Amino acids of the <i>α</i><sub>1B</sub>‐adrenergic receptor involved in agonist binding: differences in docking catecholamines to receptor subtypes

A, Cavalli; Fanelli, Francesca; Taddei, Carlo; Benedetti, Pier G. De; Cotecchia, Susanna

doi:10.1016/s0014-5793(96)01286-0

Cited by 74 publications

(73 citation statements)

References 13 publications

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“…Furthermore, Asp125 of the a 1B -AR is the homologous to the aspartate on TMDIII of b 2 -and a 2A -AR and Dopamin1-receptor (D 1 -R) involved in the interaction with the amino group of catecholamines. 16) In the present study, mutation of Asp125 to alanine totally impaired the ability of the a 1b -AR to bind both agonists and antagonists. Although prazosin is a non-selective drug for a 1b -AR and after mutation we could not determine any detectable data for mutant Asp125Ala.…”

Section: Discussionsupporting

confidence: 43%

Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of .ALPHA.1b-Adrenergic Receptor Antagonist Binding

Takahashi

Hossain

Ahmed

et al. 2007

Biological & Pharmaceutical Bulletin

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The a 1 -adrenergic receptors (a 1 -ARs) belong to the super family of G-protein coupled receptors (GPCRs) that transmit a variety of signals across cell membrane. The a 1 -ARs are important for many cardiovascular diseases such as atherosclerosis and hypertension, 1,2) restenosis after coronary dilation, 3) myocardial hypertrophy and cardiac arrhythmia. 4,5) There are three subtypes of a 1 -ARs (a 1a -, a 1b -, and a 1c -AR) and a 1b -AR is one of them. Recent experiments suggested that a 1b -ARs are involved in cardiac hypertrophy and neurodegeneration in a 1b transgenic mice. 4,6)The a 1b -ARs are structurally characterized by seven transmembrane helices connected by alternating extracellular and intracellular loops. Whereas ligand binding involves extracellular portion of the receptor, the intracellular regions mediate the interaction of the receptor with G proteins as well as other signaling and regulatory proteins. Stimulation of the a 1b -ARs by catecholamines activates proteins of the Gq family, resulting in the production of inositol phosphates via the activation of phospholipase C (PLC). 7)It is of now-a-days interest to give more emphasis for identification of more selective ligands to fully elucidate the functional role of different subtypes. Molecular modeling and site-directed mutagenesis studies are helpful to determine the activity of currently existing agents and the development of completely novel therapeutic agents. 8)Prazosin, the prototype of a family of agents that contains a piperazinyl quinazoline nucleus, is a very potent and selective a 1 -adrenergic antagonist. Its affinity for a 1 receptor is about 1000-fold greater than that for a 2 receptors. Interestingly, the drug also is a relatively potent inhibitor of cyclic nucleotide phosphodiesterases, and it was originally synthesized for this purpose.9) Prazosin appears to depress baroreflex function in hypertensive patients.10) Based on radio-ligand binding studies, it was shown that prazosin has relatively lower affinity towards a 1a -and a 1b -AR receptor than a 1c -AR receptors. 11)However, the molecular mechanism by which prazosin binds to a 1b -AR receptors remains undetermined. Molecular modeling of prazosin to a 1b -AR receptors predicted that made strong electrostatic interactions towards Asp125 in transmembrane helix 3 and hydrogen bonding with Thr130 in transmembrane helix 3 of a 1b -AR receptor.11) We, therefore, constructed and characterized alanine mutations of these amino acid residues by site-directed mutagenesis. Our findings are consistent with the molecular modeling data that these mutations are important for prazosin binding which have been evident from the markedly decrease in affinity of prazosin for the mutant receptors. MATERIALS AND METHODSDrugs Niguldipine(ϩ), tamsulosin, prazosin, and ketanserin were obtained from RBI (Research Biochemical Incorporated, Natick, MA, U.S.A.).DNA Constructs A cDNA clone encoding human a 1b -AR receptor in the plasmid vector (pCR3) was introduced into the DH5a strain of E. coli by th...

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Section: Discussionsupporting

confidence: 43%

Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of .ALPHA.1b-Adrenergic Receptor Antagonist Binding

Takahashi

Hossain

Ahmed

et al. 2007

Biological & Pharmaceutical Bulletin

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“…1), and this residue has been shown to play a role in ligand binding and receptor activation in the ␣ 1A , ␣ 2A , and ␣ 1B adrenergic (5)(6)(7)(8) and the dopamine D 1 , D 2 , and D 3 receptors (9 -13). In the rat ␣ 1A AR, in which a Ser is absent at 5.43, Ser-188 5.42 (aligned with ␤ 2 AR Ser-203 5.42 ) interacts with the mOH of catecholamines, and it is this H bond that is critical for ligand binding and receptor activation (5).…”

