Mepyramine, a Histamine H1 Receptor Inverse Agonist, Binds Preferentially to a G Protein-coupled Form of the Receptor and Sequesters G Protein

Fitzsimons, Carlos P.; Monczor, Federico; Fernández, Natalia Cristina; Shayo, Carina; Davio, Carlos

doi:10.1074/jbc.m400738200

Cited by 54 publications

(59 citation statements)

References 42 publications

(48 reference statements)

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“…We have previously described that certain inverse agonists acting on histaminergic receptors interfere with the signaling of other receptors that share common G␣ subunits (14,15). This interference is thought to be caused by the ligand-induced stabilization of a G protein-coupled form of the receptor, which is unable to evoke a response.…”

Section: Discussionmentioning

confidence: 99%

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Expression of a G Protein-coupled Receptor (GPCR) Leads to Attenuation of Signaling by Other GPCRs

Tubio

Fernández

Fitzsimons

et al. 2010

Journal of Biological Chemistry

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The idea of G protein-coupled receptors (GPCRs) coupling to G protein solely in their active form was abolished when it was found that certain ligands induce a G protein-coupled but inactive receptor form. This receptor form interferes with signaling of other receptors by sequestering G protein. However, the spontaneous existence of this receptor species has never been established. The aim of the present work was to evaluate the existence of the spontaneous conformation of the receptor inactively coupled to G protein able to interfere with the response of other GPCRs. According to the law of mass action, receptor overexpression should lead to increased amounts of all spontaneously occurring species. Based on this, we generated Chinese hamster ovary (CHO-K1)-derived cell lines expressing various amounts of the human histamine H2 receptor. In these systems, the signaling of other endogenously and transiently expressed GPCRs was attenuated proportionally to human H2 receptor expression levels. G protein transfection specifically reverted this attenuation, strongly suggesting hijacking of the G protein from a common pool. Similar attenuation effects were observed when the ␤ 2 -adrenergic receptor was overexpressed, suggesting that this is a more general phenomenon. Moreover, in human mammary MDA-MB-231 cells, a consistent increase in the response of other GPCRs was observed when endogenous expression of ␤ 2 -adrenergic receptor was knocked down using specific small interfering RNAs. Our findings show that GPCRs may interact with the signaling of other receptors by modulating the availability of the G protein and suggest the existence of GPCR spontaneous coupling to G proteins in an inactive form. G protein-coupled receptors (GPCRs)2 form a large and functionally diverse superfamily of proteins that transduce signals across cell membranes. Although much is known about structural features of GPCRs involved in ligand recognition and G protein binding, the actual mechanism underlying GPCR signaling remains unclear.Traditionally, agonist occupancy of GPCRs is believed to result in a conformational change in the receptor, leading to activation of G proteins (1). However, in genetically engineered systems where receptors can be expressed at high densities, Costa and Herz (2) noted that high levels of receptor expression uncovered the existence of a population of spontaneously (unliganded) active receptors, resulting in an elevated basal response in the system.The histamine H2 receptor (H2R) is an extensively characterized member of the GPCR family, which in most systems couples to G s proteins to activate adenylyl cyclase (3-6). Compared with other GPCRs, the H2R is unique in that the wildtype receptor possesses a remarkably high degree of constitutive activity. With a receptor density of 300 fmol/mg protein, constitutive H2 receptor activity could be detected in Chinese hamster ovary (CHO-K1) cells (7).The notion that GPCRs also signal without an external chemical trigger, i.e. in a constitutive or spontaneous manner, res...

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Section: Discussionmentioning

confidence: 99%

“…Inverse agonists may act by binding to an inactive, G protein-coupled form of the receptor, decreasing basal activity of the specific GPCR of interest but also in some cases the activity of other GPCRs that signal through the same G protein, via a proposed "molecular kidnapping mechanism" (13)(14)(15).…”

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confidence: 99%

Expression of a G Protein-coupled Receptor (GPCR) Leads to Attenuation of Signaling by Other GPCRs

Tubio

Fernández

Fitzsimons

et al. 2010

Journal of Biological Chemistry

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“…Stabilization of an inactive CB 1 receptor-G i/o complex by the inverse agonist SR141716A inhibited insulin-and insulin-like growth factor 1-mediated activation of mitogenactivated protein kinase through G i/o (Bouaboula et al, 1997). Likewise, purinergic receptor-stimulated G q/11 activation and subsequent Ca 2ϩ mobilization is attenuated in the presence of the guinea pig histamine H 1 receptor inverse agonist mepyramine, presumably by stabilization of an H 1 receptor-G q/11 complex (Fitzsimons et al, 2004). Taken together, these findings provide experimental evidence for a ligand-occupied inactive G protein-coupled state of the receptor.…”

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confidence: 91%

Activation of Adenylyl Cyclase by Endogenous G_s-Coupled Receptors in Human Embryonic Kidney 293 Cells Is Attenuated by 5-HT₇Receptor Expression

