) and 125I-cyanopindolol (125I-CYP) labeled specific sites on rat pineal gland membranes with equilibrium dissociation constants (KD) of 48 (.+-. 5) pM and 30 (.+-. 5) pM, respectively. Binding site maxima were 481 (.+-. 63) and 1,020 (.+-. 85) fmol/mg protein. The sites labeled by 125I-HEAT had the pharmacological characteristics of .alpha.1-adrenergic receptors. 125I-CYP labeled .beta.-adrenergic receptors were characterized as a homogenous population of .beta.1-adrenergic receptors. The .alpha.1- and .beta.1-adrenergic receptors were covalently labeled with the specific photoaffinity probes 4-amino-6,7-dimethoxy-2-{4-[5-(4-azido-3-(125I]iodophenyl) pentanoyl]-1-piperazinyl}quinazoline (125I-APDQ) and 125I-p-azidobenzylcarazolol (125I-pABC). 125I-APDQ labeled an .alpha.1-adrenergic receptor peptide of Mr = 74,000 (.+-. 4,000), which was similar to peptides labeled in rat cerebral cortex, liver, and spleen. 125I-pABC labeled a single .beta.1-adrenergic receptor peptide with a Mr = 42,000 (.+-. 1,500), which differed from the 60-65,000 peptide commonly seen in mammalian tissues. Possible reasons for these differences are discussed.