key: cord-0007848-tt1u6ugy
authors: Schneider, Erich H.; Seifert, Roland
title: Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutants in the Sf9 Cell Expression System
date: 2017-02-24
journal: Histamine and Histamine Receptors in Health and Disease
DOI: 10.1007/164_2016_124
sha: eef81233541c4504a9dc542873a5a8dca4a313e6
doc_id: 7848
cord_uid: tt1u6ugy

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 [(35)S]GTPγS assays). The human H(1)R was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hH(1)R and insect cell Gα(q). By contrast, functional expression of the hH(2)R required the generation of an hH(2)R-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hH(2)R to the long (Gsα(L)) or short (Gsα(S)) splice variant of Gα(s) resulted in comparable constitutive hH(2)R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH(1)R/hH(2)R and their guinea pig orthologues gpH(1)R/gpH(2)R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH(3)R with Gα(i1), Gα(i2), Gα(i3), and Gα(i/o) 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 hH(3)R state. Potential sodium binding sites in the hH(3)R protein were analyzed by expressing specific hH(3)R mutants in Sf9 cells. In contrast to the hH(3)R, the hH(4)R preferentially couples to co-expressed Gα(i2) in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hH(4)R shows structural instability and adopts a G protein-independent high-affinity state. A detailed characterization of affinity and activity of a series of hH(4)R antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hH(4)R. 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 H(x)R pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general.

an hH 2 R-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hH 2 R to the long (Gsα L ) or short (Gsα S ) splice variant of Gα s resulted in comparable constitutive hH 2 R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH 1 R/hH 2 R and their guinea pig orthologues gpH 1 R/ gpH 2 R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH 3 R with Gα i1 , Gα i2 , Gα i3 , and Gα i/o 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 À , Br À , I À ) revealed that anions with large radii most efficiently stabilize the inactive hH 3 R state. Potential sodium binding sites in the hH 3 R protein were analyzed by expressing specific hH 3 R mutants in Sf9 cells. In contrast to the hH 3 R, the hH 4 R preferentially couples to co-expressed Gα i2 in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hH 4 R shows structural instability and adopts a G proteinindependent high-affinity state. A detailed characterization of affinity and activity of a series of hH 4 R antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hH 4 R. 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 H x R pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general. N α -methylhistamine NgpChH 2 R-Gsα S Fusion protein of Gsα S with a chimeric receptor (Nterminus to transmembrane domain 3 from guinea pig H 2 R plus transmembrane domain 4 to C-terminus from human H 2 R) NhCgpH 2 R-Gsα S Fusion protein of Gsα S with a chimeric receptor (Nterminus to transmembrane domain 3 from human H 2 R plus transmembrane domain 4 to C-terminus from guinea pig H 2 R) pEC 50 Negative decadic logarithm of the agonist concentration that causes 50% of the maximum effect pIC 50 Negative decadic logarithm of the antagonist concentration that causes 50% inhibition pK b

Negative decadic logarithm of a dissociation constant determined in a functional assay PKC Protein kinase C pK i Negative decadic logarithm of a dissociation constant determined in a competition binding assay PLC Phospholipase C PTX Pertussis toxin r Rat (prefix) RAMH (R)-α-methylhistamine RGS4

Regulator of G protein signaling 4 SAR Structure-activity relationship S49

Murine lymphoma cell line Sf9, Sf21

Insect cell lines originating from ovarian cells of Spodoptera frugiperda Th1, Th2

Differentially polarized T helper cell subgroups TM Transmembrane helix of a G protein-coupled receptor TMN Tuberomamillary nucleus U373 MG Human astrocytoma cell line V max Maximum enzymatic reaction speed in the presence of saturating substrate concentrations 1 Principles of GPCR Analysis in the Sf9 Cell Expression System

Pharmacological characterization of GPCRs is commonly performed in transfected mammalian cells or in cells that endogenously express the receptor of interest (Kenakin 1996) . There are, however, several problems of mammalian cell systems.

First, mammalian cells normally express various additional GPCRs, which may result in GPCR heteromerization or signaling crosstalk (Breitwieser 2004; Prezeau et al. 2010; Gomes et al. 2016) . For example, signaling crosstalk between GPCRs has been described for the Gα i -coupled GABA B R and the Gα q -coupled mGlu 1A R (Rives et al. 2009 ). Another example is ACKR1 (atypical chemokine receptor 1), which has been shown to functionally antagonize CCR5 by forming ACKR1/CCR5 heterodimers (Chakera et al. 2008) . Second, the presence of other constitutively active receptors may interfere with the analysis of agonist-independent activity of the receptor of interest. For example, the inverse FPR1 agonist cyclosporin H failed to inhibit basal Gα i protein activity in HL-60 cells, indicating that these cells additionally express other constitutively active receptors different from FPR1 (Wenzel- Seifert and Seifert 1993; . Third, promiscuous G protein coupling of GPCRs in the presence of several G protein subtypes may preclude the analysis of GPCR-G protein selectivity (Woehler and Ponimaskin 2009) . Finally, some GPCRs are only expressed at low levels in mammalian cells, rendering it difficult to obtain a sufficiently high signal-to-noise ratio in functional and ligand binding assays. As discussed in a comprehensive review article (Schneider and Seifert 2010c) , the problems listed above are effectively addressed by using the Sf9 cell expression system. Sf9 cells are derived from the Sf21 cell line, which had been originally isolated from the pupal ovarian tissue of the American fall army worm (Spodoptera frugiperda). The protein of interest is expressed by infecting Sf9 cells with baculoviruses encoding the corresponding gene. Although Sf9 cells express Gα i -, Gα q -, and Gα s -like proteins, insect cell Gα i is not activated by mammalian GPCRs. This renders Sf9 cells a functionally "Gα i -free" system and permits the analysis of Gα i -coupled receptors without the necessity of pertussis toxin (PTX)-mediated GPCR/Gα i uncoupling. Also, PTX would not be active in Sf9 cells, because it does not enter the cells (Wenzel-Seifert et al. 1998) . By contrast, uncoupling of Gα i -coupled GPCRs by PTX in mammalian cells is problematic. Despite entering mammalian cells, PTX is not capable of completely inactivating all Gα i proteins (Wenzel-Seifert and Seifert 1990) .

Moreover, Sf9 cells do not express constitutively active GPCRs and therefore provide a low-background environment for the analysis of agonist-independent receptor activity. Furthermore, the highly efficient baculovirus promoters lead to very high expression levels of GPCRs in Sf9 cells. This results in high signal-tonoise ratios in binding assays and allows the purification of receptor protein, e.g. for crystallization purposes. Finally, as explained below, Sf9 cell membranes expressing large amounts of GPCRs and G proteins can be used to study G protein activation in steady-state GTPase assays and experiments with [ 35 S]GTPγS ([ 35 S]labeled guanosine 5 0 -O-[γ-thio]triphosphate).

For the preparation of baculoviruses encoding the gene of interest, several straightforward methods are established. The Sf9 cell studies discussed in this chapter were performed by using the BaculoGold™ kit from Invitrogen. As explained in Fig. 1 , the gene of interest (in this example hH 4 R) is cloned into a pVL1392 baculovirus transfer vector, which is transfected into Sf9 cells together with the missing part of the baculovirus genome (BaculoGold™ DNA).

After that, the full baculovirus genome with the integrated receptor gene is reconstituted in the host cells by homologous recombination. The cell then releases virus particles into the surrounding medium which is harvested and used for further infections. A detailed protocol for the production and maintenance of genetically modified baculoviruses was published in Methods in Enzymology . Numerous examples of the characterization of Gα q -, Gα s -, and Gα icoupled receptors reconstituted in Sf9 insect cells were documented by Schneider and Seifert (2010c) . In this chapter, an in-depth discussion of the pharmacological characterization of histamine receptors in Sf9 cell membranes is provided.

Methods for the Characterization of Histamine Receptors in Sf9 Cell Membranes

The G protein activation cycle (Gilman 1987; Oldham and Hamm 2008) , which is explained in the following, is the basis for the methods used to generate the functional histamine receptor data discussed in this chapter. When histamine binds to the hH 4 R, the receptor protein undergoes a conformational change and interacts with an inactive GDP-bound heterotrimeric G protein ( Fig. 2 step 1 ). This induces GDP release and the formation of the so-called ternary complex, which contains agonist, receptor and guanine-nucleotide-free G protein (Fig. 2 GPCR and G protein promotes GTP binding to the Gα-subunit. This weakens the intermolecular interactions in the G protein and in the ternary complex, breaking the complex up into agonist and GPCR as well as Gα-and Gβγ subunit (Fig. 2, step 3) . After their dissociation from the receptor, the active GTP-loaded Gα subunit and the Gβγ part interact with various effector proteins (Fig. 2, step 4) and induce numerous biochemical processes. Such effects include activation (Gα s ) or inhibition (Gα i ) of membranous adenylyl cyclase (AC), modulation of ion channel activity (Gβγ, Gα i ) or stimulation of phospholipase C (PLC) activity followed by intracellular Ca 2+ mobilization (Gβγ, Gα q ). As long as GTP is bound to Gα, the Gα and Gβγ subunits are active. To terminate signaling, the Gα subunit inactivates itself by its intrinsic GTPase activity, resulting in conversion of the bound GTP to GDP and release of inorganic phosphate (Fig. 2 , step 5). The inactive GDP-bound Gα subunit re-associates with Gβγ and becomes available for another cycle (Fig. 2 , step 6).

High affinity radioligand binding with histamine receptors is performed with radiolabeled agonists, e.g. tritiated histamine ([ 3 H]histamine). Normally, agonists show their highest affinity to the ternary complex (Fig. 2, step 2 ) and stabilize the active receptor conformation. Thus, agonistic radioligands preferentially label the G protein-coupled high-affinity receptor population. When two populations of GPCRs with different G protein coupling states occur simultaneously, the saturation or competition curves with agonistic radioligands may become biphasic, which allows the determination of high-affinity and a low-affinity binding constants. This was, e.g., demonstrated for histamine H 2 R (Houston et al. 2002) as well as for the β 2 -adrenergic receptor (β 2 AR) or the dopamine D 1 R (Gille and Seifert 2003) . By contrast, inverse agonists interact preferentially with the inactive receptor state and therefore show increased affinity to uncoupled GPCRs. Neutral antagonists do not discriminate between active and inactive receptor states and label both receptor conformations with comparable affinity.

For some experiments it may be required to convert GPCRs to their inactive conformation by disrupting receptor-G protein interactions. This is achieved by addition of GTPγS (guanosine 5 0 -O-[γ-thio]triphosphate), which binds to the Gα-subunit like GTP (Fig. 2, step 3 ), but cannot be hydrolyzed by the Gα subunit (Gilman 1987) . Thus, no GDP-loaded G protein is available anymore for the formation of new ternary complexes resulting in uncoupling of the entire GPCR population. This is normally reflected by a dramatic reduction in the binding affinity of agonistic radioligands. A detailed protocol for high-affinity agonist binding assays as well as example data for various receptor/G protein systems is provided in book chapters about GPCR/G protein co-expression and fusion protein systems in Sf9 cell membranes (Schneider and Seifert 2010a, b) .

In steady-state GTPase assays, the intrinsic GTPase activity (Fig. 2 , step 5) of the active GTP-bound Gα-subunit is determined (Gilman 1987; . This is achieved by quantitating radioactive inorganic phosphate released after Gα-mediated hydrolysis of [γ-32 P]GTP. The steady-state GTPase assay represents a very proximal readout of GPCR activation, which directly reflects GPCR-mediated G protein stimulation. By contrast, functional assays analyzing more distal parameters (e.g., Ca 2+ -, cAMP-or reporter gene assays) are often influenced by signal amplification processes, making valid conclusions about the original extent of receptor activation difficult. Technical details of the steady-state GTPase assay were explained in two book chapters about GPCR/G protein co-expression and fusion protein systems in Sf9 cells (Schneider and Seifert 2010a, b) .

Steady-state GTPase assays can be used for the functional characterization of ligands in medicinal chemistry projects. In addition, these assays provide information about the efficacy of receptor-G protein interactions. In Michaelis-Menten kinetics experiments with increasing concentrations of the substrate [γ-32 P]GTP, the K M and V max value of the Gα-GTPase can be determined Seifert 2009, 2010a) . Subtraction of the GTPase activity in the presence of a full inverse agonist from the activity elicited by a full agonist yields the total receptor-regulated GTPase activity (ΔV max ). Dividing the ΔV max value by B max (maximum number of radiolabeled receptor proteins) provides the so-called turnover number, which signifies the number of GTP molecules hydrolyzed per minute, resulting from the activation of a single GPCR protein ).

The [ 35 S]GTPγS binding assay is another method to determine the functional effect of a ligand at a very proximal level of GPCR signal transduction. As depicted in Fig. 2 , GTPγS binds to the activated Gα-subunit instead of GTP, resulting in the dissociation of the ternary complex (Fig. 2, step 3) . Unlike GTP, however, GTPγS cannot be hydrolyzed by the intrinsic GTPase activity of Gα (Gilman 1987) , resulting in an accumulation of GTPγS-bound Gα-subunits. When radiolabeled [ 35 S]GTPγS is used, the amount of activated Gα subunits can be quantitated by scintillation counting, allowing the characterization of Gα activation kinetics (time course of [ 35 S]GTPγS-Gα accumulation) and the determination of agonist-and inverse-agonist modulated Gα activation. When the total ligand-regulated Gα activation (maximum effect of full agonist minus activation level in the presence of a full inverse agonist) is divided by the B max value from radioligand binding, the so-called coupling factor is obtained. Similar to the aforementioned turnover number, the coupling factor provides information about the number of Gα subunits stimulated by a single GPCR protein.

Furthermore, saturation binding experiments with increasing concentrations of [ 35 S] GTPγS yield information about alterations of Gα affinity to [ 35 S]GTPγS under various conditions (e.g., constitutive receptor activity, agonist-or inverse agonist-induced effects). Finally, [ 35 S]GTPγS binding assays are useful to pharmacologically characterize new ligands synthesized during the course of medicinal chemistry projects. A detailed experimental protocol of [ 35 S]GTPγS binding assays as well as an explanation of how to analyze and interpret the data is provided in comprehensive book chapters about the characterization of GPCR/Gα co-expression and fusion protein systems in Sf9 cell membranes (Schneider and Seifert 2010a, b) .

Mammalian GPCRs and G protein Gα and Gβγ subunits can be readily coexpressed in the baculovirus/Sf9 cell system yielding useful systems for the pharmacological characterization of GPCR ligands and receptor-G protein interactions. However, sometimes co-expression systems produce only insufficient GPCRmediated Gα activation (Seifert et al. 1998a; Gille and Seifert 2003) . Specifically, Gα s proteins rapidly dissociate from the plasma membrane (Yu and Rasenick 2002) and therefore cannot be efficiently activated by a co-expressed GPCR. This problem is solved by constructing GPCR-Gα fusion proteins (Fig. 3 ) that guarantee close proximity of receptor and G protein.

This approach was successfully used for the pharmacological characterization of Gα s -coupled receptors like the β 2 AR (Bertin et al. 1994; Seifert et al. 1998a) or the histamine H 2 R (Wenzel- Seifert et al. 2001) . GPCR-Gα fusion proteins of β 2 AR, FPR1 or dopamine D 1 R allowed a detailed examination of Gα-isoform specificity of these receptors (Wenzel-Seifert et al. 1999; Wenzel-Seifert and Seifert 2000; Gille and Seifert 2003) . GPCR-Gα fusion proteins are also useful controls to exclude activation of Sf9 cell G proteins by a specific mammalian GPCR. Normally, the turnover number from steady-state GTPase assays or the coupling factor from [ 35 S]GTPγS binding experiments should be around unity in fusion protein systems, corresponding to linear signaling. A coupling factor >1 in a GPCR-Gα fusion protein system, however, indicates additional activation of insect cell proteins.

