Photoaffinity cross-linking of a radioiodinated probe, 125I-A55453, into alpha 1-adrenergic receptors

KE Dickinson, LM Leeb-Lundberg, SL Heald, JE Wikberg, JF DeBernardis, MG Caron and RJ Lefkowitz

We have synthesized and characterized a high-affinity alpha 1- adrenergic receptor probe, 4-amino-6,7-dimethoxy-2[4'- [5"(3"'-125I- iodo-4"'-aminophenyl)pentanoyl]-1'-piperazinyl] quinazoline (125I- A55453). This ligand binds reversibly to rat hepatic plasma membranes with high affinity (KD = 77 +/- 6 pM), and it labels the same number of "specific" prazosin-competable sites as the alpha 1-adrenergic receptor- selective radioligand [125I] iodo-2-[beta-(4-hydroxyphenyl)- ethylaminomethyl]tetralone. Specific binding is stereoselective and competed for by alpha-adrenergic agents with an alpha 1-adrenergic receptor specificity. 125I-A55453 can be covalently photoincorporated into peptides of rat hepatic and splenic membranes using the bifunctional photoactive cross-linker, N-succinimidyl-6- (4'-azido-2'- nitrophenylamino)hexanoate. Following photolysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled hepatic membranes reveals a major "specifically" labeled peptide of Mr = 82,000 (+/- 1,000) with minor peptides at Mr = 50,000 (+/- 500), and 40,000 (+/- 300). Covalent incorporation of 125I-A55453 into the Mr = 82,000 peptide is inhibited by adrenergic drugs with an alpha 1-adrenergic receptor specificity. Labeled splenic membranes demonstrate a broad band of photoincorporated radioactivity centered at Mr = 82,000, and covalent incorporation into this peptide is also attenuated with an alpha 1-adrenergic receptor specificity. This new high-affinity radioiodinated probe has features which should make it useful for the molecular characterization of alpha 1-adrenergic receptors in tissues.

Volume 26, Issue 2, pp. 187-195, 09/01/1984
Copyright © 1984 by American Society for Pharmacology and Experimental Therapeutics