Inverse Agonist Activity at the α<sub>2A</sub>-Adrenergic Receptor

Wade, Susan M.; Lan, Keng Li; Moore, D; Neubig, Richard R.

doi:10.1124/mol.59.3.532

Cited by 50 publications

(58 citation statements)

References 30 publications

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“…Altered affinities of agonists and inverse agonists in constitutively active mutated recep- (Costa and Herz, 1989;Samama et al, 1994;Wurch et al, 1999;Wade et al, 2001). Our binding studies using several H3 receptor agonists and antagonists certainly corroborated with this model.…”

Section: Discussionsupporting

confidence: 74%

Generation and Characterization of Highly Constitutive Active Histamine H3 Receptors

Takahashi

Tokita²,

Kotani³

2003

J Pharmacol Exp Ther

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Constitutive activation of G-protein-coupled receptors is a well recognized phenomenon, and G-protein-coupled receptor antagonists have been found to possess inverse agonist activity. Constitutive activation of histamine H3 receptor is recently documented in in vivo as well as in recombinant receptor systems in vitro. Several H3 antagonists have been shown to act as inverse agonists and such profiles of H3 antagonists have been implicated in their pharmacological functions. Here we report the construction and characterization of a highly constitutive active H3 receptor (MT6), in which the 357 alanine residue was converted to lysine (A357K). We generated a series of mutated H3 receptors and their functions were examined in human embryonic kidney (HEK) 293 cells. Among them, induced mutation at the amino acid 357 position (A357K) showed a dramatically enhanced response to thioperamide-induced cAMP accumulation compared with the cells expressing wildtype (WT) H3 receptors, suggesting that the mutation rendered receptors to high constitutive activity. We further characterized by ligand binding assays using membrane fractions, and K i values of imetit (agonist) and proxyfan (partial agonist) against the MT6 receptors were lower compared with those observed in WT H3 receptors. In contrast, H3 antagonists (thioperamide, ciproxifan, and GT2016) with inverse agonism displayed increased K i values against the MT6 receptors (2.5-to 5.8-fold), demonstrating more a prominent effect of inverse agonists to the constitutive active receptor. Taken together, these data suggested that A357K mutation in the H3 receptor increased the population of active state receptors that preferably binds to agonists than inverse agonists, which could be termed as a constitutively active mutant of H3 receptor.

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Section: Discussionsupporting

confidence: 74%

Generation and Characterization of Highly Constitutive Active Histamine H3 Receptors

Takahashi

Tokita²,

Kotani³

2003

J Pharmacol Exp Ther

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“…Previous studies have also failed to observe differences in the binding affinity of inverse agonists between wild-type and CAM receptors (Kjelsberg et al, 1992;Ren et al, 1993;Ford et al, 2002). Theoretical analysis predicts that even when 50% of the total receptor population is present in the active state, a shift of only 2-fold might be observed in the binding affinity of an inverse agonist at a CAM receptor (Wade et al, 2001;Strange, 2002); therefore, it is possible that the differences in the magnitude of constitutive activity between wildtype and CAM receptors are insufficient for differences in antagonist affinities to be detected in radioligand binding assays. However, it remains unclear why darifenacin (and to a lesser extent oxybutynin) exhibits such a reduced affinity for the CAM receptor (6-fold).…”

Section: Discussionmentioning

confidence: 98%

“…According to the extended ternary complex model, inverse agonists may exert their effects via a selectively higher affinity for the inactive (R i ) than for the active (R a ) receptor species and/or by reducing the affinity of the ligandbound receptor for its cognate G-protein (Samama et al, 1993;Strange, 2002). Thus, any perturbation of the system in favor of R a and/or R a G will result in a reduction in the apparent binding affinity of an inverse agonist for the receptor population (Costa and Herz, 1989;Samama et al, 1993;Huang et al, 1998;Wade et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Constitutive Activity and Inverse Agonism at the M₂Muscarinic Acetylcholine Receptor