The fusion protein approach can also be applied to generate GPCR-RGS fusion proteins. RGS proteins (regulators of G protein signaling) activate the intrinsic GTPase activity of Gα proteins. GPCR-RGS fusion proteins bring the RGS protein in close proximity to receptor and G protein. This may enhance signal intensity in steady-state GTPase assays. The first GPCR-RGS fusion proteins were constructed in 2003 (Bahia et al. 2003) . A detailed discussion of various aspects of co-expression and fusion protein systems was provided in Methods in Enzymology (Schneider and Seifert 2010a, b) .

The biogenic amine histamine is formed by histidine decarboxylase (HDC)mediated decarboxylation of the precursor amino acid histidine. Histamine is stored in granula of mast cells and basophils and occurs in enterochromaffin-like cells of the stomach (Panula et al. 2015) . Moreover, by means of a highly sensitive HPLC-MS/MS-based detection method, histamine was identified in lymph nodes and thymus of C57Bl/6 and Balb/c mice (Zimmermann et al. 2011 ). In the central nervous system (CNS), histamine occurs as a neurotransmitter. It is synthesized in histaminergic neurons that emerge from the tuberomamillary nucleus (TMN) in the posterior hypothalamus and spread to numerous regions throughout the brain (Schneider et al. 2014a, b; Panula et al. 2015) . The distribution of histamine in the body indicates its most important functions, namely the regulation of inflammatory/allergic reactions, stimulation of gastric acid secretion and neurotransmission. Most of the histamine effects are mediated by four G protein-coupled receptors, H 1 R, H 2 R, H 3 R, and H 4 R Panula et al. 2015) . Additionally histamine acts on some non-histaminergic targets, e.g. at NMDA The GPCR is N-terminally tagged with a FLAG epitope, which allows detection by an anti-FLAG antibody, and connected to the N-terminus of a Gα-subunit via a His6 linker. Gα proteins are anchored in the plasma membrane via their acylation sites. The interaction between Gα s proteins and the plasma membrane is only weak in co-expression systems, but can be significantly improved in GPCR-Gαs fusion proteins receptors (Vorobjev et al. 1993; Panula et al. 2015) , which, however, is not in the focus of mainstream histamine research. This section addresses the results obtained from the pharmacological characterization of the four human histamine receptor isoforms in Sf9 cells. Other species variants will only be mentioned, when this is required by the context (e.g., comparisons of human and guinea pig H 1 R or H 2 R). Moreover, data from the characterization of ligands in medicinal chemistry projects will only be discussed, when they lead to new insights about structure and conformation of the corresponding receptor. Finally, publications that contain only "in silico" results without experimental verification will be omitted, since the purpose of this chapter is specifically the expression and characterization of human histamine receptors in the Sf9 cell system. For detailed information on the analysis of histamine receptor species variations in Sf9 cells or for the characterization of histamine receptor subtypes in cellular systems other than insect cells, the reader is referred to comprehensive review articles Strasser et al. 2013; Panula et al. 2015 ).

The Histamine H 1 Receptor 2.1.1 General Information About the Histamine H 1 R The H 1 R is ubiquitously expressed, specifically in lung, CNS, and blood vessels. It preferentially couples to Gα q/11 proteins, causing PLC and protein kinase C (PKC) activation as well as inositol-1,4-5-trisphosphate (IP 3 ) formation and intracellular Ca 2+ release Panula et al. 2015) . The typical signs of a type I allergic reaction like pruritus, increased vascular permeability, and edema are caused by H 1 R activation. Therefore, administration of H 1 R antagonists (so-called antihistamines) belongs to the most important anti-allergic therapeutic interventions (Simons and Simons 2011) , e.g. for the treatment of allergic rhinitis. The H 1 R is expressed on various types of immune cells, specifically on T cell subsets and dendritic cells and influences T cell polarization . Moreover, as indicated by results from H 1 R-deficient mice, the H 1 R plays a role in various models of inflammatory diseases, e.g. nasal allergy, Th2-driven allergic asthma, atopic dermatitis or experimental autoimmune encephalitis (EAE) ). In the CNS, H 1 R is involved in the regulation of locomotor activity, emotions, cognitive functions, arousal, sleep and circadian rhythm or pain perception (Schneider et al. 2014a ). Moreover, the H 1 R participates in the modulation of energy consumption, food intake, and respiration. H 1 R blockade with antagonists increases susceptibility to seizures (Schneider et al. 2014a ). Sedation, the most important side effect of brainpenetrating first-generation antihistamines, is caused by antagonism at H 1 R in the CNS (Simons and Simons 2011; Neumann et al. 2014) . The human H 1 R (hH 1 R) is endogenously expressed by various human cell lines. HeLa cervix carcinoma cells as well as U373 MG astrocytoma cells are used since more than two decades to study hH 1 R pharmacology and signal transduction ). In the following, the results from the characterization of the human H 1 R in the Sf9 insect cell expression system will be discussed.

The hH 1 R was extensively characterized in Sf9 cells with regard to ligand pharmacology, and activation of G proteins. Moreover, the pharmacological differences between the hH 1 R and its guinea pig orthologue (gpH 1 R) were addressed by mutational and molecular modeling studies. An overview of the most important results is provided in Table 1 . Although Sf9 cells contain an endogenous PLC-stimulating Gα q -like protein (Hepler et al. 1993 ), histamine does not induce a significant rise in steady-state GTPase activity in Sf9 cell membranes expressing the hH 1 R alone (Houston et al. 2002) . Only co-expression of the hH 1 R with the regulators of G protein signaling RGS4 and GAIP (G-alpha-interacting protein, RGS19) unmasks an interaction of hH 1 R with insect cell Gαq, resulting in histamine-induced stimulatory effects of 142% (RGS4) and 126% (GAIP) (Houston et al. 2002) . These results indicate that the intrinsic GTPase activity of Sf9 cell Gα q is rate-limiting for hH 1 R-mediated G protein activation in Sf9 cell membranes. This is probably due to a low number of G proteins relative to hH 1 R molecules. RGS proteins commonly accelerate the intrinsic GTPase activity of Gα proteins, which results in a higher turnover and in increased availability of inactive GDP-bound Gα subunits (Fig. 2) .

Due to its favorable properties, the Sf9 cell hH 1 R/RGS protein co-expression system is routinely used to characterize affinity (radioligand binding), activity (steady-state GTPase assays), and binding mode of hH 1 R ligands in medicinal chemistry projects. This revealed major pharmacological differences between H 1 R species isoforms. Specifically, some agonistic bulky 2-phenylhistamines and histaprodifens exhibited increased efficacy and up to tenfold higher potency at gpH 1 R as compared to hH 1 R . Such differences were also observed for antagonists. Most notably, the potency of several arpromidine-type H 1 R antagonists was up to tenfold higher at gpH 1 R than at hH 1 R . Mutagenesis experiments were performed to elucidate the molecular basis of these pharmacological species differences. Basing on the hypothesis that smaller amino acid substitutions render the gpH 1 R binding pocket more flexible than the corresponding site at the hH 1 R, the amino acids 153 or 433 of the hH 1 R were mutated into "gpH 1 R direction" (Phe-153 ! Leu 153 or Ile-433 ! Val 433) . Although this attempt was unsuccessful in terms of generating gpH 1 R-like pharmacology, the mutations dramatically decreased hH 1 R receptor expression, function, electrophoretic mobility as well as [ 3 H]mepyramine (tritiated 2-((2-(Dimethylamino)ethyl)(p-methoxybenzyl)amino)-pyridine) affinity, suggesting that these amino acid positions are essential for correct folding and expression of the H 1 R . In addition, the hH 1 R-F153L/I433V double mutant was studied. Although this protein was excellently expressed in Sf9 cell membranes, there were only partial changes in pharmacology. Thus, Phe-153 and Ile-433 cannot fully explain the species difference between hH 1 R and gpH 1 R . • Interaction of hH 1 R with insect cell Gα q unmasked • Intrinsic GTPase activity of Sf9 cell Gα q is ratelimiting for hH 1 R-mediated G protein activation in Sf9 cell membranes.

• Histamine-induced stimulation in steady-state GTPase assay: 142% with RGS4 and 126% with GAIP hH 1 R, gpH 1 R + RGS4 or GAIP • Higher efficacy and up to tenfold higher potency of bulky 2-phenylhistamines and histaprodifens at gpH 1 R than at hH 1 R • Potency of several arpromidine-type H 1 R antagonists up to tenfold higher at gpH 1 R than at hH 1 R hH 1 R-F153L a hH 1 R-I433V a + RGS4 or GAIP Compared to wild-type hH 1 R:

• Dramatic reduction of expression, function and [ 3 H]mepyramine affinity, altered electrophoretic mobility • Mutated amino acid positions required for correct folding and expression of the H 1 R hH 1 R-F153L/I433V a double mutant + RGS4 or GAIP • Excellent expression, but only partial change of pharmacological properties (compared to wild-type hH 1 R)

• Mutated amino acid positions not solely responsible for the pharmacological difference between hH 1 R and gpH 1 R hH 1 R, gpH 1 R, rH 1 R, bH 1 R + RGS4

• Differential interaction of chiral histaprodifens with hH 1 R, gpH 1 R, rH 1 R, and bH 1 R • Two compounds showed agonism at gpH 1 R, but antagonism at hH 1 R, bH 1 R, and rH 1 R.

• Potency rank order of histaprodifens: hH 1 R < bH 1 R < rH 1 R < gpH 1 R; structure and pharmacology of hH 1 R similar to bH 1 R; gpH 1 R resembles rH 1 R • Docking studies (active-state model of gpH 1 R): multiple interaction sites between dimeric histaprodifen and gpH 1 R (Asp-116, Ser-120, Lys-187, Glu-190, and Tyr-432)

• Higher maximum G q -activation and lower potency of histamine at h(gpNgpE2)H 1 R as compared to hH 1 R or h(gpE2)H 1 R • Differences between hH 1 R and gpH 1 R in N-terminus and ECL2 not responsible for pharmacological species differences • Unexpected reduction of pK i and pEC 50 in the series hH 1 R > h(gpE2)H 1 R > h(gpNgpE2)H 1 R for three phenoprodifens (change of ligand orientation?) A series of chiral histaprodifens was pharmacologically characterized at hH 1 R and gpH 1 R as well as rat (r) and bovine (b) H 1 R, revealing differential interaction with H 1 R species isoforms. Two of the compounds showed agonism at gpH 1 R, but were antagonists at hH 1 R, bH 1 R, and rH 1 R. The histaprodifens followed the rank order of potency hH 1 R < bH 1 R < rH 1 R < gpH 1 R. The hH 1 R was pharmacologically and structurally similar to bH 1 R, while gpH 1 R resembled rH 1 R (Strasser et al. 2008a) . Docking studies with an active-state model of the gpH 1 R and dimeric histaprodifen revealed multiple interaction sites, involving hydrogen bonds and electrostatic interactions with Asp-116, Ser-120, Lys-187, Glu-190 and Tyr-432 (Strasser et al. 2008a) .

Since the amino acid sequence of the N-terminus and the second extracellular loop (ECL2) exhibit major differences between hH 1 R and gpH 1 R, it was hypothesized that these structures may be responsible for the preferred binding of bulky agonists to gpH 1 R as compared to hH 1 R. To address this hypothesis, wild-type hH 1 R and gpH 1 R as well as the chimeric receptors h(gpE2)H 1 R (hH 1 R with ECL2 from gpH 1 R) and h(gpNgpE2)H 1 R (hH 1 R with N-terminus and ECL2 from gpH 1 R) were co-expressed with RGS4 in Sf9 cells and compared in radioligand binding and steady-state GTPase assays (Strasser et al. 2008b) . A small inverse agonistic effect of mepyramine suggests that all four receptors show only low constitutive activity. Histamine potency in steady-state GTPase assays decreased in the series hH 1 R > h(gpE2)H 1 R > h(gpNgpE2)H 1 R. Maximum G q -protein activation by histamine and the ΔV max /B max ratio (turnover number) was significantly enhanced at • Association rate constants for h(gpNgpE2)H 1 R significantly different from the constants for hH 1 R and gpH 1 R.

• Extracellular surface of the H 1 R influences ligand binding and recognition and guiding of the ligand into the binding pocket. Wittmann et al. (2011) hH 1 R, gpH 1 R, bH 1 R, rH 1 R + RGS4

• Identification of bulky phenylhistamines with higher potency and affinity at hH 1 R than at gpH 1 R • Molecular modeling: higher hH 1 R potency possibly due to a more effective van der Waals interaction with Asn 2.61 of hH 1 R as compared to Ser 2.61 of gpH 1 R • Two distinct binding modes of phenoprodifens cause Trp 6.48 (part of the rotamer toggle switch activation mechanism) to assume either an active or an inactive conformation Strasser et al. (2009) hH 1 R, gpH 1 R + RGS4 or GAIP N G -acylated imidazolylpropylguanidines are partial H 1 R agonists with higher efficacies at hH 1 R than at gpH 1 R Xie et al. (2006a, b) a Mutations were performed to make the hH 1 R "more similar" to gpH 1 R and to investigate the resulting alterations of receptor pharmacology b Human H 1 R with N-terminus and ECL2 of guinea pig H 1 R c Human H 1 R with ECL2 of the guinea pig H 1 R h(gpNgpE2)H 1 R as compared to hH 1 R, gpH 1 R, and h(gpE2)H 1 R, despite a very low expression level of h(gpNgpE2)H 1 R. This indicates that histamine induces a h(gpNgpE2)H 1 R conformation which is specifically efficient at activating G proteins (Strasser et al. 2008b) . Molecular dynamics simulations suggest that the replacement of N-terminus and ECL2 affect the network of hydrogen bonds between N-terminus, ECL1 and ECL2 and alter the conformation and flexibility of ECL2. Thus, either the replacement of the N-terminus or the combined exchange of N-terminus and ECL2 induces conformational alterations that increase the stimulatory effect of histamine and reduce its potency (Strasser et al. 2008b ).

The hypothesis that major differences of N-terminus and ECL2 cause the distinct pharmacology of hH 1 R and gpH 1 R, however, had to be rejected, since neither binding assays nor steady-state GTPase assays revealed more pronounced "gpH 1 R-like" properties of h(gpNgpE2)H 1 R and h(gpE2)H 1 R (Strasser et al. 2008b ). Instead, three members of a new class of histaprodifens (phenoprodifens) even exhibited a reduction of pK i and pEC 50 values in the series hH 1 R > h(gpE2)H 1 R > h(gpNgpE2) H 1 R (Strasser et al. 2008b) . Previous molecular dynamics simulations with these compounds had suggested that they can adopt two distinct orientations in the gpH 2 R binding pocket (Strasser et al. 2008a) . Thus, the data may be explained by a change in ligand orientation in the series hH 1 R -h(gpE2)H 1 R -h(gpNgpE2)H 1 R. Such changes, however, are probably determined early in ligand binding, which can only be addressed by kinetic binding studies (Strasser et al. 2008b) .

Such experiments were performed with the antagonist [ 3 H]mepyramine and the partial agonist phenoprodifen using Sf9 cell membranes expressing RGS4 together with hH 1 R, gpH 1 R as well as the chimeric receptors h(gpNgpE2)H 1 R and h(gpE2)H 1 R (Wittmann et al. 2011) . With regard to the association rate constant, h(gpNgpE2)H 1 R significantly differed from both hH 1 R and gpH 1 R. Molecular dynamics simulations helped to explain, how the extracellular surface of the H 1 R influences ligand binding kinetics, recognition of the ligand and guiding of the ligand into the binding pocket (Wittmann et al. 2011) .