Andressen¹,

Norum²,

Levy³

et al. 2005

Mol Pharmacol

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Human 5-hydroxytryptamine 7 (5-HT 7 ) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with G i/o and G q/11 are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT 7 receptor expression was sufficient to limit signaling of endogenously expressed G s -coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT 4(b) and 5-HT 7(a,b,d) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous G s -coupled ␤-adrenergic (␤AR) and prostanoid EP (EPR) receptors. ␤AR-and EPR-stimulated AC activity was attenuated by 5-HT 7 receptor expression in both membrane preparations and intact HEK293 cells. ␤AR-and EPR-stimulated AC activity was unaffected by expression of the G s -coupled 5-HT 4 receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) ␤AR-and EPR-stimulated AC activity was not restored to control values when G␣ s was overexpressed; and 2) ␤ 1 AR and ␤ 2 AR activation of G␣ s was unaffected by the expression of 5-HT 7 receptors. In addition, overexpression of AC isoforms was unable to rescue ␤AR-and EPR-stimulated AC activity. Therefore, 5-HT 7 receptors probably limit access and/or impede activation of AC by ␤AR and EP receptors. Although the 5-HT 7 receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.Serotonin (5-hydroxytryptamine, 5-HT) mediates its diverse physiological effects through at least 14 different receptor subtypes, of which 13 belong to the G protein-coupled receptor family (Hoyer et al., 1994). Among the human 5-HT receptors, three different subtypes, 5-HT 4 , 5-HT 6 , and 5-HT 7 , are coupled to G s and at least the 5-HT 4 and 5-HT 7 receptors are expressed as several different functional splice variants (Gerald et al., 1995;Heidmann et al., 1997). The functional significance of 5-HT 7 splice variants, which differ only in the carboxyl terminus (Heidmann et al., 1997), remains unknown (Krobert et al., 2001Krobert and Levy, 2002), whereas among the 5-HT 4 splice variants, constitutive activation of AC is dependent on the different carboxyl termini (Bockaert et al., 2004). We have shown previously that the 5-HT 4(b) and 5-HT 7(a) signaling properties differ fundamentally. The potency of 5-HT to stimulate AC increased with increasing receptor density in clones expressing 5-HT 4(b) but not 5-HT 7(a) receptors, even though 5-HT-stimulated AC activity in clones expressing 5-HT 7(a) receptors had reached asymptotic levels (Bruheim et al., 2003). This indicates that...

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“…Total inositol phosphate production was assessed as previously described (Fitzsimons et al, 2004). Briefly, cells were incubated with myo-[ 3 H]-inositol (5 mCi/ml) in DMEM with calf serum and cultured for 6 hours.…”

Section: H]-inositol Phosphate Productionmentioning

confidence: 99%

Cross-Desensitization and Cointernalization of H1 and H2 Histamine Receptors Reveal New Insights into Histamine Signal Integration

et al. 2013

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G protein-coupled receptor signaling does not result from sequential activation of a linear pathway of proteins/enzymes, but rather from complex interactions of multiple, branched signaling routes, i.e., signaling networks. In this work we present an exhaustive study of the cross-talk between H1 and H2 histamine receptors (H1R and H2R) in U937 cells and Chinese hamster ovary-transfected cells. By desensitization assays we demonstrated the existence of a crossdesensitization between both receptors independent of protein kinase A or C. H1R-agonist stimulation inhibited cell proliferation and induced apoptosis in U937 cells following treatment of 48 hours. H1R-induced antiproliferative and apoptotic response was inhibited by an H2R agonist suggesting that the cross-talk between both receptors modifies their function. Binding and confocal microscopy studies revealed cointernalization of both receptors upon treatment with the agonists. To evaluate potential heterodimerization of the receptors, sensitized emission fluorescence resonance energy transfer experiments were performed in human embryonic kidney 293T cells using H1R-cyan fluorescent protein and H2R-yellow fluorescent protein. To our knowledge these findings may represent the first demonstration of agonistinduced heterodimerization of the H1R and H2R. In addition, we also show that the inhibition of the internalization process did not prevent receptor crossdesensitization, which was mediated by G protein-coupled receptor kinase 2. Our study provides new insights into the complex signaling network mediated by histamine and further knowledge for the rational use of its ligands.

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Mepyramine, a Histamine H1 Receptor Inverse Agonist, Binds Preferentially to a G Protein-coupled Form of the Receptor and Sequesters G Protein

Cited by 54 publications

References 42 publications

Expression of a G Protein-coupled Receptor (GPCR) Leads to Attenuation of Signaling by Other GPCRs

Expression of a G Protein-coupled Receptor (GPCR) Leads to Attenuation of Signaling by Other GPCRs

Activation of Adenylyl Cyclase by Endogenous G_s-Coupled Receptors in Human Embryonic Kidney 293 Cells Is Attenuated by 5-HT₇Receptor Expression

Cross-Desensitization and Cointernalization of H1 and H2 Histamine Receptors Reveal New Insights into Histamine Signal Integration

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Mepyramine, a Histamine H1 Receptor Inverse Agonist, Binds Preferentially to a G Protein-coupled Form of the Receptor and Sequesters G Protein

Cited by 54 publications

References 42 publications

Expression of a G Protein-coupled Receptor (GPCR) Leads to Attenuation of Signaling by Other GPCRs

Expression of a G Protein-coupled Receptor (GPCR) Leads to Attenuation of Signaling by Other GPCRs

Activation of Adenylyl Cyclase by Endogenous Gs-Coupled Receptors in Human Embryonic Kidney 293 Cells Is Attenuated by 5-HT7Receptor Expression

Cross-Desensitization and Cointernalization of H1 and H2 Histamine Receptors Reveal New Insights into Histamine Signal Integration

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Activation of Adenylyl Cyclase by Endogenous G_s-Coupled Receptors in Human Embryonic Kidney 293 Cells Is Attenuated by 5-HT₇Receptor Expression