Nelson¹,

Nahorski²,

Challiss³

2005

J Pharmacol Exp Ther

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Introduction of a single-point mutation (Asn to Tyr) at position 410 at the junction between transmembrane domain 6 and the third extracellular loop of the human M 2 muscarinic acetylcholine (mACh) receptor generated a mutant receptor (N410Y) that possesses many of the hallmark features of a constitutively active mutant receptor. These included enhanced agonist binding affinity and potency, in addition to agonist-independent accumulation of [ 3 H]inositol phosphates in cells coexpressing the chimeric G␣ qi5 protein and the N410Y mutant M 2 mACh receptor. Constitutive activity was sensitive to inhibition by a range of muscarinic ligands, including those used clinically in the management of overactive bladder (oxybutynin, tolterodine, and darifenacin), indicating that these ligands behave as inverse agonists at the M 2 mACh receptor. Long-term (24-h) treatment of Chinese hamster ovary cells expressing the N410Y mutant M 2 mACh receptor with certain mACh receptor inverse agonists (atropine, darifenacin, and pirenzepine) elicited a concentration-dependent up-regulation of cell surface receptor expression. However, not all ligands possessing negative efficacy in the [ 3 H]inositol phosphate accumulation assays were capable of significantly up-regulating receptor expression, perhaps indicating a spectrum of negative efficacies among ligands traditionally defined as mACh receptor antagonists. Finally, structurally distinct agonists exhibited differences in their relative potencies for the activation of G␣ i/o versus G␣ s , consistent with agonist-directed trafficking of signaling at the N410Y mutant, but not at the wild-type M 2 mACh receptor. This indicates that the N410Y mutation of the M 2 mACh receptor alters receptor-G-protein coupling in an agonist-dependent manner, in addition to generating a constitutively active receptor phenotype.A crucial development in our understanding of G-proteincoupled receptor (GPCR) function has been the identification of the ability of receptors to activate their cognate G-proteins in the absence of an agonist (Costa and Herz, 1989). Thus, certain ligands (termed inverse agonists and previously characterized as competitive antagonists) can inhibit agonist-independent receptor activity (Costa and Herz, 1989). Subsequent research has identified significant agonist-independent (constitutive) activity at a wide variety of both endogenously and recombinantly expressed GPCRs (for review, see Seifert and Wenzel-Seifert, 2002).One of the most powerful tools used by researchers in this area has been the development of GPCRs harboring specific mutations known to enhance the agonist-independent coupling of receptor and G-protein [so-called constitutively active mutant (CAM) receptors] (Seifert and Wenzel-Seifert, 2002). Mutations in a number of well conserved domains, including the D/ERY motif at the intracellular interface of the third transmembrane domain (TM3) and the BBXXB motif (where B is Arg or Lys) toward the C-terminal end of the third intracellular loop, have been reported to ...

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“…Combining expression of human a 2A -receptors (particularly with a mutation at Thr 373 ) with rat G á0 protein gives rise to a constitutively active receptor that tonically suppresses production of cyclic AMP (87,113,116). Under these rather artificial circumstances, RX 821002, in common with yohimbine/rauwolscine, usually behaves as an inverse agonist at á 2A -adrenoceptors (87,113). More pertinently, a similar result has been obtained with native á 2A -adrenoceptors in rat brain slices treated with 100 ìM GTP (78).…”

Section: Rx 821002 As An Inverse Agonist At á 2 -Adrenoceptorsmentioning

confidence: 99%

RX 821002 as a Tool for Physiological Investigation of α₂‐Adrenoceptors

Clarke

Harris

2002

CNS Drug Reviews

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RX 821002 is the 2-methoxy congener of idazoxan. In binding and tissue studies it behaves as a selective antagonist of á 2 -adrenoceptors, with at least 5 times greater affinity for these receptors than any other binding site. It does not select between the different types of á 2 -receptor. Although this drug probably has no future as a therapeutic agent, it remains a good probe for physiological activity at á 2 -adrenoceptors in animal experiments. A particularly useful feature of this compound is its lack of binding at I 1 and I 2 imidazoline receptors. However, it has relatively high affinity for 5-HT 1A receptors (at which it acts as an antagonist) and a tendency to behave as an inverse agonist at á 2A -adrenoceptors in some cell culture systems. These potential drawbacks may be overcome by careful design of experiments, and the greater selectivity of RX 821002 renders it much superior to yohimbine or idazoxan as a tool for probing physiological actions at á 2 -receptors. It can be compared favorably with other selective antagonists such as atipamezole.In physiological studies, RX 821002 augments norepinephrine release in the frontal cortex and increases drinking behavior in rat. In rabbit, intrathecal administration of this drug enhances somatic and autonomic motor outflows, showing that tonic adrenergic descending inhibition of withdrawal reflexes and sympathetic pre-ganglionic neurons is strong in this species. The potentiation of reflexes may be considered a pro-nociceptive action. In the same model, RX 821002 antagonizes the inhibitory effects of the ì opioid fentanyl, indicating that exogenous opioids synergize with endogenously released norepinephrine in the spinal cord. Thus, the careful use of RX 821002 has revealed several aspects of the physiological activity of á 2 -adrenoceptors in rabbit spinal cord and rat brain. We recommend that RX 821002 and/or compounds with similar selectivity for á 2 -adrenoceptors (atipamezole, MK-912, RS-79948) should be used in preference to yohimbine or idazoxan in all future studies of this type.

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Inverse Agonist Activity at the α_2A-Adrenergic Receptor

Cited by 50 publications

References 30 publications

Generation and Characterization of Highly Constitutive Active Histamine H3 Receptors

Generation and Characterization of Highly Constitutive Active Histamine H3 Receptors

Constitutive Activity and Inverse Agonism at the M₂Muscarinic Acetylcholine Receptor

RX 821002 as a Tool for Physiological Investigation of α₂‐Adrenoceptors

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Inverse Agonist Activity at the α2A-Adrenergic Receptor

Cited by 50 publications

References 30 publications

Generation and Characterization of Highly Constitutive Active Histamine H3 Receptors

Generation and Characterization of Highly Constitutive Active Histamine H3 Receptors

Constitutive Activity and Inverse Agonism at the M2Muscarinic Acetylcholine Receptor

RX 821002 as a Tool for Physiological Investigation of α2‐Adrenoceptors

Contact Info

Product

Resources

About

Inverse Agonist Activity at the α_2A-Adrenergic Receptor

Constitutive Activity and Inverse Agonism at the M₂Muscarinic Acetylcholine Receptor

RX 821002 as a Tool for Physiological Investigation of α₂‐Adrenoceptors