There are also exceptions, where bulky agonists do not interact more efficiently with gpH 1 R than with hH 1 R. Specifically, N G -acylated imidazolylpropylguanidines (AIPGs) are partial H 1 R agonists that exhibit higher efficacies at hH 1 R as compared to gpH 1 R (Xie et al. 2006a, b) . Moreover, another study addressing the pharmacology of phenylhistamines and phenoprodifens at human, guinea pig, bovine, and rat H 1 R identified bulky phenylhistamines with higher potency and affinity at hH 1 R as compared to gpH 1 R (Strasser et al. 2009) . A comparison of the hypothesized binding modes of these compounds with the binding mode of the previously characterized N G - (Xie et al. 2006b ) suggests that the higher potency at the hH 1 R is caused by a more pronounced van der Waals interaction with Asn 2.61 of hH 1 R as compared to Ser 2.61 of gpH 1 R (Strasser et al. 2009 ). Moreover, phenoprodifens seem to adopt two distinctly oriented binding modes that cause the highly conserved Trp 6.48 , which is part of the toggle switch mechanism of GPCR activation , to assume either an active or an inactive conformation (Strasser et al. 2009 ).

The Histamine H 2 Receptor 2.2.1 General Information About the Histamine H 2 R The H 2 R is ubiquitously expressed, most importantly in stomach, heart, and CNS Schneider et al. 2014a; Panula et al. 2015) . Agonist binding to this receptor results in activation of Gα s -proteins that stimulate the adenylylcyclase-mediated production of the second messenger cAMP (Panula et al. 2015) . The central role of the H 2 R in the regulation of gastric acid production is the basis for the therapeutic use of H 2 R antagonists to treat gastroesophageal reflux disease (Schubert and Peura 2008) . The function of the H 2 R in the brain is less well documented as for H 1 R, but includes, e.g. modulation of cognitive processes and of circadian rhythm (Schneider et al. 2014a) . Moreover, H 2 R influences glucose metabolism and food intake (Schneider et al. 2014a) .

Experiments with knockout mice have revealed that the histamine H 2 R is involved in the regulation of immune responses, specifically in the modulation of Th1-or Th2-cell polarization. It should be noted, however, that the analysis of H 2 Rdeficient mice yields conflicting results, probably because of the variability of the disease models studied ). The human histamine H 2 R (hH 2 R) has been pharmacologically characterized in both human cells and in the Sf9 cell expression system . Neutrophils are specifically well suited for the analysis of hH 2 R pharmacology, because they are primary cells that can be easily isolated from human blood in large numbers. The hH 2 R inhibits superoxide anion production induced by chemotactic peptides in neutrophils (Burde et al. 1989 (Burde et al. , 1990 Reher et al. 2012a ) and eosinophils (Reher et al. 2012a) . Moreover, H 2 R activation induces functional differentiation of HL-60 promyelocytes (Klinker et al. 1996) . Furthermore, it is discussed that decreased hH 2 R function may contribute to inflammation in bronchial asthma ).

The hH 2 R was extensively characterized in Sf9 cells with regard to ligand pharmacology, and activation of G proteins. Moreover, the pharmacological differences between the hH 2 R and its guinea pig orthologue (gpH 2 R) were addressed by mutational and molecular modeling studies. An overview of the most important results is provided in Table 2 .

Functional expression of the human hH 2 R in Sf9 cells requires Gα s proteins as intracellular coupling partners. Indeed, Sf9 cells express endogenous Gα s proteins and activation of Sf9 cell Gα s has been reported for mammalian GPCRs, e.g. the bradykinin B2 receptor (Shukla et al. 2006) , the LH/CG receptor (Narayan et al. 1996) , or the histamine H 2 R (Kühn et al. 1996) . Mostly, however, the interaction of mammalian GPCRs with Sf9 cell Gα s shows only low productivity, which is most likely due to rapid dissociation of the activated Gα s subunit from the plasma membrane. Redistribution of stimulated Gα s proteins has been investigated in more detail in S49 lymphoma cells treated with the β-AR agonist isoproterenol (Ransnäs et al. 1989 ). • Only hH 2 R: no agonist-induced signal in steadystate GTPase assays, not even with GAIP • AC activation by hH 2 R (Sf9 Gαs) and by hH 2 R-Gsα S

• No activation of insect cell or co-expressed mammalian Gα q by hH 2 R in Sf9 cells • Mammalian Gα q most likely inactive in Sf9 cells Houston et al. (2002) hH 2 R-Gsα S gpH 2 R-Gsα S • Affinity of large guanidine-type agonists in [ 3 H]tiotidine binding: hH 2 R-Gsα S < gpH 2 R-Gsα S • Disruption of guanidine-type agonist highaffinity binding by GTPγS more effective at hH 2 R-Gsα S than at gpH 2 R-Gsα S • Potencies and efficacies of guanidines in steadystate GTPase assays: gpH 2 R-Gsα S > hH 2 R-Gsα S Kelley et al. (2001) 

• Higher (more "gpH 2 R-like") potencies of guanidines in steady-state GTPase assays at hH 2 R-A271D-Gsα S and NhCgpH 2 R-Gsα S than at hH 2 R-Gsα S • Efficacies of guanidine-type agonists at hH 2 R-Gsα S , hH 2 R-A271D-Gsα S , NgpChH 2 R-Gsα S and NhCgpH 2 R-Gsα S are lower than at gpH 2 R-Gsα S • Potency and efficacy are independent H 2 R properties hH 2 R-Gsα S gpH 2 R-Gsα S hH 2 R-C17Y-Gsα S hH 2 R-C17Y-A271D-Gsα S • Potencies and efficacies of guanidines in steadystate GTPase assays with hH 2 R-C17Y-A271D-Gsα S : higher than at hH 2 R-Gsα S, but lower than at gpH 2 R-Gsα S ! Tyr-17/Asp-271 interaction not solely responsible for h/gp species differences • Possibly stabilization of ligand-specific receptor conformations

• hH 2 R-C17Y-Gsα S : basal AC activity and agonist-induced steady-state GTPase activity reduced (impaired G protein coupling or degradation of Gsα S ?) Preuss et al. (2007b) (continued) Fusion of a GPCR to Gα s keeps the G protein at the cell membrane and largely enhances G protein activation. This approach was used for the human histamine H 2 R, which was expressed in Sf9 cells as a fusion protein with the long (G sαL ) or short (G sαS ) splice variant of Gα s (Wenzel- Seifert et al. 2001 ). Both fusion proteins were expressed at a similar level in Sf9 cell membranes and the affinity of the radiolabeled H 2 R agonist [ 3 H]tiotidine (tritiated 1-cyano-3-[2-[[2-(diaminomethylideneamino)-1,3thiazol-4-yl]methylsulfanyl]ethyl]-2-methyl-guanidine) was comparable (~32 nM) for hH 2 R-Gsα L and hH 2 R-Gsα S (Wenzel- Seifert et al. 2001) . Unexpectedly, the B max values of ligand-regulated [ 35 S]GTPγS binding for hH 2 R-Gsα L or hH 2 R-Gsα S exceeded the B max value from [ 3 H]tiotidine binding by~tenfold, which suggests that a large subpopulation of fusion proteins is not labeled by the radioligand (Wenzel- Seifert et al. 2001) .

G sαL exhibits lower GDP affinity than Gsα S , and therefore, the β 2 AR-G sαL fusion protein shows higher constitutive activity than β 2 AR-Gsα S (Seifert et al. 1998b) . Similarly, the hH 2 R-Gsα L fusion protein exhibited a faster GDP/GTPγS exchange than hH 2 R-Gsα S . Surprisingly, however, unlike the corresponding β 2 AR fusion proteins, hH 2 R-Gsα L and hH 2 R-Gsα S showed similar constitutive activity and comparable pharmacological properties of partial agonists and inverse agonists in steady-state GTPase and [ 35 S]GTPγS binding assays (Wenzel- Seifert et al. 2001) . This illustrates that the GDP affinity of G proteins does not influence the constitutive activity of all GPCRs to the same extent (Wenzel- Seifert et al. 2001) .

It has been reported that the rH 2 R couples to insect cell Gα q and increases intracellular Ca 2+ in Sf9 cells (Kühn et al. 1996) . However, this effect could not be confirmed and was also not observed with hH 2 R or gpH 2 R (Houston et al. 2002) . Moreover, co-expressed GAIP did not unmask a potential interaction of hH 2 R with insect cell Gα q (steady-state GTPase assays) although this approach was successful • Significantly lower histamine-induced steadystate GTPase signals of hH 2 R-K173A-Gsα S or hH 2 R-K175A-Gsα S ! Lys173 and Lys175 important for Gsα S activation? a Sequence from N-terminus to TM3 from gpH 2 R and sequence from TM4 to C-terminus from hH 2 R b Sequence from N-terminus to TM3 from hH 2 R and sequence from TM4 to C-terminus from gpH 2 R c Four e2 amino acids of hH 2 R exchanged by the corresponding residues of gpH 2 R d Four e2 amino acids of gpH 2 R exchanged by the corresponding residues of hH 2 R with hH 1 R (Houston et al. 2002) . The hH 2 R did not even activate mammalian Gα q co-expressed in Sf9 cells or fused to the hH 2 R (Ca 2+ assays, high-affinity agonist binding and [ 35 S]GTPγS binding) (Houston et al. 2002) . Surprisingly, not even the hH 1 R was able to activate co-expressed mammalian Gα q in Sf9 cells. Thus, mammalian Gα q was probably inactive in Sf9 cells, despite high expression levels, and therefore, Sf9 cells are not suited to investigate the interaction of GPCRs with mammalian Gα q (Houston et al. 2002) .

When only hH 2 R was expressed in Sf9 cells, no ternary complex formation with insect cell Gα s was observed in high-affinity agonist binding with [ 3 H]tiotidine (effect of GTPγS on histamine competition curve) and in [ 35 S]GTPγS binding (characterization of the stimulatory effect of histamine). Surprisingly, however, AC assays clearly indicated hH 2 R-mediated activation of insect cell Gα s . Thus, AC assays probably exhibit higher sensitivity than [ 3 H]tiotidine high-affinity agonist binding or [ 35 S]GTPγS binding and detect even very low insect cell Gα s stimulation (Houston et al. 2002) . Co-expression of hH 2 R with mammalian Gsα S resulted in efficient G protein interaction (high-affinity agonist binding, [ 35 S]GTPγS binding, AC assays). A further increase in interaction efficiency was observed for the hH 2 R-Gsα S fusion protein (Houston et al. 2002) .

The fusion protein approach was also used for the pharmacological comparison of hH 2 R and gpH 2 R ). In [ 3 H]tiotidine radioligand binding assays, the hH 2 R-Gsα S fusion protein expressed in Sf9 cells bound large guanidine-type agonists with lower affinity than gpH 2 R-Gsα S . Moreover, GTPγS disrupted highaffinity binding of guanidine-type agonists at hH 2 R-Gsα S more efficiently than at gpH 2 R-Gsα S . This indicates that the guanidine-stabilized conformation of gpH 2 R interacts more tightly with the tethered G protein than the corresponding conformation of hH 2 R . In steady-state GTPase assays, the potencies and efficacies of guanidines were also higher with gpH 2 R-Gsα S than with hH 2 R-Gsα S . However, the species isoforms did not differ in case of small agonists or antagonists .

Based on molecular modeling data (bovine rhodopsin-based alignment), it was hypothesized that the high potency of guanidine-type agonists at gpH 2 R is caused by the non-conserved Asp-271 in TM7 (Ala-271 in hH 2 R). This hypothesis was tested by expressing the mutant hH 2 R-A271D-Gsα S as well as the chimeras NgpChH 2 R-Gsα S (N-terminus -TM3 from gpH 2 R and TM4-C-terminus from hH 2 R, containing Ala-271) and NhCgpH 2 R-Gsα S (N-terminus -TM3 from hH 2 R, and TM4-C-terminus from gpH 2 R, containing Asp-271) in Sf9 cell membranes . In fact, steady-state GTPase assay data clearly showed increased potency of guanidines at both hH 2 R-A271D-Gsα S and NhCgpH 2 R-Gsα S , confirming the importance of Asp-271 in the gpH 2 R for guanidine binding. Unexpectedly, the efficacies of guanidine-type agonists at hH 2 R-Gsα S and NgpChH 2 R-Gsα S as well as the more "gpH 2 Rlike" constructs hH 2 R-A271D-Gsα S and NhCgpH 2 R-Gsα S were lower than at gpH 2 R. This demonstrates that potency and efficacy are independent properties of the H 2 R. The modeling and experimental data suggest that an interaction between TM1 (Tyr-17) and TM7 (Asp-271) is important for the stabilization of the guanidineinduced agonistic conformation of the gpH 2 R and therefore for guanidine efficacy.

This interaction is absent in hH 2 R and in the other constructs analyzed by Kelley et al. (2001) .

The hypothesis that a Tyr-17/Asp-271 interaction in the gpH 2 R molecule stabilizes an active receptor conformation and increases efficacy of guanidinetype agonists was tested by characterizing the mutant fusion proteins hH 2 R-C17Y-Gsα S and hH 2 R-C17Y-A271D-Gsα S (Preuss et al. 2007b) . As expected, the potencies and efficacies of guanidines in the steady-state GTPase assay were higher at the hH 2 R-C17Y-A271D-Gsα S double mutant as compared to the wildtype hH 2 R-Gsα S fusion protein, but they were still below the values determined for wild-type gpH 2 R-Gsα S . Thus, the Tyr-17/Asp-271 interaction is probably not solely responsible for the different pharmacology of hH 2 R and gpH 2 R (Preuss et al. 2007b) . Moreover, the data suggest the stabilization of ligand-specific receptor conformations by agonists and inverse agonists in wild-type and mutant hH 2 R-Gsα S fusion proteins (Preuss et al. 2007b) .

The results from the analysis of the hH 2 R-C17Y-Gsα S single mutant support the notion that an H-bond between Tyr-17 and Asp-271 stabilizes an active receptor conformation (Preuss et al. 2007b ). The hH 2 R-C17Y-Gsα S fusion protein exhibits lower basal AC and decreased agonist-induced GTPase activities (Preuss et al. 2007b) , indicating impaired G protein coupling. One possible explanation may be degradation of the hH 2 R-C17Y-Gsα S fusion protein in the Sf9 cells. This is suggested by the apparent molecular mass of 40 kDa instead of the expected 80 kDa in Western blots (Preuss et al. 2007b ).

In bovine rhodopsin (Palczewski et al. 2000) as well as in various aminergic GPCRs, e.g. dopamine D 2 R , adenosine A 2a R (Kim et al. 1996) , or muscarinic M 3 receptor (Scarselli et al. 2007) , residues in the second extracellular loop, ECL2, probably contribute to ligand binding. Thus, it was hypothesized that differences in e2 may also determine the distinct pharmacology of hH 2 R-Gsα S and gpH 2 R-Gsα S (Preuss et al. 2007c ). This hypothesis was addressed by generating mutant fusion proteins with the four e2 amino acids of hH 2 R exchanged by the corresponding residues of gpH 2 R (hH 2 R-gpE2-Gsα S ) and vice versa (gpH 2 R-hE2-Gsα S ). Steady-state GTPase assays, however, revealed that this exchange of ECL2 did not significantly alter the pharmacology of the receptors. Thus, the mutated residues most likely do not interact with the guanidine-binding pocket (Preuss et al. 2007c) .

In both hH 2 R and gpH 2 R, Cys-174 probably forms a disulfide bond with Cys-91 in TM3 and is framed by two lysines in position 173 and 175 (Preuss et al. 2007c ). A homology model of the hH 2 R predicted that these two lysines are located close to the binding site of guanidine-type agonists and are involved in agonist binding (Preuss et al. 2007c ). Thus, the two mutated fusion proteins hH 2 R-K173A-Gsα S and hH 2 R-K175A-Gsα S were expressed in Sf9 cells and analyzed in steady-state GTPase activity assays. The results, however, indicate that these mutations were ineffective at altering potency or efficacy of small as well as bulky H 2 R agonists (Preuss et al. 2007c) . Interestingly, the effect of histamine on steady-state GTPase activity of both hH 2 R-K173A-Gsα S and hH 2 R-K175A-Gsα S was reduced, which suggests that the lysines in positions 173 and 175 increase the efficiency of hH 2 Rcoupling to Gα s (Preuss et al. 2007c ).