mentioning

confidence: 99%

The Forgotten Serine

Liapakis¹,

Ballesteros²,

Papachristou³

et al. 2000

Journal of Biological Chemistry

173

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The ␤ 2 -adrenergic receptor (␤ 2 AR) 1 has been extensively studied and, along with rhodopsin, has served as a prototype for our understanding of the structure and function of related G-protein-coupled receptors (1, 2). In these receptors, the binding site is formed among the seven-transmembrane segments (TMs) (2) in a water-accessible binding-site crevice. A number of residues inferred to contact bound agonist in the ␤ 2 AR have been identified. Asp113 3.32 (see "Experimental Procedures" for a description of the indexing of residues) in the third TM (TM3) is thought to interact with the protonated amine of catecholamines (3) and is completely conserved in all amine receptors. A cluster of aromatic residues in TM6 that is thought to interact with the aromatic ring of catecholamines is highly conserved as well (2). A classical study by Strader et al. (4) in the ␤ 2 AR demonstrated that two serines in TM5 directly interact with the catechol hydroxyls (OHs) of agonists. Specifically, 5.43 interacts with the meta-hydroxyl (mOH) and Ser-207 5.46 with the para-hydroxyl (pOH) (4). These interactions were shown to contribute to the affinity, potency, and efficacy of various catecholamine agonists. A potential role of 5.42 in binding and activation, however, was obscured by the lack of expression of the mutant ␤ 2 AR in which 5.42 was mutated to Ala (4). Nonetheless, because the catechol ring contains two hydroxyls and because two serines were inferred to hydrogen bond to these two hydroxyls, Ser-203 5.42 has been generally assumed not to play a significant role in agonist binding to and activation of the wild-type ␤ 2 AR.Curiously, ␤ 2 AR Ser-203 5.42 is completely conserved in all catecholamine receptors (Fig. 1), and this residue has been shown to play a role in ligand binding and receptor activation in the ␣ 1A , ␣ 2A , and ␣ 1B adrenergic (5-8) ) interacts with the mOH of catecholamines, and it is this H bond that is critical for ligand binding and receptor activation (5).Using the substituted cysteine accessibility method (14, 15), we found that in the dopamine D 2 receptor each of the three aligned serines ) are accessible in the binding-site crevice (16). This is consistent with experimental results in the D 2 receptor implicating each of the three serines in the binding of various ligands, although different serines were found to be more or less critical with different ligands (11,12). More recently we have applied the substituted cysteine accessibility method to the aligned portion of TM5 in the human ␤ 2 AR, 2 and we were surprised to observe that substitution of by Cys, unlike the published mutation of this Ser to Ala in the hamster ␤ 2 AR (4), did not impair expression of the receptor but instead lowered its affinity for

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“…Furthermore, Asp125 of the a 1b -AR is the homologous to the aspartate on TMD III of b 2 -and a 2A -AR and dopamine1-receptor involved in the interaction with the amino group of catecholamines. 25) Cavalli and co-workers reported that mutation of Asp125 to alanine totally impaired the ability of the a 1b -AR to bind both agonists and antagonists. This was shown by the fact that Asp125Ala displayed no specific [ 125 I]HEAT binding and no epinephrine-induced stimulation of IP production.…”

Section: Discussionmentioning

confidence: 99%

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

Ahmed

Hossain

Bhuiyan

et al. 2008

Biological & Pharmaceutical Bulletin

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The a 1 -adrenergic receptors (a 1 -AR) belong to Class A of the super family of G protein coupled receptors (GPCRs). They comprise at least three cloned subtypes, a 1a -, a 1b -and a 1d -ARs.1,2) a 1 -ARs are of particular therapeutic interest due to their important roles in the control of blood pressure, in the contraction and growth of smooth muscle and in renal and hepatic metabolism. The a 1a -AR located in large abundance in the prostate is thought to be influential in the condition of benign prostatic hyperplasia (BPH), a considerable health problem for aging man.3) The development of high affinity a 1a -AR antagonists is of paramount importance for the treatment of BPH.To rationally design selective drugs, an understanding of subtype differences in the ligand-binding pockets would be invaluable. In this paper the attention is focused on a 1a -AR due to its role in neurotransmission processes, in pharmacological treatment of BPH. 4,5) Indeed, many researchers are involved in mutagenesis studies on the a 1a -AR, which revealed the key residues of both agonist and antagonist binding sites. 6) In contrast to the wealth of information about the agonist binding pocket of adrenergic receptors, the knowledge about the binding mode of antagonists is much more limited. Three consecutive residues set on second extracellular loop (Gln177, Ile178 and Asn179) are involved in interaction with some a 1a -AR selective antagonists. 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. 10)Pedretti and colleagues published a homology model of a 1a -AR where both the agonist and antagonist interacted with Asp106. 11)KMD-3213 is a selective a 1a -AR antagonist.12,13) a 1a -AR selective antagonist would be very useful for the treatment of BPH without any untoward effect on the blood pressure. By homology modeling of a 1a -AR with KMD-3213, we identified a key amino acid residue responsible for higher binding affinity to a 1a -AR. The study revealed that carboxamide nitrogen of KMD-3213 forms an ionic bond with the carboxylate side chain of Gln167 in TMD IV.9) Prazosin and tamsulosin are proposed to interact with the side chain of Asp106 in TMD III.The objectives of this study are both the construction of mutant a 1a -ARs such as Asp106Ala, double mutants Asp106Ala-Gln167Phe and Gln167Phe by site-directed mutagenesis and the analysis of their interac...

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Amino acids of the α_1B‐adrenergic receptor involved in agonist binding: differences in docking catecholamines to receptor subtypes

Cited by 74 publications

References 13 publications

Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of .ALPHA.1b-Adrenergic Receptor Antagonist Binding

Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of .ALPHA.1b-Adrenergic Receptor Antagonist Binding

The Forgotten Serine

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

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Amino acids of the α1B‐adrenergic receptor involved in agonist binding: differences in docking catecholamines to receptor subtypes

Cited by 74 publications

References 13 publications

Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of .ALPHA.1b-Adrenergic Receptor Antagonist Binding

Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of .ALPHA.1b-Adrenergic Receptor Antagonist Binding

The Forgotten Serine

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

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Product

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Amino acids of the α_1B‐adrenergic receptor involved in agonist binding: differences in docking catecholamines to receptor subtypes