Phenoxybenzamine Binding Reveals the Helical Orientation of the Third Transmembrane Domain of Adrenergic Receptors

Abstract

In the adrenergic receptor subfamily, the seven ␣-helical transmembrane (TM) 1 domains form a crevice for the recognition and binding of ligands (1). The amino acid sequences are highly conserved within the seven hydrophobic TM domains of the three human ␣ 2 -adrenoreceptor (␣ 2 -AR) subtypes (ϳ 75% amino acid identity) (2-5). The ␣ 2 -AR subtypes also share significant structural identity within the TM domains with other members of the adrenoreceptor family (2, 6). For example, of the 182 amino acids comprising the TM domains of the human ␣ 2A -AR, about 40% are identical with the human ␤ 2 -AR. The conserved regions are known to contain structural determinants responsible for recognizing and binding the endogenous hormones/neurotransmitters adrenaline and noradrenaline and other receptor ligands (2, 7-9).Phenoxybenzamine (PB) is an irreversible, subtype-nonselective ␣-AR antagonist. PB has been used as a pharmacological tool to study ␣-AR subpopulations in tissue preparations. PB was also the first ␣-AR antagonist to be therapeutically evaluated in humans. It produces long lasting ␣-AR blockade and reduces blood pressure, but its clinical use was limited by severe side effects (10 -12). Although the pharmacology of PB has been studied quite extensively, the molecular basis of its interaction with ␣-ARs has not been examined in detail. It is known that ␤-haloalkylamines, such as PB, cyclize in aqueous solution to form an unstable aziridinium ion, which can bind to target proteins with a strong ionic bond. The aziridinium ion then opens to create a reactive intermediate, with the consequence that a covalent bond between the drug molecule and the binding site can be formed. Side chains of amino acid residues that can be alkylated by haloalkylamines include -SH, -OH, ϭNH, and -COOH (13). Of the susceptible amino acid residues, cysteine (-SH) is the most reactive (14).The helical arrangement of the TM domains indicated by receptor modeling (15,16), in conjunction with analysis of sequence alignments (see Fig. 1), suggested to us an interaction between PB and the TM3 region of the ␣-ARs. The amino acid sequence alignment of TM3 of adrenergic receptors presented in Fig. 1 is validated by the position of the conserved aspartate residue (D 3.32 according to the nomenclature of Ballesteros and Weinstein (17) or position 113 in the ␣ 2A -AR), known to be crucial for binding the charged nitrogen present in adrenergic phenethylamine ligands (7, 9). According to our hypothesis, the reactive aziridinium derivative of PB forms a covalent bond with C 3.36 in TM3 of the ␣ 2A -, ␣ 2B -and ␣ 2C -AR (Fig. 2) (corresponding to amino acid residues 117, 96 and 135, respectively). Also the three ␣ 1 -AR subtypes have a cysteine in this position, but the three ␤-AR subtypes have a valine residue in its place (Fig. 1). To test our hypothesis, we determined the irreversible binding of PB to the three human ␣ 2 -AR subtypes and constructed and tested an ␣ 2A -AR mutant lacking the Cys 117 (Cys 117 was substituted with valine; ␣ 2A -C117V...

Homology Modelling and Docking Studies of Human α2-Adrenergic Receptor Subtypes

“…F (3.35) [18,21,22] was projecting away from binding site and hence may not have any role in binding. This observation is in agreement with Frang et al [60]. This group proved, by experimental studies, that F (3.35) in α2a is not exposed in the binding pocket and thus not accessible for Phenoxybenzene and other receptor ligands.…”

“…This group proved, by experimental studies, that F (3.35) in α2a is not exposed in the binding pocket and thus not accessible for Phenoxybenzene and other receptor ligands. However, the orientation of C (3.36), important in ligand binding and earlier predicted by Frang et al [60], was towards binding site proving its involvement in ligand binding. This observation supports experimental finding [60] and theoretical finding [22] hence proving that our models are suitable for further studies.…”

Model structures of α-2 adrenoceptors in complex with automatically docked antagonist ligands raise the possibility of interactions dissimilar from agonist ligands

“…Five ligands were enough to select a suitable model for human 2A -AR and human 2B -AR: a suitably large cavity was found and all ligands were well clustered. Seven additional ligands were then docked to the selected 2A -AR and 2B -AR models: BAM1303, prazosin, spiroxatrine, abanoquil, doxazosin, dibozane, and phenoxybenzamine (phenoxybenzamine forms a covalent bond with C117(3.36), an added constraint on its mode of binding (Frang et al, 2001)). These seven ligands were found to dock to the same location (top scoring solutions) as the previous set of Wve ligands.…”

“…Nonetheless, in 2 -ARs, studies with alkylating reagents targeted to speciWc residues at the proposed binding site Salminen et al, 1999;Frang et al, 2001), by sitedirected mutagenesis (Suryanarayana et al, 1991;Wang et al, 1991), and by competitive experiments where labeled antagonists are displaced by other antagonists and agonist ligands (Nyrönen et al, 2001;Peltonen et al, 2003) suggest that antagonists bind to the same cavity as agonists, as is the case for 1 -ARs and -ARs (reviewed in Shi and Javitch, 2002). An antagonist-receptor ion pair is commonly predicted for biogenic amine receptors based on mutants at D3.32 (see Shi and Javitch, 2002 for a review).…”

Phenoxybenzamine and Benextramine, but Not 4-Diphenylacetoxy-N-[2-chloroethyl]piperidine Hydrochloride, Display Irreversible Noncompetitive Antagonism at G Protein-Coupled Receptors

“…Phenoxybenzamine is known to irreversibly bind to ␣-adrenergic, H 1 -histamine, and muscarinic acetylcholine (mACh) receptors (Timmermans et al, 1985;Eglen et al, 1994;Amobi and Smith, 1995;Giardinà et al, 1995Giardinà et al, , 2002Van der Graaf and Danhof, 1997;Ruffolo and Hieble, 1999;Van der Graaf and Stam, 1999;Frang et al, 2001). Benextramine has been known to irreversibly bind to ␣ 2 -adrenoceptors (Melchiorre, 1981;Belleau et al, 1982b;Lew and Angus, 1984;Hieble et al, 1985;Timmermans et al, 1985;Taouis et al, 1987;McKernan et al, 1988;Brink et al, 2000;Umland et al, 2001), 5-HT 1A -serotonergic receptors (Stanton and Beer, 1997), H 2 -histaminergic receptors (Belleau et al, 1982a), and neuropeptide Y receptors (Melchiorre et al, 1994).…”