The Histamine H 3 Receptor 2.3.1 General Information About the hH 3 R The Gα i/o -coupled histamine H 3 R is mainly expressed on neurons and acts as a presynaptic auto-and heteroreceptor. It inhibits the release of histamine (Arrang et al. 1983 (Arrang et al. , 1985 , but also of other neurotransmitters such as acetylcholine, noradrenaline, dopamine, or glutamate (Haas et al. 2008) . Additionally, there is increasing evidence that H 3 R is expressed postsynaptically (Ellenbroek and Ghiabi 2014) , where it regulates, e.g. dopamine D 1 R signaling (Ferrada et al. 2008; Brabant et al. 2009 ). Knockout mouse models demonstrate that the H 3 R regulates numerous behaviors like locomotor activity, pain perception, food intake, memory, circadian rhythm, cognition, and anxiety (Schneider et al. 2014b) . Moreover, H 3 Rdeficiency reduces addictive behavior in mouse models of ethanol consumption, which is probably due to the reward-inhibiting function of an increased histamine release (Vanhanen et al. 2013; Schneider et al. 2014b ). This renders the H 3 R an interesting target for the treatment of alcohol addiction (Nuutinen et al. 2012) .

Despite the decade-long research on H 3 R pharmacology, only the inverse H 3 R agonist pitolisant is currently used as an orphan drug to treat narcoleptic patients (Dauvilliers et al. 2013) . Mouse models suggest that, in contrast to the other three histamine receptor subtypes, the H 3 R does not seem to play a major role in immunological processes and inflammation ).

There is no standard human cell culture model available that endogenously expresses hH 3 R. Thus, expression and characterization of hH 3 R and its species orthologues in the Sf9 insect cell system is of major importance (Schnell et al. 2010a, b; Schnell and Seifert 2010; Seifert et al. 2013; Strasser et al. 2013 ). Sf9 cells do not express endogenous Gαi-like protein that could interact with the corresponding mammalian GPCRs. It is, therefore, required to co-express the receptor of interest with mammalian Gα i and Gβγ subunits. This, however, provides the unique opportunity to freely combine Gα i -coupled receptors with any Gα i/o isoform, allowing the characterization of Gα i isoform specificity of GPCRs. As described in the following sections, the pharmacology of the hH 3 R was extensively characterized in Sf9 cells. An overview of the most important results is provided in Table 3 .

The hH 3 R was co-expressed in Sf9 cells with Gβ 1 γ 2 and Gα i1, Gα i2, Gα i3 , or Gα o . All hH 3 R/G protein combinations could be readily expressed in Sf9 cells, and a semiquantitative analysis of expression levels by Western blot (purified Gα i2 and Pharmacological Characterization of Human Histamine Receptors and Histamine. . . • No species selectivity of histamine, Nα-methylhistamine, (R)-α-methylhistamine, imetit, and clobenpropit • Striking species selectivity of imoproxifan: nearly full agonist at hH 3 R, but inverse agonist at rH 3 R • Imoproxifan: pEC 50 > pK i (hH 3 R and rH 3 R) ! conformations with low partial/inverse agonist affinity, but efficient Gα interaction? hH 3 R + Gα i2 + Gβ 1 γ 2 Influence of ions on hH 3 R properties [ 3 H]NAMH radioligand binding: Increase in radioligand B max and no significant reduction of binding affinity by 100 mM of NaCl Effect of NaCl (100 mM) in steady-state GTPase assays:

• Increase in efficacy and reduction of potency of histamine • Reduction of efficacy and increase in potency of thioperamide ! stabilization of hH 3 R inactive state by NaCl Comparison of various cations and anions: Rank order of efficacy at inhibiting hH 3 R constitutive activity: Li +~N a +~K+ < Cl À < Br À < I À Schnell and Seifert (2010) hH 3 R + Gα i1, Gα i2, Gα i3 or Gα o + Gβ 1 γ 2 NaCl effect on hH 3 R basal activity: strongest NaCl-mediated reduction of constitutive activity in the presence of Gα i3 (continued) Gα o as reference) yielded receptor-to-G protein ratios between 1:50 and 1:100 ). The receptor expression levels determined by Western blot were confirmed by radioligand saturation binding assays with the antagonist [ 3 H]JNJ-7753707 ((4-Fluorophenyl)(1-methyl-2-{[1-(1-methylethyl)piperidin-4-yl] methoxy}-1H-imidazol-5-yl)methanone). By contrast, quantitation of the total number of activated Gα i/o proteins in [ 35 S]GTPγS binding assays revealed a much lower amount of [ 35 S]GTPγS binding sites as compared to the Western blot results, yielding hH 3 R/Gα i isoform coupling ratios between 1:2 (hH 3 R/Gα i1 ) and 1:11 (hH 3 R/Gα o ) .

Potencies and efficacies of the physiological agonist histamine and the inverse agonist thioperamide (N-Cyclohexyl-4-(imidazol-4-yl)-1-piperidinecarbothioamide) were determined in steady-state GTPase assays for all hH 3 R/Gα i/o combinations ). When hH 3 R was expressed in Sf9 cell membranes without any mammalian G protein, the signals induced by histamine and thioperamide were only small, indicating that hH 3 R-mediated stimulation of insect cell G proteins was virtually absent . A comparison of all five expression systems (hH 3 R alone and combined with Gα i1 , Gα i2 , Gα i3 , or Gα o ) revealed that the relative stimulatory signal of histamine and the relative inhibitory signal of thioperamide were comparable, indicating that the constitutive activity of hH 3 R does not depend on the type of co-expressed Gα i/o protein . Overall, the constitutive activity of the hH 3 R was similar to the basal activity of the hH 4 R ) (see following section).

Steady-state GTPase experiments were also performed with various hH 3 R standard ligands in all hH 3 R/Gα i/o co-expression systems. N α -methylhistamine (NAMH) and (R)-α-methylhistamine (RAMH) turned out to be full agonists under all conditions and imetit almost reached full efficacy. Proxyfan (4-[3-(Phenylmethoxy)propyl]-1Himidazole) and impentamine (4-(5-Aminopentyl)imidazole) were partial agonists with comparable efficacy under all conditions. Ciproxifan (cyclopropyl-(4-(3-(1Himidazol-4-yl)propyloxy)phenyl) ketone), clobenpropit (N-(4-Chlorobenzyl)-S-[3-(4 (5)-imidazolyl)propyl]isothiourea), and thioperamide exhibited inverse agonism in all systems, but efficacies were significantly different between the various Gα i/o proteins. Nevertheless, the rank orders of potency and efficacy of the ligands remained unaltered. Taken together, these experiments again confirm the notion that the hH 3 R • Constitutive activity (steady-state GTPase assays) completely eliminated • Stimulatory effect of histamine still NaClsensitive Schnell and Seifert (2010) hH 3 R-D2.50N + Gα i1, Gα i2, Gα i3 or Gα o + Gβ 1 γ 2 Surprising G protein selectivity of hH 3 R-D2.50N mutation: no interaction with Gα i3 , but activation of Gα i1 , Gα i2 and Gα o1 exhibits similar pharmacological properties independently of the co-expressed Gα i/o isoforms ).

As mentioned above, the hH 3 R/G protein ratios ranged between 1:2 and 1:11, indicating that it is difficult to exactly control the expression levels of receptor and G proteins. Thus, the fusion protein approach was used to ensure a 1:1 coupling ratio of hH 3 R and Gα subunit. The hH 3 R was fused to Gα i2 and Gα o , because these two Gα i/o isoforms exhibit the lowest structural similarity. The pharmacological properties of the standard ligands histamine, imetit, proxyfan, clobenpropit, and thioperamide were similar in steady-state GTPase assays with hH 3 R-Gα i2 and hH 3 R-Gα o . This indicates again that the hH 3 R pharmacology is largely independent of the type of co-expressed or fused Gα subunit .

Previously published studies about hH 3 R pharmacology had reported that, depending on the expression system and the functional readout, proxyfan can be a full, a partial, or even an inverse agonist (Gbahou et al. 2003; Krueger et al. 2005 ). This was explained by the phenomenon of "protean agonism," which is the ability of a ligand to induce GPCR conformations with lower G protein-coupling efficiency than the agonist-stimulated or constitutively active receptor (Gbahou et al. 2003) . It has been hypothesized that protean agonism of proxyfan is due to functional selectivity, i.e. G protein coupling of the proxyfan-bound hH 3 R differentiates between various Gα i/o isoforms. The data reported by Schnell et al. (2010a) , however, strongly suggest that neither proxyfan nor any other of the tested hH 3 R ligands exhibits this kind of functional selectivity, at least when the hH 3 R is co-expressed with or fused to various Gα i/o isoforms in Sf9 cell membranes. One reason for this discrepancy could be the influence of different types of Gβγ subunits, which was not systematically investigated in Sf9 cells, because in the experiments performed by Schnell et al. (2010a) all hH 3 R/Gα i/o combinations were uniformly co-expressed with Gβ 1 γ 2 . Moreover, specific combinations of various Gα i/o isoforms or cross-talk between signaling pathways could have influenced the results reported by Gbahou et al. (2003) and Krueger et al. (2005) .

As discussed in the preceding section, the study of Gbahou et al. (2003) suggested that proxyfan shows protean agonism, which, however, was not confirmed in the Sf9 cell system ). One of the reasons for this discrepancy could be a pharmacological difference in H 3 R isoforms. Gbahou et al. (2003) used rat H 3 R (rH 3 R), while the experiments of Schnell et al. (2010a) were performed with hH 3 R. To test this hypothesis, both species isoforms were directly compared in the Sf9 cell expression system (Schnell et al. 2010b ).

Similar to the human isoform ), the rH 3 R was also coexpressed with Gβ 1 γ 2 and the Gα i/o isoforms Gα i1 , Gα i2 , Gα i3 , or Gα o . A quantitation of rH 3 R binding sites by radioligand binding with [ 3 H]JNJ-7753707 and of receptorcoupled Gα subunits by [ 35 S]GTPγS binding revealed a rH 3 R/G protein stoichiometry between 1:2 and 1:7 (Schnell et al. 2010b) , which is comparable to the properties of the corresponding hH 3 R membranes . Moreover, similar to the hH 3 R, the rH 3 R showed similar high constitutive activity with each of the four Gα i/o subunits as indicated by comparable relative effects of the agonist histamine and the inverse agonist thioperamide (Schnell et al. 2010b ). The independence of rH 3 R pharmacology of the co-expressed Gα i/o type was confirmed by steady-state GTPase experiments.

Several H 3 R standard ligands were characterized at rH 3 R (+ Gα i2 Gβ 1 γ 2 ) and hH 3 R (+ Gα i2 Gβ 1 γ 2 ) in [ 3 H]NAMH radioligand binding assays. The affinities of histamine, N α -methylhistamine, (R)-α-methylhistamine, imetit, proxifan, and clobenpropit did not differ between species isoforms, while the affinities of impentamine, imoproxifan, ciproxifan, and thioperamide were increased at the rH 3 R (Schnell et al. 2010b ). The radioligand binding results were largely confirmed on the functional level by steady-state GTPase experiments. Histamine, N αmethylhistamine, RAMH, imetit, and clobenpropit did not show species selectivity. Impentamine, however, was more potent at rH 3 R than at hH 3 R. Additionally, ciproxifan and thioperamide exhibited higher potency but less efficacy at rH 3 R as compared to hH 3 R (Schnell et al. 2010b) . The hypothesis that the protean agonism of proxyfan reported by Gbahou et al. (2003) was characteristic for the rat H 3 R orthologue had to be rejected, because proxyfan acted as a strong partial agonist at rH 3 R expressed in Sf9 cells, independently of the co-expressed G protein (Schnell et al. 2010b) .

A striking difference between hH 3 R and rH 3 R was observed for the H 3 R ligand imoproxifan, which acted as a nearly full agonist at the hH 3 R, but exhibited inverse agonism at the rat orthologue (Schnell et al. 2010b ). To explain this switch in quality of action, molecular modelling studies were performed by docking imoproxifan into the binding site of the active hH 3 R and the inactive rH 3 R. The simulations revealed different electrostatic surfaces between TM V and TM III. While the hH 3 R shows a positive surface potential in this region (NH moiety of Trp 6.48 ), the corresponding part of the rH 3 R is slightly negatively charged (OH moiety of Thr 6.52 ), which results in different orientations of the ligand at both receptors. Moreover, hH 3 R differs from rH 3 R in amino acid position 3.37. Thr 3.37 of the hH 3 R interacts with Glu 5.46 , making Glu 5.46 pointing away from the binding pocket, which creates a binding site for the imoproxifan methyl moiety (Schnell et al. 2010b) . By contrast, an alanine in position 3.37 of the rH 3 R precludes any electrostatic interaction between Glu 5.46 and position 3.37.

Ala 3.40 of hH 3 R is replaced by the bulkier Val 3.40 in rH 3 R. Thus, the imoproxifan oxime moiety points downward towards Ala 3.40 in hH 3 R and stabilizes Trp 6.48 in its horizontal conformation via a hydrogen bond. By contrast, the oxime moiety is directed upwards in rH 3 R and interacts with Thr 6.52 , while the methyl group of imoproxifan fits into a pocket between Val 3.40 and Trp 6.48 . This stabilizes Trp 6.48 of rH 3 R in its vertical conformation. According to the rotamer toggle switch mechanism of GPCR activation , the horizontal conformation of Trp 6.48 corresponds to the active state, while the vertical conformation stabilizes the inactive receptor state. Thus, this model explains the different quality of action of imoproxifan at hH 3 R and rH 3 R (Schnell et al. 2010b) .

Interestingly, in case of imoproxifan, a comparison of steady-state GTPase assay and [ 3 H]NAMH radioligand binding data revealed that the pEC 50 values at hH 3 R and rH 3 R were significantly higher than the corresponding pK i values. This suggests that both hH 3 R and rH 3 R can adopt conformations with low affinity to partial/inverse agonists that nevertheless exhibit efficient G protein interaction (Schnell et al. 2010b ).

According to the (simplifying) two-state model of receptor activation (Fig. 4) , GPCRs can adopt an active or an inactive conformation (Leff 1995) . The equilibrium between both receptor states is shifted to the active side by (partial) agonists and/or interaction with G proteins. The inactive state, however, is stabilized by (partial) inverse agonists Sato et al. 2016) . The degree of constitutive activity depends on the intrinsic tendency of the receptor protein to occur in the active state. It is well established that ions are able to modulate GPCR function (Strasser et al. 2015) . Specifically, sodium represents an allosteric stabilizer of the inactive receptor conformation and inhibits constitutive activity, which was, e.g., demonstrated for chemoattractant receptors (Seifert and Wenzel-Seifert 2001, 2003) .

