Phe-308 and Phe-312 in Transmembrane Domain 7 Are Major Sites of α1-Adrenergic Receptor Antagonist Binding

Abstract

Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there are few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-protein coupled receptor. It has been speculated that antagonist binding is quite diverse depending upon the chemical structure of the antagonist, which can be quite different from agonists. We now report the identification of two phenylalanine residues in transmembrane domain 7 of the ␣ 1a -adrenergic receptor (Phe-312 and Phe-308) that are a major site of antagonist affinity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4 -1200-fold) for the antagonists prazosin, WB4101, BMY7378, (؉) niguldipine, and 5-methylurapidil, with no changes in affinity for phenethylamine-type agonists such as epinephrine, methoxamine, or phenylephrine. Interestingly, both residues are involved in the binding of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and may be more antagonist-like, which may explain their partial agonist properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed toward transmembrane domain 7.Adrenergic receptors (ARs) 1 (␣ 1a , ␣ 1b , ␣ 1d , ␣ 2a , ␣ 2b , ␣ 2c , ␤ 1 , ␤ 2 , and ␤ 3 ) are members of the G-protein coupled receptor superfamily of membrane proteins that mediate the actions of the endogenous catecholamines, the neurotransmitter norepinephrine, and the hormone epinephrine. Similar to rhodopsin, these proteins are proposed to traverse the plasma membrane in a series of seven transmembrane-spanning ␣-helical domains linked by three intracellular and three extracellular loops (1). In accordance with the observation that the greatest structural conservation is localized to the transmembrane helical domains of the receptor, the catecholamine binding pocket is also localized to these regions. Mutagenesis studies in our laboratory and others have identified that the endogenous agonist in biogenic amine receptors is stabilized in the binding pocket by ionic, hydrogen bond, and aromatic/hydrophobic interactions involving residues on TM3, TM5, and TM6, although there are various modulations of these interactions between the families (2-5). We have also recently shown that ␣ 1 -ARs and perhaps some other biogenic amine receptors have additional aromatic/ hydrophobic interactions with the endogenous agonist to residues in TM4 and TM5 (6).However, our knowledge of how antagonists bind to the adrenergic receptor family is limited. Mutagenesis studies in our laboratory have identified that the subtype selectivity of two ␣ 1a -AR antagonis...

Characterization and functional divergence of the α1-adrenoceptor gene family: insights from rainbow trout (Oncorhynchus mykiss)

“…This study was unable to differentiate between these two possibilities. With the use of isolated rings from the ABA and EBA, both BMY-7378 and RS-17053 increased the EC 50 value for norepinephrine-elicited (37). These two phenylalanine residues are found in the fish, but only Ile 178 exists within the second extracellular loop (Fig.…”

The Crystallographic Model of Rhodopsin and Its Use in Studies of Other G Protein–Coupled Receptors

“…In the case of α 1 -adrenergic receptor (122), an antagonist binding site has been shown to involve residues F7. 35[308] and F7.39 [312].…”

“…Hydropathy plots based on the sequences of human melanocortin receptors were used to map mutations onto the rhodopsin model to ensure that the sites were in the transmembrane region (125). Residues D3.25 [122] and D3.29 [126] in helix III and F6. 51[261] and H6.54 [264] in helix VI decreased the binding of the melanocyte-stimulating hormone.…”

Mutational Analysis of the .ALPHA.1a-Adrenergic Receptor Binding Pocket of Antagonists by Radioligand Binding Assay

“…8,9,19) To verify the modeling data and the involvement of corresponding amino acids (Asp106 and Gln167) in the human a 1a -AR, these amino acids were mutated to alanine and phenylalanine. (Table 2).…”

“…7) Two conserved phenylalanine residues on transmembrane domain (TMD) VII (Phe308 and Phe312) are involved in p-p stacking with almost all a 1a -AR antagonists. 8) Previous molecular modeling studies of a 1a -AR in our laboratory predicted that the protonated amino group of prazosin, tamsulosin and KMD-3213 make ionic interactions with the carboxylate side chain of Asp106 in TMD III, which is highly conserved in all GPCR-binding amine ligands. 9) Docking study and subsequent molecular simulation studies with a 1a -AR antagonist/receptor complex by other investigators have also revealed the same aspartate (Asp106) to be interacting with doxazosin (a quinazoline based antagonist, with a similar basic structure of prazosin) and tamsulosin.…”