Activation of the α1b-Adrenergic Receptor Is Initiated by Disruption of an Interhelical Salt Bridge Constraint

Porter, James E.; Hwa, John; Perez, Dianne M.

doi:10.1074/jbc.271.45.28318

Cited by 119 publications

(121 citation statements)

References 18 publications

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“…However, this result is consistent with previous mutagenesis of Asp-125 in the ␣ 1b -AR, in which antagonist binding was not affected as greatly as agonist binding (5). Modeling of the other antagonists produced similar results, with one end of the molecule interacting with Phe-308 and Phe-312, whereas the other end was orientated more toward TM6 than TM4 -5.…”

Section: Discussionsupporting

confidence: 90%

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

Waugh¹,

Gaivin

Zuscik

et al. 2001

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 90%

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

Waugh¹,

Gaivin

Zuscik

et al. 2001

Journal of Biological Chemistry

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“…However, binding of agonists activates all other GPCRs. In the amine receptors, agonist binding disrupts the salt bridge between TM3 and TM7, similar to opsins [10]. In the peptide-hormone receptors salt-bridge disruption is not common, but displacement of residues in TM3 leads to activation [11,12].…”

Section: Family Smentioning

confidence: 99%

Activation of G-protein-coupled receptors: a common molecular mechanism

Karnik

Gogonea

Patil

et al. 2003

Trends in Endocrinology and Metabolism

172

149

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“…Charged residues in other domains within GPCRs have been shown to be important for signaling also. For example, Perez and coworkers (31,32) presented evidence that disruption of a salt-bridge between an Asp in TMH3 and a Lys in TMH7 may be involved in ␣ 1b -adrenergic receptor activation. Donohue et al (33) developed support for the idea that a saltbridge between an Asp at the extracellular loop 1/TMH2 boundary and an Arg at the extracellular loop 3/TMH7 boundary constrained the gastrin-releasing peptide receptor in conformation that is needed for coupling to G proteins.…”

Section: Viral G Protein-coupled Receptor Activationmentioning

confidence: 99%

Charged Residues at the Intracellular Boundary of Transmembrane Helices 2 and 3 Independently Affect Constitutive Activity of Kaposi's Sarcoma-associated Herpesvirus G Protein-coupled Receptor

Ho¹,

Ganeshalingam²,

Rosenhouse‐Dantsker³

et al. 2001

Journal of Biological Chemistry

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Because charged residues at the intracellular ends of transmembrane helix (TMH) 2 and TMH3 of G protein-coupled receptors (GPCRs) affect signaling, we performed mutational analysis of these residues in the constitutively signaling Kaposi's sarcoma-associated herpesvirus GPCR (KSHV-GPCR). KSHV-GPCR contains the amino acid sequence Val-Arg-Tyr rather than the Asp/Glu-Arg-Tyr ((D/ E)RY) motif at the intracellular end of TMH3. Mutation of Arg-143 to Ala (R143A) or Gln (R143Q) abolished constitutive signaling whereas R143K exhibited 50% of the basal activity of KSHV-GPCR. R143A was not stimulated by agonist, whereas R143Q was stimulated by growth-related oncogene-␣, and R143K, similar to KSHV-GPCR, was stimulated further. These findings show that Arg-143 is critical for signal generation in KSHV-GPCR. In other GPCRs, Arg in this position may act as a signaling switch by movement of its sidechain from a hydrophilic pocket in the TMH bundle to a position outside the bundle. In rhodopsin, the Arg of Glu-Arg-Tyr interacts with the adjacent Asp to constrain Arg outside the TMH bundle. V142D was 70% more active than KSHV-GPCR, suggesting that an Arg residue, which is constrained outside the bundle by interacting with Asp-142, leads to a receptor that signals more actively. Because the usually conserved Asp in the middle of TMH2 is not present in KSHV-GPCR, we tested whether Asp-83 at the intracellular end of TMH2 was involved in signaling. D83N and D83A were 110 and 190% more active than KSHV-GPCR, respectively. The double mutant D83A/V142D was 510% more active than KSHV-GPCR. That is, cosubstitutions of Asp-83 by Ala and Val-142 by Asp act synergistically to increase basal signaling. A model of KSHV-GPCR predicts that Arg-143 interacts with residues in the TMH bundle and that the sidechain of Asp-83 does not interact with Arg-143. These data are consistent with the hypothesis that Arg-143 and Asp-83 independently affect the signaling activity of KSHV-GPCR.

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Activation of the α1b-Adrenergic Receptor Is Initiated by Disruption of an Interhelical Salt Bridge Constraint

Cited by 119 publications

References 18 publications

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

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

Activation of G-protein-coupled receptors: a common molecular mechanism

Charged Residues at the Intracellular Boundary of Transmembrane Helices 2 and 3 Independently Affect Constitutive Activity of Kaposi's Sarcoma-associated Herpesvirus G Protein-coupled Receptor

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