As discussed above, the hH 3 R exhibits high constitutive activity. Thus, hH 3 R represents an interesting model for the detailed investigation of the activitymodulating effects of ions. The hH 3 R was co-expressed with Gα i2 and Gβ 1 γ 2 in Sf9 cells and the influence of 100 mM of NaCl on [ 3 H]NAMH high-affinity agonist binding and on GTP hydrolysis in the steady-state GTPase assay was investigated. Unexpectedly, in contrast to the data reported for other Gα i/o -coupled receptors like FPR1 , the affinity of the hH 3 R to the radioligand R R * full inverse agonist partial inverse agonist neutral antagonist partial agonist full agonist sodium G protein Fig. 4 Two-state model of receptor activation and factors stabilizing the active (R*) and inactive (R) receptor conformation. Every GPCR population exists in an equilibrium of active and inactive receptor conformations. Full agonists produce a maximum shift towards the active side, while inverse agonists cause a maximum stabilization of the inactive GPCR conformation. Partial agonists and partial inverse agonists induce only an incomplete shift towards either side. Neutral antagonists bind to all receptor states with the same affinity and therefore do not change the equilibrium. G proteins stabilize the active conformation, while sodium ions usually uncouple GPCRs from their G proteins by shifting the equilibrium towards the inactive side. It should be noted that, despite its usefulness, the two-state model is very simplistic and does not account for the numerous distinct ligand-and G protein-specific receptor conformations occurring in reality. Adapted from was not significantly reduced by NaCl. Moreover, most surprisingly, the B max value was even increased by NaCl. The NaCl resistance of the hH 3 R in the [ 3 H]NAMH radioligand binding assays is not fully explained yet, but may be caused by the extremely high constitutive activity of the hH 3 R (Schnell and Seifert 2010) . The resistance of the hH 3 R to the effect of NaCl in radioligand binding was not reflected by the data from steady-state GTPase experiments. In the presence of 100 mM of NaCl, the efficacy of histamine (full agonist) was increased and the pEC 50 value of histamine was reduced from 8.01 to 7.53. By contrast, the pIC 50 value of thioperamide (inverse agonist) was increased from 7.15 to 7.43 by NaCl, while the efficacy of thioperamide was reduced. This clearly indicates that NaCl stabilizes the inactive state of the hH 3 R and reduces the constitutive activity of the system, which agrees with the predictions of the two-state model system of receptor activation (Schnell and Seifert 2010) .

Since NaCl does not only contain sodium cations but also chloride anions, it is not clear if the effect of NaCl on hH 3 R constitutive activity is mediated by Na + , by Cl À or by both ions. To address this question, a profile of the effects of various monovalent cations (Li + , Na + , and K + ) as well as of different anions (Cl À , Br À , and I À ) was determined in steady-state GTPase assays with membranes expressing hH 3 R plus Gα i2 and Gβ 1 γ 2 . The rank order of efficacy was Li +~N a +~K+ < Cl À < Br À < I À . This indicates a direct proportionality between anion radii and reduction of basal hH 3 R activity and shows that anions contribute more to the salt-induced reduction of constitutive activity than cations. Moreover, the different efficacies of the anions exclude the possibility that an increased osmolality may be responsible for the effect on constitutive activity (Schnell and Seifert 2010) . Similar results had been previously obtained with the hβ 2 AR-Gsα L fusion protein, and it had been hypothesized that anions may enhance GDP affinity to the G protein, reducing the ability of the receptor to promote GDP dissociation (Seifert 2001) . Interestingly, a comparison of the NaCl effect on hH 3 R basal activity in membranes co-expressing Gβ 1 γ 2 and various Gα i/o subunits (Gα i1 , Gα i2 , Gα i3 or Gα i/o ) revealed the strongest NaCl-mediated reduction of constitutive activity in the presence of Gα i3 (Schnell and Seifert 2010) .

It is generally assumed that the highly conserved Asp 2.50 acts as a Na + binding site in GPCRs (Horstman et al. 1990; Wittmann et al. 2014) . Thus, the functional consequences of a charge-neutralizing mutation from Asp 2.50 to Asn 2.50 in the hH 3 R protein were investigated. In the absence of sodium, the D2.50N mutant (coexpressed with Gα i2 and Gβ 1 γ 2 ) exhibited a reduced number of [ 3 H]NAMH binding sites and an affinity reduction of [ 3 H]NAMH by about 90% as compared to the wild-type hH 3 R (Schnell and Seifert 2010) . Constitutive activity in steady-state GTPase assays was completely eliminated by the D2.50N mutation (co-expressed with Gα i2 and Gβ 1 γ 2 ) and consequently, neither thioperamide nor NaCl further inhibited basal activity. Interestingly, however, the stimulatory effect of histamine at the D2.50N mutant was highly sensitive to NaCl and was completely eliminated at NaCl concentrations > 90 mM. Most surprisingly, the D2.50N mutation introduced G protein selectivity, as the mutant did not productively interact any more with Gα i3 , but still activated Gα i1 , Gα i2 , and Gα o1 . Thus, Asp 2.50 seems to play a decisive role in the hH 3 R/Gα i3 -interaction (Schnell and Seifert 2010) . In summary, the characterization of the hH 3 R in the Sf9 cell expression system by Schnell and Seifert (2010) revealed that Gα i3 interacts with hH 3 R in a very distinct manner as compared to the other tested Gα i/o isoforms (stronger NaCl effect on activity of wild-type hH 3 R and complete inactivity of the hH 3 R-D2.50N mutant). Interestingly, the D2.50N mutant was not completely NaCl-insensitive, which indicates that the interaction between ions and hH 3 R is more complex and cannot be explained by a single interaction site (Schnell and Seifert 2010) .

In contrast to the hH 3 R, the structurally similar hH 4 R (see Sect. 2.4) exhibits completely NaCl-resistant constitutive activity . A potential explanation for this discrepancy was recently offered by Wittmann et al. (2014) . A comparison of various human aminergic GPCRs revealed that in the majority of receptors, glycine is the most abundant (80%) amino acid in the sodium binding channel between the ligand binding site and the sodium binding region (Wittmann et al. 2014) . This is, however, not the case for hH 3 R and hH 4 R. Moreover, in hH 4 R the glutamine in position 7.42 disrupts a water chain, which is extending from Asp 3.32 (orthosteric binding site) to Asp 2.50 (allosteric binding site). This might kinetically prevent sodium from binding to the allosteric binding site (Wittmann et al. 2014 ).

The fourth histamine receptor couples to PTX-sensitive Gα i proteins, specifically to Gα i2 and shows high constitutive activity ). The H 4 R is a chemotactic receptor mainly expressed on hematopoietic cells, specifically on eosinophils (O'Reilly et al. 2002; Buckland et al. 2003; Reher et al. 2012b) .

Human eosinophils belong to the best characterized primary cells endogenously expressing hH 4 R, but it is difficult to isolate this rare cell type in sufficiently high purity and numbers from healthy volunteers . Moreover, H 4 R is expressed on mast cells (Hofstra et al. 2003; Jemima et al. 2014 ) as well as dendritic cells (Gutzmer et al. 2005; Damaj et al. 2007; Bäumer et al. 2008; Gschwandtner et al. 2011 ) and expression on natural killer cells has been reported, too (Damaj et al. 2007) . The presence of the H 4 R on monocytes is discussed controversially (Damaj et al. 2007; Gschwandtner et al. 2013; Werner et al. 2014) . Data from a comprehensive analysis of hH 4 R expression on various myeloid cell types have been published very recently (Capelo et al. 2016) . H 4 R knockout mouse models suggest that this receptor plays a role in the pathophysiology of itch, experimental asthma and EAE . The H 4 R represents an interesting target for anti-inflammatory drugs. For example, the H 4 R regulates eosinophilic inflammation in a mouse model of ovalbumininduced allergic asthma (Hartwig et al. 2015) . Moreover, the hH 4 R seems to be a key player in pruritus during inflammatory reactions (Bell et al. 2004; Dunford et al. 2007; Rossbach et al. 2011) . However, studies with mouse models should be interpreted with caution, because H 4 R pharmacology strongly differs between various species . For example, the "prototypical" hH 4 R antagonist JNJ7777120 (1-[(5-Chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) is an inverse agonist at the hH 4 R, but a partial agonist at the rat, mouse, and canine orthologues Strasser et al. 2013 ). Another caveat is H 4 R-induced G proteinindependent β-arrestin signaling. Although JNJ-7777120 is an inverse H 4 R agonist with regard to G protein activation, it exhibits agonistic effects on H 4 R-dependent β-arrestin signaling (Rosethorne and Charlton 2011; Seifert et al. 2011; Nijmeijer et al. 2013) . Recently, the H 4 R antagonist JNJ 39758979 ((R)-4-(3-amino-pyrrolidin-1-yl)-6-isopropyl-pyrimidin-2-ylamine) was shown to be safe and efficacious at reducing histamine-induced pruritus in a phase 1 clinical study (Kollmeier et al. 2014 ).

The N-terminally FLAG-tagged and C-terminally His-tagged wild-type hH 4 R was co-expressed with Gαi 2 and Gβ 1 γ 2 in Sf9 cells. Binding studies with [ 3 H]histamine revealed a K D value of~10 nM ), which fits well to the literature range (5-20 nM). Steady-state GTPase and [ 35 S]GTPγS binding experiments confirmed the high constitutive activity of the hH 4 R, which was effectively inhibited by the inverse agonist thioperamide . Surprisingly, thioperamide was not able to suppress [ 35 S]GTPγS binding in the co-expression system (hH 4 R + Gαi 2 + Gβ 1 γ 2 ) to the level of control membranes expressing only Gαi 2 and Gβ 1 γ 2 ). This strongly indicates that thioperamide is only a partial H 4 R inverse agonist and not, as originally suggested in the literature (Lim et al. 2005) , a full inverse agonist. The Sf9 cell system provides a "clean" background devoid of mammalian Gα i proteins and their cognate GPCRs. Thus, expression of mammalian G proteins without GPCRs in Sf9 cells provides a valid control for baseline Gα activity and for the maximum possible effect of a full inverse agonist. In the following, the most important results from the pharmacological characterization of the hH 4 R in Sf9 cell membranes are discussed. An overview of the most important results is provided in Table 4 .

According to the ternary complex model (De Lean et al. 1980 ), a GPCR shows its highest agonist affinity, when it is part of the ternary complex (Sect. 1.2.1, Fig. 2 ). Ternary complex formation, however, is prevented in the presence of GTPγS which binds to the Gα subunit like GTP (Gilman 1987 ), but cannot be hydrolyzed. Thus, GTPγS disrupts the G protein cycle, resulting in the accumulation of uncoupled inactive GPCRs with reduced agonist affinity. Surprisingly the hH 4 R shows an active state which is completely independent of G proteins ). This is supported by the following four observations: First, high-affinity [ 3 H]histamine binding (K D and B max ) to membranes expressing hH 4 R, Gα i2 , and Gβ 1 γ 2 was retained in the presence of GTPγS. Second, [ 3 H]histamine binding affinity was almost identical in the hH 4 R/Gα i2 /Gβ 1 γ 2 co-expression system and in Sf9 cell membranes expressing hH 4 R in the absence of mammalian G proteins. Third, the K i values of the inverse hH 4 R agonists thioperamide and JNJ-7777120 were unaltered in membranes expressing only hH 4 R, although the two-state model of receptor activation (Fig. 4) suggests that inverse agonist affinity increases, when Pharmacological Characterization of Human Histamine Receptors and Histamine. . . • Compared to co-expression system (hH 4 R + Gα i2 + Gβ 1 γ 2 ):

• Increased expression level • Unaltered histamine affinity • More efficient hH 4 R/Gα i2 interaction • Increased constitutive activity • Linear signaling hH 4 R-A6.30E + Gα i2 + Gβ 1 γ 2

Compared to wild-type (hH 4 R + Gα i2 + Gβ 1 γ 2 ):

• Slight (non-significant) reduction of constitutive activity and G protein coupling efficiency

• Unaltered K D of histamine • G protein-independent high-affinity binding retained hH 4 R-R3.50A + Gα i2 + Gβ 1 γ 2 Compared to wild-type (hH 4 R + Gα i2 + Gβ 1 γ 2 ):

• G protein coupling eliminated • Affinity of thioperamide increased • Affinity of histamine reduced hH 4 R + RGS4 + Gα i2 + Gβ 1 γ 2 Compared to wild-type co-expression system (+ Gα i2 Gβ 1 γ 2 ):

• No significant change of histamine effect and baseline steady-state GTPase activity • Significant increase of thioperamide inverse agonistic effect hH 4 R-RGS4 + Gα i2 + Gβ 1 γ 2 Compared to wild-type co-expression system (+ Gα i2 Gβ 1 γ 2 ):

• Significant increase of baseline steadystate GTPase activity and of thioperamide inverse agonistic effect • Significantly increased EC 50 -values of histamine and JNJ-7777120 (~twofold)

• Significantly increased apparent K M value of Gα i2 intrinsic GTPase activity in the presence of histamine (continued) the receptor is not coupling to G proteins and assumes an inactive state. Finally, steady-state GTPase assays with membranes co-expressing hH 4 R, Gα i2 and Gβ 1 γ 2 revealed that the constitutive activity of the hH 4 R is insensitive to sodium ions. According to the standard two-state model of receptor activation depicted in Fig. 4 , however, it is expected that Na + stabilizes the inactive state of a GPCR. This has been shown previously, e.g. for FPR-26 (Wenzel-Seifert et al. 1998; Seifert and Wenzel-Seifert 2001) or the α 2 -adrenoceptor (Tian and Deth 2000) .

Analysis of hH 4 R activation in the steady-state GTPase assay in membranes co-expressing hH 4 R with Gβ 1 γ 2 and a specific Gα subunit (Gα i1 , Gα i2 , Gα i3 or Compared to wild-type co-expression system (+ Gα i2 Gβ 1 γ 2 ):

• No significant change of signal range and baseline activity in steady-state GTPase assays • Significantly increased apparent K M value of Gα i2 intrinsic GTPase activity in the presence of histamine hH 4 R-GAIP + Gα i2 + Gβ 1 γ 2 Compared to wild-type co-expression system (+ Gα i2 Gβ 1 γ 2 ):

• Basically identical pharmacological properties

• Significantly increased relative histamine-and thioperamide-induced signals in steady-state GTPase assays hH 4 R-GAIP + Gα i1 , Gα i2 , Gα i3 or Gα o + Gβ 1 γ 2 Identical G protein selectivity of hH 4 R-GAIP and hH 4 R ! G protein coupling is mainly determined by the GPCR, but not by the RGS protein hH 4 R-F169V+S179A or hH 4 R-F169V+S179M hH 4 R-F169V hH 4 R-S179A hH 4 R-S179M + Gα i2 + Gβ 1 γ 2 Compared to wild-type hH 4 R: constitutive activity not affected by the S179A or S179M single mutations. Constitutive activity slightly reduced in F169V single mutant; stronger reduction in double mutants. S179A single mutant: increased potency and affinity of JNJ-7777120 Wifling et al. (2015b) mH 4 R-V171F mH 4 R-V171F+M181S + Gα i2 + Gβ 1 γ 2 No constitutive activity of wild-type mH 4 R and mH 4 R-V171F mutant, but weak constitutive activity of mH 4 R-V171F +M181S double mutant hH 4 R-F168A + Gα i2 + Gβ 1 γ 2 Total loss of hH 4 R constitutive activity. Wifling et al. (2015a) Gα o ) revealed that Gα i2 was most effectively stimulated by the hH 4 R. By contrast, the hH 4 R hardly activated Gα o proteins ). Since Gα o is the main G protein subtype in the brain, this result suggests that the hH 4 R is not of major importance in the CNS. We have seriously questioned the widespread but largely unfounded notion of functional hH 4 R expression on neurons (Schneider and Seifert 2016) . The stoichiometry of the receptor-G protein interaction can be calculated by dividing the total number of receptor-regulated G proteins (from GTPγS binding assays) by the number of receptors per cell (B max from radioligand binding or from Western blot). When co-expressed with Gα i2 and Gβ 1 γ 2 in Sf9 cell membranes, the hH 4 R catalytically activates up to five Gα i2 subunits simultaneously ). The affinity of [ 35 S]GTPγS to the Gα subunit (K D value) reflects efficiency of G protein activation. The inverse agonistic character of thioperamide was confirmed in [ 35 S]GTPγS assays with membranes co-expressing hH 4 R, Gα i2 and Gβ 1 γ 2 . While the [ 35 S]GTPγS K D value was 3.4 nM in the presence of histamine, it was about threefold increased by thioperamide ), indicating reduced [ 35 S]GTPγS affinity of the Gα subunit due to uncoupling from the hH 4 R.

