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

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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.