Photoaffinity label for the alpha 1-adrenergic receptor: synthesis and effects on membrane and affinity-purified receptors.

Abstract

An azide analog, 2-[4-(4-azidobenzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline (CP59,430), of the highly selective a1-adrenergic receptor antagonist prazosin was synthesized and its effects on rat hepatic membrane and affinity-purified a1-adrenergic receptor preparations were examined. CP59,430 behaved as a competitive antagonist before photolysis. When the membrane or purified preparations pretreated with CP59,430 were irradiated with UV While considerable progress has been made with the isolation, purification, and molecular characterization of a number of hormone and drug receptors (1-3), studies of the a-adrenergic receptor have been hampered by the lack of suitable techniques for the isolation and characterization of the minute quantities of this receptor present in most cells. In recent years, photoaffinity labels for several hormone and drug receptors have been developed and have aided greatly in the identification and biophysical characterization of these receptors (4-7). Such photoaffinity probes offer the advantage over affinity labels ofbeing completely reversible until photoactivated. Reliable affinity constants can thus be obtained, and the labels can be characterized prior to their photolysis and concomitant irreversible binding. We have previously described the development and application of an affinity matrix synthesized with an analog of the highly selective a1-adrenergic receptor antagonist prazosin, which permitted purification of the a1-adrenergic receptor (8-10). To further aid in the molecular characterization of this receptor, a specific photoaffinity label has been developed. We report here on the synthesis of this compound, 2-[4-(4-azidobenzoyl)piperazin-1-yl]-4 -amino-6, 7-dimethoxyquinazoline (CP59,430), which is also an analog of prazosin (2-[4-(2-furoyl)piperazin-1-yl]4amino-6,7-dimethoxyquinazoline), and on its characterization as a photoaffinity label for the membranebound and purified a1-adrenergic receptor. MATERIALS AND METHODS Materials. [3H]Yohimbine was purchased from New England Nuclear and p-aminobenzoic acid from Sigma. The sources of all other reagents and compounds were as described (8).Synthesis of CP59,430. A solution of 2-(1-piperazin-1-yl)-4-amino-6,7-dimethoxyquinazoline (838 mg, 2.90 mmol) prepared as described (11) 2102The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

LNP 906, the first high‐affinity photoaffinity ligand selective for I1 imidazoline receptors

“…We report here that LNP 906 exhibited high affinity for I 1 ‐binding sites of PC12 membrane preparations. Photoaffinity labelling has been useful in the identification of α 1 ‐adrenoreceptors ( Hess et al ., 1983 ; Leeb‐Lundberg et al ., 1984 ), α 2 ‐adrenoreceptors ( Regan et al ., 1985 ), β ‐adrenoreceptors ( Lavin et al ., 1981 ; Rashidbaigi & Ruoho, 1981 ) or I 2 receptors ( Lanier et al ., 1993 ; Dontenwill et al ., 1997 ; Coates et al ., 2000 ). Irreversible ligands allow complete knockout of one particular site.…”

Photoaffinity labeling of the .alpha.1-adrenergic receptor using an 125I-labeled aryl azide analog of prazosin

“…Both groups labeled rat hepatic membranes with [3H]phenoxybenzamine. However, this agent lacks selectivity for the a, receptor as it also binds to other monoaminergic receptors (Hess et al, 1983).…”

“…Image-intensifying screens were from Du Pont. The 0006-2960/84/0423-3765S01.50/0 © 1984 American Chemical Society source of all other reagents and compounds were as previously described (Graham et al, 1982a,b;Hess et al, 1983).…”

“…Binding Studies. Typical binding experiments employed 25-50 pg of protein per assay in 100 pL of buffer B. Membranes were incubated with shaking, in test tubes containing 25 pL of [125I]CP63,789, [125I]CP65,526, or [3H]prazosin and either 25 pL of buffer B or 25 pL of competing ligand. After 30 min at room temperature, the reaction mixture was washed with 3X4 mL aliquots of buffer B onto glass 24-mm filters and bound ligand separated by vacuum filtration as previously described (Hess et al, 1983). Filters were counted directly for 125I in a 7 spectrometer (Micromedic) at 80% efficiency or dissolved in 10 mL of Hydrofluor scintillation fluid for the 3H samples and counted in a liquid scintillation spectrometer (Packard) with a counting efficiency of 50%.…”

The alpha-1 Adrenergic Receptors

“…However, the subsequent discovery of alpha-2 adrenergic receptors located postsynaptically in vascular smooth muscle made it necessary to define receptor subtypes based not on anatomical location or function, but rather on a pharmacologic basis in which the affinity for a well-defined series of agonists and antagonists is used for classification (Langer, 1974;Berthelsen and Pettinger, 1977;Wikberg, 1979). Although many laboratories are directing efforts toward isolating and characterizing, on a molecular level, the actual chemical constituents of the alpha-adrenergic receptors in order to define them chemically and differentiate their structures (Venter et al, 1984;Dickinson et al, 1984;Seidman et al, 1984;Hess et al, 1983;Regan et al, 1982Regan et al, , 1984Regan et al, , 1985, this work is still in its infancy, and we are at present left with an indirect chemical definition of the alpha-adrenergic receptor subtypes based on structure-activity relationships. Through the discovery of molecules that interact selectively with the alpha-1 and alpha-2 adrenergic receptor subtypes, we have been able to define the receptors that mediate many important physiologic events.…”