As demonstrated for the constitutively active mutant of the β 2 -adrenoreceptor (β 2 AR CAM ) (Gether et al. 1997) , constitutive activity of a GPCR increases conformational flexibility and favors denaturation. By contrast, ligand binding reduces conformational flexibility and stabilizes the receptor. Thus, addition of ligands to a cell culture expressing β 2 AR CAM increased the B max value of this receptor (Gether et al. 1997) . This effect was caused by both agonists and inverse agonists, suggesting that it is the switch between different activation states rather than the nature of the activation state, which destabilizes the receptor.

The high constitutive activity of the hH 4 R prompted us to investigate its conformational stability and the stabilizing effect of ligands. In fact, addition of histamine (10 μM) or thioperamide (1 μM) to Sf9 cells co-expressing hH 4 R, Gα i2 and Gβ 1 γ 2 significantly increased the B max value in histamine high-affinity agonist binding assays ). Interestingly, this effect was not visible in immunoblots, indicating that histamine and thioperamide mainly support the correct folding of hH 4 R in the cell membrane, but not during intracellular protein synthesis. This was confirmed in experiments, where denaturation of hH 4 R (co-expressed with Gα i2 and Gβ 1 γ 2 ) was induced by incubation of the membranes at 37 C. After 120 min, almost 70% of the histamine binding sites in the ligand-free control were lost, but only 35% in the presence of histamine. Most surprisingly, however, thioperamide increased the B max by 30-40%, suggesting that it did not only prevent hH 4 R denaturation, but even re-folded a priori misfolded receptors. This intriguing "refolding" effect of the inverse agonist thioperamide was confirmed in a two-step assay, during which the receptor was first denatured and then incubated with thioperamide.

To analyze the interaction of the hH 4 R with Gα i2 , the C-terminus of the receptor was fused to the N-terminus of the G protein by using a His 6 linker (Fig. 3) . The hH 4 R-Gα i2 protein co-expressed with Gβ 1 γ 2 in Sf9 cell membranes exhibited linear signaling with a coupling factor of~1 in [ 35 S]GTPγS binding assays and a turnover number of~1 in steady-state GTPase assays. Thus, hH 4 R exclusively activates the tethered mammalian G protein but not the insect cell G proteins ). This was additionally supported by the lack of [ 35 S]GTPγS binding in membranes expressing non-fused hH 4 R in the absence of mammalian G proteins ). The K D value of [ 35 S]GTPγS in the presence of the full hH 4 R agonist histamine or the inverse agonist thioperamide in membranes co-expressing hH 4 R-Gα i2 and Gβ 1 γ 2 was significantly reduced as compared to the coexpression system, indicating enhanced efficiency of G protein activation ). A higher GTP affinity of Gα i2 in the fusion protein was also reflected by a significantly decreased K M value in the presence of histamine in steady-state GTPase assays. Moreover, a slight increase of constitutive activity in steady-state GTPase assays additionally demonstrates the increased efficiency of G protein activation in the fusion protein system ). Interestingly, the B max value of the hH 4 R-Gα i2 fusion protein in immunoblots and [ 3 H]histamine binding assays was increased as compared to the non-fused receptor ). This suggests a chaperone-like stabilizing effect of Gα i2 , favoring membrane insertion of the receptor protein. Incubation of the cell culture with histamine or thioperamide did not further enhance the B max value of the fusion protein in [ 3 H]histamine binding ), suggesting that the fusion of hH 4 R to Gα i2 induces already the maximum possible number of correctly folded receptors. An overview of the most important features of the hH 4 R-Gα i fusion protein in comparison to the co-expression system (hH 4 R + Gα i2 + Gβ 1 γ 2 ) is provided in Table 4 .

Western blotting of hH 4 R-expressing Sf9 cell membranes revealed two bands at 43 and 46 kDa. Incubation of the baculovirus-infected Sf9 cell culture with the glycosylation inhibitor tunicamycin removed the 46 kDa band, indicating that this is most likely a glycosylated H 4 R species ). Although the total protein amount on the Western blot was comparable for both untreated and tunicamycin-treated H 4 R protein (2.5-3 pmol/mg as assessed by using FLAGβ 2 AR standard membranes with known receptor expression levels), the B max value in [ 3 H]histamine binding was reduced by 75% after tunicamycin treatment. Nevertheless, the K D value of [ 3 H]histamine remained unchanged ). Thus, hH 4 R deglycosylation does not significantly affect the [ 3 H]histamine binding site of functional hH 4 R, although it significantly reduces the amount of correctly folded receptor protein.

The activation of Gα i2 proteins by deglycosylated hH 4 R was investigated in [ 35 S]GTPγS saturation binding and steady-state GTPase assays. Even in the presence of histamine, the deglycosylated hH 4 R in the tunicamycin-treated membranes activated Gα i2 less efficiently than the glycosylated H 4 R (increased K D value of [ 35 S] GTPγS) ). Thus, proper glycosylation of hH 4 R seems to be a prerequisite for efficient G protein coupling. By contrast, determination of the K M value of GTP at the Gα i2 subunit in steady-state GTPase assays only revealed a non-significant trend towards an increased K M -value in the tunicamycin-treated membranes ).

In [ 35 S]GTPγS binding assays, deglycosylation reduced the constitutive activity of H 4 R coexpressed with Gα i2 and Gβ 1 γ 2 from 70 to 40% ). Neither the coupling factor from [ 35 S]GTPγS binding assays nor the turnover number from steady-state GTPase assays changed significantly, when hH 4 R was deglycosylated ). This suggests that deglycosylation of hH 4 R reduces efficacy of Gα activation without affecting the total number of activated G proteins.

The inactive state of GPCRs is established by intramolecular interactions that conformationally restrain the receptor. Data obtained from the rhodopsin molecule have led to the assumption that the so-called ionic lock is highly important for the inactivation of GPCRs (Palczewski et al. 2000; Vogel et al. 2008) . The ionic lock is a salt bridge between a highly conserved glutamate in position 6.30 of TM6 and the arginine of the DRY motif located on the bottom of TM3 (position 3.50). The importance of the ionic lock for the regulation of odorant GPCR activity has been shown recently (de March et al. 2015) . However, some receptors do not form an ionic lock, despite the presence of the required amino acids. This has been reported, e.g. for the human β 2 AR Rasmussen et al. 2007; Rosenbaum et al. 2007) or the human A 2A adenosine receptor (Jaakola et al. 2008) , both of which show considerable constitutive activity.

The hH 4 R is the only histamine receptor with an alanine in position 6.30, which precludes ionic lock formation ) and possibly explains the observed high G protein-independent activity of the hH 4 R ). To test this hypothesis, the TM6 part of the potential ionic lock was reconstituted by introducing the A6.30E mutation, and the resulting mutant was analyzed in the Sf9 cell expression system. Immunoblots and [ 3 H]histamine saturation binding indicated comparable expression levels of the mutant and the wild-type hH 4 R. Unexpectedly, the pharmacological properties of hH 4 R-A6.30E (co-expressed with Gα i2 and Gβ 1 γ 2 ) in radioligand binding, steady-state GTPase assay and [ 35 S]GTPγS binding assays were basically unaltered as compared to the wild-type hH 4 R ). The replacement of alanine 6.30 by glutamate resulted in a slight but non-significant reduction of coupling factor ([ 35 S]GTPγS binding), turnover number (steady-state GTPase assay) and constitutive activity ([ 35 S]GTPγS binding and steady-state GTPase assay). This indicates that the ionic lock interaction was either not fully reconstituted or not sufficient to stabilize the inactive conformation of hH 4 R ). An overview of the most important features of the hH 4 R-A6.30E mutation in comparison to the wild-type hH 4 R is provided in Table 4 . Molecular modeling studies revealed potential interactions that may stabilize the active conformation despite the presence of the reconstituted ionic lock. The hH 4 R active state was modeled in complex with the C terminus of Gα i2 by using the crystal structures of the turkey β 1 AR (Warne et al. 2008 ) and the human adenosine A 2A receptor (Jaakola et al. 2008 ) as templates. This revealed an additional salt bridge between D5.69 at the N-terminus of the second cytoplasmic loop (CL3) and R6.31, which may stabilize an active receptor conformation ). Since D5.69 is nearly unique among the GPCRs for biogenic amines, this salt bridge may be at least partly responsible for the high constitutive activity of hH 4 R and should be analyzed in future studies.

Recently, the reasons for the high constitutive activity of hH 4 R were further elucidated (Wifling et al. 2015a, b) . These studies made use of the large pharmacological differences between human and rodent H 4 R Strasser et al. 2013) . For example, constitutive activity of mH 4 R and rH 4 R is strongly reduced as compared to hH 4 R ) and the inverse hH 4 R agonist JNJ7777120 exhibits partial agonism at mH 4 R and rH 4 R. Moreover, the potency of the agonist histamine is lower for the rodent orthologues as compared to hH 4 R . Mutational studies indicate that position 169 of the second extracellular loop is an important determinant of the distinct agonist binding properties of human and mouse H 4 R (Lim et al. 2008) . The F169 of the hH 4 R is replaced by a V169 in the mH 4 R. Thus, Wifling et al. (2015b) performed a detailed analysis of the "mouse-like" hH 4 R-F169V mutant in the Sf9 cell system. In fact, hH 4 R-F169V exhibited decreased constitutive activity as compared to wild-type hH 4 R, resulting in an increased agonistic effect of histamine. Moreover, histamine binding affinity as well as the inverse agonistic effect of thioperamide was reduced (Wifling et al. 2015b ). The second key amino acid identified by Wifling et al. (2015b) was S179, which is replaced by methionine in the mH 4 R and by alanine in the rH 4 R. The double mutants hH 4 R-F169V+S179A and hH 4 R-F169V+S179M showed an even stronger reduction of constitutive activity as compared to the hH 4 R-F169V single mutant (Wifling et al. 2015b) . These results suggest that the constitutively active state of hH 4 R at least partly depends on hydrophobic interactions between the extracellular domains of TM 5, 6, and 7 and ECL2. A hydrogen bond between S179 and T323 additionally stabilizes the agonist-free active state of the hH 4 R (Wifling et al. 2015b) .

These mutations, however, did not completely eliminate the constitutive activity of hH 4 R. A total loss of constitutive activity was only achieved by introducing the F168A mutation (Wifling et al. 2015a ). This indicates that -despite the strong reduction of constitutive activity in the hH 4 R-F169V mutation -the adjacent amino acid in the FF motif, F168, is the key residue responsible for the high constitutive activity of hH 4 R (Wifling et al. 2015a ). An FF motif in ECL2 is also present in other GPCRs, e.g. β 2 AR, hH 3 R and M 2 R, suggesting a similar role of the ECL2 conformation on constitutive activity of these receptors.

Activation by the Human hH 4 R The arginine R3.50 of the DRY motif at the bottom of TM3 stabilizes the inactive receptor state by forming a salt bridge with the adjacent D/E3.49 residue (Nygaard et al. 2009 ). Therefore, we analyzed the effect of the hH 4 R-R3.50A mutation on constitutive activity and ligand binding in membranes co-expressing hH 4 R-R3.50A, Gα i2 and Gβ 1 γ 2 . Surprisingly, the R3.50A exchange totally eliminated G protein coupling as indicated by the complete absence of receptor-regulated steadystate GTPase activity . Moreover, the hH 4 R-R3.50A mutant adopted an inactive state with reduced affinity of the agonist histamine and increased affinity of the inverse agonist thioperamide ). However, introduction of the R3.50A mutation reduced histamine affinity only by 50% and did not affect B max . This suggests that the hH 4 R-R3.50A mutant still adopts a "residual" G protein-independent high-affinity state.

To explain the total loss of G protein coupling of the hH 4 R-R3.50A mutant, molecular modelling studies were performed using the active-state of the hH 4 R in complex with the C-terminus of Gα i2 . This analysis revealed that R3.50 of the hH 4 R may interact with the backbone oxygens of C352 and G353 in the Gα i2 C-terminus ). This supports the adoption of the Gα i2 conformation, which is required for interaction with TM6 of the receptor. Thus, the R3.50A mutation hampers G protein recognition by hH 4 R. Nevertheless, the hH 4 R-R3.50A mutant is still able to form the salt bridge between D5.69 and R6.31, which stabilizes an active state. This could explain why hH 4 R-R3.50A still exhibits relatively high histamine affinity ). However, the effect of mutations in the E/DRY motif is not disrupting G protein coupling in all GPCRs. Rovati et al. (2007) described two phenotypes P1 and P2 that are produced by mutations of the E/D3.49-or the R3.50-residue. While in P1-type receptors highaffinity agonist binding and G protein coupling are retained after mutating position R3.50, P2-type receptors show a disrupted receptor-G protein interaction and reduced agonist binding affinity (Rovati et al. 2007) . Accordingly, the hH 4 R belongs to the group of P2-type GPCRs. An overview of the most important features of the hH 4 R-R3.50A mutation in comparison to the wild-type hH 4 R is provided in Table 4 .

As explained above, co-expression of the hH 4 R and its cognate mammalian G proteins in Sf9 cells results in high constitutive activity ). This reduces the maximum available signal range, yielding a very low signal-to-noise ratio. Even in the presence of 100 mM of NaCl, the full agonist histamine produced only a signal intensity of~30% (related to baseline) ). The expression of an hH 4 R-Gα i2 fusion protein did not improve the signal-to-noise ratio, but resulted in even higher constitutive activity and reduced relative intensity of histamine-induced signals ). Thus, the properties of the hH 4 R/G protein co-expression system and the hH 4 R-Gα i2 fusion protein are rather unfavorable for the characterization of hH 4 R ligands.

This prompted us to perform a closer investigation of the effects of regulators of G protein signaling (RGS proteins). A common feature of RGS proteins is the 120 amino acid RGS domain, which interacts with Gα subunits and increases their intrinsic GTPase activity (Willars 2006) . RGS proteins are classified in eight subfamilies that differ from each other by protein size and the presence of additional functional domains. They regulate the activity of Gα i/o -or Gα q proteins, but no RGS protein-mediated activation of Gα s has been reported to date. Due to their mechanism of action, RGS proteins should enhance signal intensity in steady-state GTPase assays. In fact, fusion of the α 2 AR C-terminus to the RGS4 N-terminus significantly increased α 2 AR-mediated stimulation of GTPase activity (Bahia et al. 2003) .

For the experiments with the hH 4 R, the two RGS proteins RGS4 and GAIP (Gα-interacting protein; also known as RGS19) were selected. RGS4 and GAIP both exhibit a simple protein structure without additional functional domains. Therefore, only activation of Gα i GTPase activity is expected. Both RGS proteins were fused to the hH 4 R via a His 6 linker (Fig. 5) , very similar to the previously described hH 4 R-Gα i2 fusion protein approach (Fig. 3) . The hH 4 R-RGS fusion proteins were co-expressed with Gα i2 and Gβ 1 γ 2 in Sf9 cell membranes. The corresponding co-expression system was characterized by infecting Sf9 cells with baculoviruses encoding hH 4 R, Gα i2 , Gβ 1 γ 2 and RGS4 or GAIP.

Both RGS4 and GAIP, irrespective of whether they were co-expressed or fused to hH 4 R, increased the apparent K M value of Gα i2 in the presence of histamine in steady-state GTPase assays. This effect reached significance for the co-expressed GAIP and the hH 4 R-RGS4 fusion protein ). By contrast, there was no effect of RGS proteins on the K M value in the presence of the inverse agonist thioperamide Schneider and Seifert (2010c) Compared to the RGS4-free co-expression system (hH 4 R + Gα i2 + Gβ 1 γ 2 ), both the quadruple expression system (hH 4 R + Gα i2 + Gβ 1 γ 2 + RGS4) and the fusion protein system (hH 4 R-RGS4 + Gα i2 + Gβ 1 γ 2 ) yielded a significantly increased relative steady-state GTPase signal of the inverse agonist thioperamide, while the histamine-induced signal remained unaffected ). The only major difference between co-expressed and hH 4 R-attached RGS4 was an increased baseline steady-state GTPase activity in the hH 4 R-RGS4 fusion protein system, but an unaltered baseline, when RGS4 was co-expressed ).

