Histamine H4 receptor–RGS fusion proteins expressed in Sf9 insect cells: A sensitive and reliable approach for the functional characterization of histamine H4 receptor ligands

Schneider, Erich H.; Seifert, Roland

doi:10.1016/j.bcp.2009.05.015

Cited by 24 publications

(17 citation statements)

References 41 publications

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“…However, these ‘negative’ findings do not preclude functional selectivity of hH 3 R ligands because several variables such as distinct β x γ y complexes or different RGS proteins have yet to be examined. Lastly, for hH 4 R, the most effective analysis system is represented by a fusion protein of hH 4 R with RGS19, also referred to as GAIP, in coexpression with Gia 2 and Gβ 1 γ 2 [76]. …”

Section: Analysis Of Human Histamine Receptor Subtypes In Spodoptera mentioning

confidence: 99%

“…Fusion of hH 4 R to GAIP increases signal-to-noise ratio without changing G protein specificity [76]…”

Section: Figurementioning

confidence: 99%

“…A weak interaction with insect cell Gα q is unmasked in hH 4 R–GAIP fusion protein (system S2 ), compared with system S1 [76]…”

Section: Figurementioning

confidence: 99%

“…hH 4 R–GAIPfusion protein shows increased agonist signal-to-noise ratio in the GTPase assay, compared with hH 4 R coexpressed with Gα i2 and Gβ 1 γ 2 [76]…”

Section: Figurementioning

confidence: 99%

“…High constitutive activity leads to low signal-to-noise ratio (histamine-induced relative signal in GTPase assays ~25% above baseline in S1 [65] and ~50% in S2 [76])…”

Section: Figurementioning

confidence: 99%

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Molecular and cellular analysis of human histamine receptor subtypes

Seifert¹,

Straßer²,

Schneider³

et al. 2013

Trends in Pharmacological Sciences

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151

139

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The human histamine receptors hH1R and hH2R constitute important drug targets, and hH3R and hH4R have substantial potential in this area. Considering the species-specificity of pharmacology of HxR orthologs, it is important to analyze hHxRs. Here,we summarize current knowledge of hHxRs endogenously expressed in human cells and hHxRs recombinantly expressed in mammalian and insect cells. We present the advantages and disadvantages of the various systems. We also discuss problems associated with the use of hHxR antibodies, an issue of general relevance for G-protein-coupled receptors (GPCRs). There is much greater overlap in activity of ‘selective’ ligands for other hHxRs than the cognate receptor subtype than generally appreciated. Studies with native and recombinant systems support the concept of ligand-specific receptor conformations, encompassing agonists and antagonists. It is emerging that for characterization of hHxR ligands, one cannot rely on a single test system and a single parameter. Rather, multiple systems and parameters have to be studied. Although such studies are time-consuming and expensive, ultimately, they will increase drug safety and efficacy.

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Section: Analysis Of Human Histamine Receptor Subtypes In Spodoptera mentioning

confidence: 99%

“…Fusion of hH 4 R to GAIP increases signal-to-noise ratio without changing G protein specificity [76]…”

Section: Figurementioning

confidence: 99%

“…A weak interaction with insect cell Gα q is unmasked in hH 4 R–GAIP fusion protein (system S2 ), compared with system S1 [76]…”

Section: Figurementioning

confidence: 99%

“…hH 4 R–GAIPfusion protein shows increased agonist signal-to-noise ratio in the GTPase assay, compared with hH 4 R coexpressed with Gα i2 and Gβ 1 γ 2 [76]…”

Section: Figurementioning

confidence: 99%

“…High constitutive activity leads to low signal-to-noise ratio (histamine-induced relative signal in GTPase assays ~25% above baseline in S1 [65] and ~50% in S2 [76])…”

Section: Figurementioning

confidence: 99%

See 3 more Smart Citations

Molecular and cellular analysis of human histamine receptor subtypes

Seifert¹,

Straßer²,

Schneider³

et al. 2013

Trends in Pharmacological Sciences

Self Cite

151

139

View full text Add to dashboard Cite

show abstract

Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutants in the Sf9 Cell Expression System