Co-expression of GAIP with hH 4 R, Gα i2 and Gβ 1 γ 2 had no significant effect on baseline activity or thioperamide-and histamine-induced signals in steady-state GTPase assays. However, when GAIP was fused to hH 4 R, the histamine-induced relative signal in steady-state GTPase assays was significantly increased by~69% and the thioperamide-induced signal was enhanced by~45%. The baseline activity of the GAIP-hH 4 R fusion protein system, however, remained unaffected ). Thus, in contrast to hH 4 R-RGS4, the hH 4 R-GAIP fusion protein (co-expressed with Gα i2 and Gβ 1 γ 2 ) enhanced the absolute histamine-induced signal without changing baseline activity. Therefore, the relative stimulatory effect of histamine was increased .

The different behavior of RGS4 and GAIP in the fusion proteins is surprising, because both RGS proteins have a similar RGS domain and no additional functionalities. Possibly, the differences are caused by distinct G protein affinities of these RGS proteins. According to the UniProtKB database entry P49795, GAIP binds to Gα i proteins in the rank order Gα i3 > Gα i1 > Gα o >> Gα z /Gα i2 . Thus, among the Gα i isoforms, Gα i2 is the one with the lowest affinity to GAIP. This means that the effect of GAIP may only become visible, when the number of activated Gα i2 subunits exceeds a certain threshold. While under basal conditions the number of activated Gα i2 subunits is too low for a visible hH 4 R-GAIP-mediated effect, stimulation by histamine increases the number of active Gα i2 to a level, where the GAIP-mediated effect becomes visible. By contrast, RGS4 may exhibit a higher Gα i2 affinity than GAIP and therefore show already an effect under basal conditions. This hypothesis, however, should be tested by a side-by-side comparison of the Gα i2 protein affinity of RGS4 and GAIP.

Co-expression of the hH 4 R-GAIP fusion protein with Gα i2 and Gβ 1 γ 2 produces a system with improved signal-to-noise ratio as compared to the standard co-expression system (hH 4 R + Gα i2 + Gβ 1 γ 2 ). A comparison of hH 4 R-GAIP and wild-type hH 4 R (both co-expressed Gα i2 and Gβ 1 γ 2 ) in steady-state GTPase assays revealed comparable pharmacological properties. First, potency and efficacy of selected hH 4 R standard ligands were unaltered. Second, similar to wild-type hH 4 R, the hH 4 R-GAIP fusion protein exhibited sodium chloride-insensitive constitutive activity ). Third, the hH 4 R-GAIP fusion protein showed an unchanged G protein selectivity profile as compared to the unmodified hH 4 R protein ). The unaltered G protein profile is surprising, because GAIP shows distinct affinities to different Gα i isoforms, which should theoretically influence the interaction between hH 4 R-GAIP and the G protein. The results, however, indicate that the G-protein-specificity of the hH 4 R-GAIP fusion protein is governed by the properties of the receptor rather than by the RGS protein part. In summary, the hH 4 R-GAIP fusion protein (co-expressed with Gα i2 and Gβ 1 γ 2 ) can fully replace the standard co-expression system (hH 4 R + Gα i2 + Gβ 1 γ 2 ) in steady-state GTPase assays and allows the functional characterization of new hH 4 R ligands with higher sensitivity and signalto-noise ratio. The hH 4 R-GAIP fusion protein approach was successfully used to evaluate a new class of N G -acylated imidazolylpropyl-guanidine-derived hH 4 R agonists (Ghorai et al. 2008; Igel et al. 2009b) or of cyanoguanidine-related hH 4 R agonists (Igel et al. 2009a; Geyer et al. 2016 ). An overview of the most important features of the various H 4 R/RGS fusion protein and co-expression approaches in comparison to the "standard" co-expression system (hH 4 R + Gα i2 + Gβ 1 γ 2 ) is provided in Table 4 .

The high constitutive activity of hH 4 R significantly reduces the signal-to-noise ratio in steady-state GTPase assays and reduces the sensitivity of agonist assays. However, this feature becomes an advantage, when inverse agonists are characterized. The hH 4 R may maintain its constitutive activity under physiological conditions, because it is resistant to high sodium concentrations. As hypothesized by , on the one side, inverse agonists could be therapeutically advantageous in case of pathophysiologically increased constitutive H 4 R activity, because they may exert a stronger anti-pruritic effect than neutral antagonists. On the other side, the re-folding of misfolded H 4 R protein observed with the inverse agonist thioperamide ) (Sect. 2.4.4) may be a general effect of inverse H 4 R agonists. Thus, inverse agonist-mediated upregulation of intact H 4 R protein may result in rebound effects after drug discontinuation ). Although these hypotheses were not proven yet under physiological conditions, they illustrate the potential importance of characterizing inverse H 4 R agonism during drug development. Therefore, structure-activity relationships for hH 4 R inverse agonism should be established.

A series of 25 previously described (Venable et al. 2005 ) H 4 R ligands (indoles, benzimidazoles, and thienopyrroles; Fig. 6 ) structurally derived from the prototypical H 4 R antagonist JNJ7777120 (Thurmond et al. 2004 ) was characterized in [ 3 H]histamine binding assays and steady-state GTPase assays using membranes expressing hH 4 R + Gα i2 + Gβ 1 γ 2 . The steady-state GTPase assays were performed in the absence of sodium chloride to obtain maximum constitutive activity.

The steady-state GTPase assay data reveal that most of the compounds were inverse agonists with a lower efficacy than thioperamide. Only three of the 25 compounds (~12%) were neutral antagonists ). This confirms a previous analysis of literature data on 380 antagonists binding to 73 GPCRs. Only 15% of these compounds were neutral antagonists (Kenakin 2004) . Thus, neutral antagonism seems to be a rare phenomenon.

In general, the pK b values from steady-state GTPase assays in the presence of histamine fit very well to the pK i values from [ 3 H]histamine binding. In a subset of compounds, the pEC 50 values determined in the absence of histamine were significantly lower than the pK i and/or pK b values. Such discrepancies have been reported before for inverse agonists, e.g. at the hH 4 R (Smits et al. 2008) or the β 2 AR (Chidiac et al. 1994) . Maybe, this subset of hH 4 R antagonists discriminates between the agonist-free constitutively active receptor and the histamine-activated receptor state ). These observations confirm the insufficiency of the two-state model of receptor activation and point to the existence of ligand-specific receptor states.

The potential binding mode of inverse hH 4 R agonists of the indole series was analyzed by molecular dynamics simulations with the completely unsubstituted indole compounds (R4-7 ¼ H; Fig. 6 ). The positively charged piperazine amino group interacts electrostatically with the highly conserved Asp 3.32 . Moreover, both the carbonyl moiety and the indole NH of the ligand establish an interaction with the side chain of the uncharged Glu 5.46 . The indole moiety of the ligand shows a hydrophobic interaction with the indole part of Trp 6.48 ). Trp 6.48 is a key player in the so-called rotamer toggle switch mechanism of receptor activation, which had been previously postulated for the β 2 AR . The stabilization of Trp 6.48 in its vertical conformation by the indole-derived ligand is a typical feature of the inactive receptor conformation and may explain the inverse agonism of such compounds. The benzimidazole-related structures bind in a similar way, but, in contrast to the indole-derived compounds, they form two tautomers with distinct binding modes Replacement of the R5/R7 hydrogen of the indole derivatives by the more spacefilling chlorine increases H 4 R binding affinity. Molecular dynamics simulations suggest that two small binding pockets in the H 4 R protein may be filled by these chlorine residues, which increases the ligand-receptor contact area ). Substitution of R5 by -OCH 3 reduces binding affinity, suggesting that larger substituents may be unfavorable. However, there is no significant correlation between molar volume and affinity of a series of indole compounds, suggesting that the volume of R5 may not be the only descriptor that influences binding affinity . By contrast, the size of R5 correlates excellently with the inverse agonistic efficacy of a subset of eight indole-derived compounds with varying R5 substituents. A calculation of the descriptors logP, molar refractivity, molar volume, polarizability, refraction index and polar surface area revealed that inverse agonistic efficacy solely depended on molar volume, but not on the other factors. The inverse agonistic efficacy of these compounds was inversely correlated to the molar volume of the substituent R5 .

In summary, despite the limited number of compounds and substitution patterns available, in this study the first structure-activity relationships for inverse H 4 R agonism were identified. It was, however, not possible to predict all changes in binding mode and receptor conformation that result from small structural alterations of the ligand. Moreover, a general model that applies to structurally distinct classes of hH 4 R inverse agonists could not be established yet. In the future, the hH 4 R should be co-crystallized with various inverse agonists to elucidate the exact binding mode of these compounds. Although this would be a very ambitious project, the numerous crystallized ligand-receptor complexes published in the recent years Rasmussen et al. 2007 Rasmussen et al. , 2011 Shonberg et al. 2015) demonstrate that this is not impossible.

In this chapter, the results from the characterization of all four histamine receptor subtypes in the Sf9 insect cell system were summarized. On the one hand, it might be argued that insect cells do not represent physiological conditions as well as primary cells. On the other hand, it is difficult to isolate primary cells in sufficiently high numbers. Moreover, a side-by-side comparison of receptor isoforms or species orthologues in a defined environment is virtually impossible in primary cells. Since cells from different tissues have to be used, cell type-specific properties like crosstalk with other receptors or special features of the signaling pathways can lead to heterogeneous results, even for the same receptor isoform. Also, for some receptors like H 3 R, no suitable primary cell system is available . Thus, for a comparison of the intrinsic properties of GPCR isoforms, e.g. G protein affinity/selectivity or constitutive activity, Sf9 cells represent a superior option. As explained in this chapter, Sf9 cells do not contain background GPCR activity and do not produce endogenous agonists activating mammalian GPCRs. Moreover, Sf9 cells allow the co-expression of defined mammalian Gα s or Gα i protein subunits on a "clean" signaling background. This was demonstrated by the analysis of the hH 2 R interaction with long and short Gα s splice variants or by in-depth studies of hH 3 R/hH 4 R Gα i isoform specificity and ion sensitivity. Table 5 shows numerous aspects of histamine receptor pharmacology addressed by using the Sf9 insect cell expression system.

The ligand binding studies and the G protein activation assays discussed in this chapter were all performed with radiolabeled reagents. Radioactivity-based assays, however, are increasingly hampered by legal overregulation and growing waste disposal costs. In this situation, fluorescence-based GPCR ligand binding and G protein activation assays could represent interesting alternatives. Unfortunately, many histamine receptor ligands are rather small molecules and easily lose binding affinity when coupled to a bulky fluorophore. Nevertheless, some progress has been made during the past years. For example, a cyanine dye-labeled aminopotentidine derivative exhibited nanomolar hH 2 R potency (Xie et al. 2006c) . Moreover, fluorescent pyrylium-or cyanine-labeled dimeric carbamoylguanidines were synthesized, but these compounds failed in binding assays due to intracellular accumulation and the resulting high fluorescence background (Kagermeier et al. 2015) . A high-affinity fluorescent H 1 R antagonist was obtained by labeling mepyramine with a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-derived dye (Rose et al. 2012) . Fluorescent hH 3 R-selective ligands were developed by using the chalcone partial structure (Tomasch et al. 2012 ). Moreover, a compound named "Bodilisant," which has been reported recently, is a BODIPY-labeled non-imidazole ligand with nanomolar hH 3 R affinity (Tomasch et al. 2013) . Some progress has also been made in the field of fluorescence-based G protein activation assays. For example, a europium-labeled non-hydrolysable GDP derivative can replace [ 35 S]GTPγS in GTPγS binding assays (Koval et al. 2010 ). This enables a time-resolved fluorescence-based assay that is, e.g., suited for the functional characterization of hH 3 R ligands (Singh et al. 2012 ).

The functional assays described in this chapter focused on the determination of GPCR-mediated G protein activation (steady-state GTPase and [ 35 S]GTPγS binding assays). However, GPCRs can additionally activate G protein-independent signaling mechanisms, most importantly through β-arrestin recruitment (Lefkowitz and Shenoy 2005; Shukla et al. 2014) . The hH 4 R ligand JNJ-7777120, which acts as an inverse hH 4 R agonist in G protein activation assays ), unexpectedly turned out to be an agonist with regard to hH 4 R-mediated β-arrestin recruitment (Rosethorne and Charlton 2011) . This phenomenon is also known as "biased signaling" or "functional selectivity" and has important implications for drug development Nijmeijer et al. 2013) . In future studies, biased signaling of hH 1 R, hH 2 R, or hH 3 R and functional selectivity of the corresponding ligands should be investigated in more detail.

However, the most important, but also most ambitious, goal in future studies would be the crystallization of all four histamine receptor subtypes. To date, only the crystal structure of the hH 1 R has been resolved (Shimamura et al. 2011) . The crystal structures of the histamine receptors are required to answer several still unresolved questions. For example, exact knowledge of the hH 4 R conformation could help to explain, why this receptor shows such a high constitutive activity Table 5 Various aspects of histamine receptor pharmacology and medicinal chemistry investigated in the Sf9 insect cell expression system Wifling et al. 2015a ). Moreover, a crystal structure of the hH 3 R may provide important information about the hH 3 R-G protein interaction interface and possibly answer the question, why the hH 3 R discriminates between Gα i3 and other Gα i/o isoforms (Schnell and Seifert 2010) . Furthermore, an hH 3 R crystal may lead to the identification of the anion binding sites responsible for the monovalent anion-mediated reduction of constitutive hH 3 R activity (Schnell and Seifert 2010) . Finally, the knowledge of H x R crystal structures could lead to the development of compounds that alter H x R function as allosteric modulators. The concept of GPCR modulation by allosteric ligands is well established, and such ligands have been identified, e.g. for dopamine, muscarinic, adenosine, or chemokine receptors (Christopoulos 2014) . By contrast, to the best of our knowledge, to date nothing is known about allosteric modulation of histamine receptors.

As a prerequisite for the preparation of H x R crystals, high amounts of receptor protein have to be expressed, e.g. in Sf9 cells. After purification and solubilization, Seifert et al. (1998) the physical properties of the receptors can be investigated, e.g. with fluorescencebased methods. Such studies have been previously performed with the β 2 AR (Gether et al. 1995; Kobilka 1995; Neumann et al. 2002) and were important steps towards the final goal of receptor crystallization Rasmussen et al. 2007 Rasmussen et al. , 2011 .

Interactions of recombinant human histamine H 1 R, H 2 R, H 3 R, and H 4 R receptors with 34 antidepressants and antipsychotics

Auto-inhibition of brain histamine release mediated by a novel class (H 3 ) of histamine receptor

Autoregulation of histamine release in brain by presynaptic H 3 -receptors

Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G proteincoupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity of RGS4

Histamine H 4 receptors modulate dendritic cell migration through skin--immunomodulatory role of histamine

Involvement of histamine H 4 and H 1 receptors in scratching induced by histamine receptor agonists in Balb C mice

Cellular signaling by an agonist-activated receptor/G s α fusion protein

Effects of the H 3 receptor inverse agonist thioperamide on cocaine-induced locomotion in mice: role of the histaminergic system and potential pharmacokinetic interactions

G protein-coupled receptor oligomerization: implications for G protein activation and cell signaling

Histamine induces cytoskeletal changes in human eosinophils via the H 4 receptor

Histamine inhibits activation of human neutrophils and HL-60 leukemic cells via H 2 -receptors

Characterization of histamine H 2 -receptors in human neutrophils with a series of guanidine analogues of impromidine. Are cell type-specific H 2 -receptors involved in the regulation of NADPH oxidase?

Cellular analysis of the histamine H 4 receptor in human myeloid cells

The duffy antigen/receptor for chemokines exists in an oligomeric form in living cells and functionally antagonizes CCR5 signaling through hetero-oligomerization

High-resolution crystal structure of an engineered human β 2 -adrenergic G protein-coupled receptor

Inverse agonist activity of β-adrenergic antagonists

Advances in G protein-coupled receptor allostery: from function to structure

Functional expression of H 4 histamine receptor in human natural killer cells, monocytes, and dendritic cells

Pitolisant versus placebo or modafinil in patients with narcolepsy: a double-blind, randomised trial

A ternary complex model explains the agonistspecific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor

Conserved residues control activation of mammalian G protein-coupled odorant receptors

Histamine H 4 receptor antagonists are superior to traditional antihistamines in the attenuation of experimental pruritus

The other side of the histamine H 3 receptor

Interactions between histamine H 3 and dopamine D 2 receptors and the implications for striatal function

Protean agonism at histamine H 3 receptors in vitro and in vivo

Fluorescent labeling of purified β 2 adrenergic receptor. Evidence for ligand-specific conformational changes

Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility

Synthesis and functional characterization of imbutamine analogs as histamine H 3 and H 4 receptor ligands

Conformational restriction and enantioseparation increase potency and selectivity of cyanoguanidine-type histamine H 4 receptor agonists

Acylguanidines as bioisosteres of guanidines: N G -acylated imidazolylpropylguanidines, a new class of histamine H 2 receptor agonists

Co-expression of the β 2 -adrenoceptor and dopamine D 1 -receptor with G sα proteins in Sf9 insect cells: limitations in comparison with fusion proteins

G proteins: transducers of receptor-generated signals

Protein-coupled receptor heteromers

Pharmacological Characterization of Human Histamine Receptors and Histamine

The histamine H 4 receptor is highly expressed on plasmacytoid dendritic cells in psoriasis and histamine regulates their cytokine production and migration

Profiling of histamine H 4 receptor agonists in native human monocytes

Histamine H 4 receptor stimulation suppresses IL-12p70 production and mediates chemotaxis in human monocyte-derived dendritic cells

Histamine in the nervous system

The histamine H 4 -receptor (H 4 R) regulates eosinophilic inflammation in ovalbumin-induced experimental allergic asthma in mice

Purification from Sf9 cells and characterization of recombinant G qα and G 11α . Activation of purified phospholipase C isozymes by G α subunits

Histamine H 4 receptor mediates chemotaxis and calcium mobilization of mast cells

An aspartate conserved among G-protein receptors confers allosteric regulation of α 2 -adrenergic receptors by sodium

The human histamine H 2 -receptor couples more efficiently to Sf9 insect cell G s -proteins than to insect cell G q -proteins: limitations of Sf9 cells for the analysis of receptor/G q -protein coupling

Synthesis and structureactivity relationships of cyanoguanidine-type and structurally related histamine H 4 receptor agonists

G -acylated imidazolylpropylguanidines as potent histamine H 4 receptor agonists: selectivity by variation of the N G -Substituent

The 2.6 angstrom crystal structure of a human A 2A adenosine receptor bound to an antagonist

Functional characterization of histamine H 4 receptor on human mast cells

Dimeric carbamoylguanidine-type histamine H 2 receptor ligands: a new class of potent and selective agonists

Distinct interaction of human and guinea pig histamine H 2 -receptor with guanidine-type agonists

The classification of seven transmembrane receptors in recombinant expression systems

Efficacy as a vector: the relative prevalence and paucity of inverse agonism

Glutamate residues in the second extracellular loop of the human A 2a adenosine receptor are required for ligand recognition

G-protein-coupled receptors in HL-60 human leukemia cells

Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor

The histamine H 4 receptor antagonist, JNJ 39758979, is effective in reducing histamine-induced pruritus in a randomized clinical study in healthy subjects

Europium-labeled GTP as a general nonradioactive substitute for [ 35 S]GTPγS in high-throughput G protein studies

N G -acylated aminothiazolylpropylguanidines as potent and selective histamine H 2 receptor agonists

G protein-dependent pharmacology of histamine H 3 receptor ligands: evidence for heterogeneous active state receptor conformations

G proteins of the G q family couple the H 2 histamine receptor to phospholipase C

The two-state model of receptor activation

Transduction of receptor signals by β-arrestins

Evaluation of histamine H 1 -, H 2 -, and H 3 -receptor ligands at the human histamine H 4 receptor: identification of 4-methylhistamine as the first potent and selective H 4 receptor agonist

Phenylalanine 169 in the second extracellular loop of the human histamine H 4 receptor is responsible for the difference in agonist binding between human and mouse H 4 receptors

Expression of functional lutropin/choriogonadotropin receptor in the baculovirus system

Functional immobilization of a ligand-activated G-protein-coupled receptor

Analysis of histamine receptor knockout mice in models of inflammation

Detailed analysis of biased histamine H 4 receptor signalling by JNJ 7777120 analogues

Histamine H 3 receptor: a novel therapeutic target in alcohol dependence?

Ligand binding and micro-switches in 7TM receptor structures

Identification of a histamine H 4 receptor on human eosinophils--role in eosinophil chemotaxis

Heterotrimeric G protein activation by G-protein-coupled receptors

Crystal structure of rhodopsin: a G protein-coupled receptor

International Union of Basic and Clinical Pharmacology. XCVIII. Histamine Receptors

Constitutive activity and ligand selectivity of human, guinea pig, rat, and canine histamine H 2 receptors

Pharmacological Characterization of Human Histamine Receptors and Histamine

Mutations of Cys-17 and Ala-271 in the human histamine H 2 receptor determine the species selectivity of guanidinetype agonists and increase constitutive activity

Point mutations in the second extracellular loop of the histamine H 2 receptor do not affect the species-selective activity of guanidine-type agonists

Functional crosstalk between GPCRs: with or without oligomerization

Stimulation of β-adrenergic receptors of S49 lymphoma cells redistributes the α subunit of the stimulatory G protein between cytosol and membranes

Crystal structure of the human β 2 adrenergic G-protein-coupled receptor

Crystal structure of the β 2 adrenergic receptor-Gs protein complex

Evidence for ligand-specific conformations of the histamine H 2 -receptor in human eosinophils and neutrophils

Incomplete activation of human eosinophils via the histamine H 4 -receptor: evidence for ligand-specific receptor conformations

Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration

A novel fluorescent histamine H 1 receptor antagonist demonstrates the advantage of using fluorescence correlation spectroscopy to study the binding of lipophilic ligands

GPCR engineering yields high-resolution structural insights into β 2 -adrenergic receptor function

Agonist-biased signaling at the histamine H 4 receptor: JNJ7777120 recruits β-arrestin without activating G proteins

Histamine H 1 , H 3 and H 4 receptors are involved in pruritus

The highly conserved DRY motif of class A G proteincoupled receptors: beyond the ground state

Inverse agonism: the classic concept of GPCRs revisited

Multiple residues in the second extracellular loop are critical for M 3 muscarinic acetylcholine receptor activation

Histamine H 4 receptor-RGS fusion proteins expressed in Sf9 insect cells: a sensitive and reliable approach for the functional characterization of histamine H 4 receptor ligands

Coexpression systems as models for the analysis of constitutive GPCR activity

Fusion proteins as model systems for the analysis of constitutive GPCR activity

Sf9 cells: a versatile model system to investigate the pharmacological properties of G protein-coupled receptors

The histamine H 4 -receptor and the central and peripheral nervous system: a critical analysis of the literature

High constitutive activity and a G-proteinindependent high-affinity state of the human histamine H 4 -receptor

Impact of the DRY motif and the missing "ionic lock" on constitutive activity and G-protein coupling of the human histamine H 4 receptor

Structural requirements for inverse agonism and neutral antagonism of indole-, benzimidazole-, and thienopyrrole-derived histamine H 4 receptor ligands

Modulation of behavior by the histaminergic system: lessons from H 1 R-and H 2 R-deficient mice

Modulation of behavior by the histaminergic system: lessons from HDC-, H 3 R-and H 4 R-deficient mice

Modulation of histamine H 3 receptor function by monovalent ions

No evidence for functional selectivity of proxyfan at the human histamine H 3 receptor coupled to defined G i /G o protein heterotrimers

Comparison of the pharmacological properties of human and rat histamine H 3 -receptors

Expression and functional properties of canine, rat, and murine histamine H 4 receptors in Sf9 insect cells

Control of gastric acid secretion in health and disease

Monovalent anions differentially modulate coupling of the β 2 -adrenoceptor to G sα splice variants

Unmasking different constitutive activity of four chemoattractant receptors using Na + as universal stabilizer of the inactive (R) state

The human formyl peptide receptor as model system for constitutively active G-protein-coupled receptors

Reconstitution of β 2 -adrenoceptor-GTPbinding-protein interaction in Sf9 cells--high coupling efficiency in a β 2 -adrenoceptor-G sα fusion protein

Different effects of G s α splice variants on β 2 -adrenoreceptor-mediated signaling. The β 2 -adrenoreceptor coupled to the long splice variant of G s α has properties of a constitutively active receptor

Multiple differences in agonist and antagonist pharmacology between human and guinea pig histamine H 1 -receptor

Paradoxical stimulatory effects of the "standard" histamine H 4 -receptor antagonist JNJ7777120: the H 4 receptor joins the club of 7 transmembrane domain receptors exhibiting functional selectivity

Molecular and cellular analysis of human histamine receptor subtypes

Pharmacological Characterization of Human Histamine Receptors and Histamine

The binding site of aminergic G protein-coupled receptors: the transmembrane segments and second extracellular loop

2 adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch

Structure of the human histamine H 1 receptor complex with doxepin

GPCR crystal structures: medicinal chemistry in the pocket

Functional overexpression and characterization of human bradykinin subtype 2 receptor in insect cells using the baculovirus system

Visualization of arrestin recruitment by a Gprotein-coupled receptor

Histamine and H 1 -antihistamines: celebrating a century of progress

Development of time-resolved fluorescent based [EU]-GTP binding assay for selection of human Histamine 3 receptor antagonists/inverse agonist: a potential target for Alzheimer's treatment

Discovery of quinazolines as histamine H 4 receptor inverse agonists using a scaffold hopping approach

Pharmacological profile of histaprodifens at four recombinant histamine H 1 receptor species isoforms

Ligand-specific contribution of the N terminus and E2-loop to pharmacological properties of the histamine H 1 -receptor

Molecular basis for the selective interaction of synthetic agonists with the human histamine H 1 -receptor compared with the guinea pig H 1 -receptor

Species-dependent activities of G-protein-coupled receptor ligands: lessons from histamine receptor orthologs

Modulation of GPCRs by monovalent cations and anions

A potent and selective histamine H 4 receptor antagonist with anti-inflammatory properties

Differences in efficacy and Na + sensitivity between α 2B and α 2D adrenergic receptors: implications for R and R* states

Novel chalcone-based fluorescent human histamine H 3 receptor ligands as pharmacological tools

Bodilisant-a novel fluorescent, highly affine histamine H 3 receptor ligand

Histamine is required for H 3 receptor-mediated alcohol reward inhibition, but not for alcohol consumption or stimulation

Preparation and biological evaluation of indole, benzimidazole, and thienopyrrole piperazine carboxamides: potent human histamine H 4 antagonists

Functional role of the "ionic lock"--an interhelical hydrogen-bond network in family A heptahelical receptors

Histamine potentiates N-methyl-Daspartate responses in acutely isolated hippocampal neurons

Structure of a β 1 -adrenergic G-protein-coupled receptor

Nucleotide-, chemotactic peptide-and phorbol ester-induced exocytosis in HL-60 leukemic cells

Cyclosporin H is a potent and selective formyl peptide receptor antagonist

Molecular analysis of β 2 -adrenoceptor coupling to G s -, G i -, and G q -proteins

High constitutive activity of the human formyl peptide receptor

Quantitative analysis of formyl peptide receptor coupling to G i α 1 , G i α 2 , and G i α 3

Similar apparent constitutive activity of human histamine H 2 -receptor fused to long and short splice variants of G sα

No evidence for histamine H 4 receptor in human monocytes

The extracellular loop 2 (ECL2) of the human histamine H 4 receptor substantially contributes to ligand binding and constitutive activity

Molecular determinants for the high constitutive activity of the human histamine H 4 receptor: functional studies on orthologues and mutants

Mammalian RGS proteins: multifunctional regulators of cellular signalling

Influence of the N-terminus and the E2-loop onto the binding kinetics of the antagonist mepyramine and the partial agonist phenoprodifen to H 1 R

Sodium binding to hH 3 R and hH 4 R--a molecular modeling study

G protein--mediated signaling: same receptor, multiple effectors

Probing ligand-specific histamine H 1 -and H 2 -receptor conformations with N G -acylated imidazolylpropylguanidines

-cyclohexylbutanoyl)-N 2 -[3-(1H-imidazol-4-yl)propyl]guanidine (UR-AK57), a potent partial agonist for the human histamine H 1 -and H 2 -receptors

Pharmacological Characterization of Human Histamine Receptors and Histamine

Synthesis and pharmacological characterization of novel fluorescent histamine H 2 -receptor ligands derived from aminopotentidine

Real-time visualization of a fluorescent Gα s : dissociation of the activated G protein from plasma membrane

Systematic analysis of histamine and N-methylhistamine concentrations in organs from two common laboratory mouse strains: C57Bl/6 and Balb/c

Example ReferenceSpecies-specificity of receptor pharmacology H 1 R• Efficacy/potency of some agonistic bulky 2-phenylhistamines and histaprodifens: gpH 1 R > hH 1 R • Potency of several arpromidine-type H 1 R antagonists: gpH 1 R > hH 1 R H 2 R • Affinity of large guanidinetype agonists in [ 3 H]tiotidine binding: hH 2 R-Gsα S < gpH 2 R-Gsα S• GTPγS-sensitivity of highaffinity agonist binding: hH 2 R-Gsα S > gpH 2 R-Gsα S • potencies and efficacies of guanidines (steady-state GTPase): gpH 2 R-Gsα S > hH 2 R-Gsα S Kelley et al. (2001) H 3 R Imoproxifan: nearly full agonist at hH 3 R, but inverse agonist at rH 3 R (steady-state GTPase) Schnell et al. (2010b) H 4 R Constitutive activity of mH 4 R and rH 4 R < hH 4 R; inverse hH 4 R agonist JNJ7777120 is a partial agonist at cH 4 R, mH 4 R and rH 4 R; histamine potency at cH 4 R, mH 4 R and rH 4 R < hH 4 R Schnell et al. (2011) Studies with chimeric receptor proteins H 1 R N-terminus and ECL2 of hH 1 R replaced by guinea-pig sequences (h(gpNgpE2)H 1 R): higher maximum G q -activation and lower histamine potency as compared to hH 1 R or h(gpE2) H 1 R; extracellular surface of the H 1 R influences ligand binding, recognition and guiding into the binding pocket Strasser et al. (2008b) and Wittmann et al. (2011) H 2 R Comparison of hH 2 R-A271D-Gsα S , NhCgpH 2 R-Gsα S , NgpChH 2 R-Gsα S , hH 2 R-Gsα S and gpH 2 R-Gsα S to investigate the causes for the pharmacological differences between hH 2 R and gpH 2 R with regard to large guanidine-type agonists Kelley et al. (2001) (continued) Pharmacological Characterization of Human Histamine Receptors and Histamine. . . H 4 R hH 4 R-RGS4 fusion protein: increase of baseline steady-state GTPase activity and of thioperamide inverse agonistic effect (compared to hH 4 R); hH 4 R-GAIP: pharmacological properties unchanged (compared to hH 4 R), but significantly increased relative histamine-and thioperamide-induced GTPase signals Protean agonism: Steady-state GTPase assays with membranes co-expressing hH 3 R with Gβ 1 γ 2 plus Gα i1 , Gα i2 , Gα i3 , or Gα o1 as well as with membranes co-expressing hH 3 R-Gα i2 or hH 3 R-Gα o1 did not confirm the previously reported protean agonism of proxyfan Schnell et al. (2010a) Comparison of H 4 R mutants/ species orthologues with different constitutive activities: JNJ-7777120 is a protean agonist Wifling et al. (2015b) (continued)