Schneider

Seifert

2017

Handbook of Experimental Pharmacology

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A large problem of histamine receptor research is data heterogeneity. Various experimental approaches, the complex signaling pathways of mammalian cells, and the use of different species orthologues render it difficult to compare and interpret the published results. Thus, the four human histamine receptor subtypes were analyzed side-by-side in the Sf9 insect cell expression system, using radioligand binding assays as well as functional readouts proximal to the receptor activation event (steady-state GTPase assays and [S]GTPγS assays). The human HR was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hHR and insect cell Gα. By contrast, functional expression of the hHR required the generation of an hHR-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hHR to the long (Gsα) or short (Gsα) splice variant of Gα resulted in comparable constitutive hHR activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hHR/hHR and their guinea pig orthologues gpHR/gpHR. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hHR with Gα, Gα, Gα, and Gα in Sf9 cells revealed high constitutive activity and comparable interaction efficiency with all G protein isoforms. A comparison of various cations (Li, Na, K) and anions (Cl, Br, I) revealed that anions with large radii most efficiently stabilize the inactive hHR state. Potential sodium binding sites in the hHR protein were analyzed by expressing specific hHR mutants in Sf9 cells. In contrast to the hHR, the hHR preferentially couples to co-expressed Gα in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hHR shows structural instability and adopts a G protein-independent high-affinity state. A detailed characterization of affinity and activity of a series of hHR antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hHR. In summary, the Sf9 cell system permitted a successful side-by-side comparison of all four human histamine receptor subtypes. This chapter summarizes the results of pharmacological as well as medicinal chemistry/molecular modeling approaches and demonstrates that these data are not only important for a deeper understanding of HR pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general.

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Establishment of recombinant cannabinoid receptor assays and characterization of several natural and synthetic ligands

Geiger

Nickl

Schneider

et al. 2010

Naunyn-Schmied Arch Pharmacol

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Cannabinoid receptors (CBR) are important drug targets for the treatment of various inflammatory, metabolic and neurological diseases. Therefore, sensitive test systems for the assessment of ligands are needed. In this study, a steady-state GTPase assay for human CBR subtypes 1 and 2 was developed to characterize the pharmacological property of ligands at a very proximal point of the signal transduction cascade. Establishing these in vitro test sytems, we studied cell or tissue membranes heterogenously or endogenously expressing CBR, such as CBR-infected Human Embryonic Kidney (HEK) 293 cells, rat cerebellum and spleen cells. The lack of effects in the GTPase assay and in [(35)S]GTPgammaS binding experiments in these expression system, directed us to use Spodoptera frugiperda (Sf9) cells. Co-expressing CBR, different Galpha-subunits, Gbetagamma heterodimer, and RGS (Regulator of G-protein signaling)-proteins in Sf9 cell membranes greatly improved the sensitivity of the assay, with highest GTPase activation in the CBR + Galpha(i2) + Gbeta(1)gamma(2) + RGS4 system. We examined exogenous and endogenous standard ligands as well as secondary metabolites as Delta(9)-tetrahydrocannabinol (Delta(9)-THC), dodeca-2E,4E-dienoic acid isobutylamide, an alkylamide from Echinacea purpurea, and an E. purpurea hexane extract according their agonistic and antagonistic properties. The suitability of the assay for screening procedures was also proven by detecting the activity of Delta(9)-THC in a matrix of other less active compounds (Delta(9)-THC-free Cannabis sativa extract). In conclusion, we have developed highly sensitive test systems for the analysis of CBR ligands.

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Histamine H4 receptor–RGS fusion proteins expressed in Sf9 insect cells: A sensitive and reliable approach for the functional characterization of histamine H4 receptor ligands

Abstract: H receptor-RGS fusion proteins expressed in Sf9 insect cells: A sensitive and reliable approach for the functional characterization of histamine H receptor ligands.

Cited by 24 publications

References 41 publications

Molecular and cellular analysis of human histamine receptor subtypes

Molecular and cellular analysis of human histamine receptor subtypes

Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutants in the Sf9 Cell Expression System

Establishment of recombinant cannabinoid receptor assays and characterization of several natural and synthetic ligands

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