[Frontiers in Bioscience 17, 2089-2106, June 1, 2012]

Molecular pharmacology of histamine H4 receptors

Saskia Nijmeijer1, Chris de Graaf1, Rob Leurs1, Henry F. Vischer1

1VU University Amsterdam, Leiden-Amsterdam Center for Drug Research, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands

TABLE OF CONTENTS

1. Abstract
2. Introduction/history
3. H4R gene
4. H4R expression profile
5. H4R protein structure and post-translational modifications
5.1. H4R assembly in the cell membrane
6. H4R ligands
6.1. H4R agonists
6.2. H4R antagonists / inverse agonists
6.3. H4R radioligands
7. Elucidation of ligand binding modes in H4R
7.1. Species differences
7.2. Binding pockets in H4R
7.3. Binding mode of histamine
7.4. Binding mode of JNJ 7777120
8. Signaling of H4R
8.1. Calcium mobilization
8.2. Migration
8.3. Modulation of protein expression and physiological effects regulated by H4R
8.4. Beta-arrestin recruitment to H4R / Biased agonism
9. Final remarks
10. Acknowledgements
11. References

1. ABSTRACT

The histamine H4 receptor (H4R) is the youngest member of the histamine receptor family. Based on its predominant expression pattern in hematopoietic cells, the H4R is considered to be an interesting drug target for inflammatory disorders such as allergy and asthma. Since the identification and cloning of the H4R in 2000, drug discovery programs boosted the development of various H4R (specific) ligands. Differences between H4R orthologs in combination with available three-dimensional G protein-coupled receptor (GPCR) models have guided site-directed mutagenesis studies to gain insight in ligand binding and receptor activation. In addition, ongoing characterization of H4R-mediated signaling in transfected and native cells contributes to further unravel the (patho-) physiological functions of H4Rs.

2. INTRODUCTION / HISTORY

The small biogenic amine histamine was identified as an important mediator in a variety of physiological processes in the beginning of the 20th century (1-3). Histamine is produced de novo by L-histidinedecarboxylase (HDC)-catalyzed decarboxylation of the amino acid L-histidine, and is stored in cytoplasmic granules in immune cells (e.g. eosinophils and mast cells), enterochromaffin-like cells in the stomach and in neurons. Contact of eosinophils or mast cells with an allergen results in immunoglobulin cross-linking and release of stored histamine from the granules (4). Histamine causes the well-known allergic responses, such as sneezing, coughing, rhinorrea and vasoconstriction. Drugs inhibiting these allergic symptoms were initially classified as antihistamines and are now known to act through a receptor protein called histamine H1 receptor (H1R).

In the stomach, histamine stimulation increases gastric acid secretion. The different pharmacology that was found for some histaminergic ligands in airway smooth muscle, heart, uterus and stomach, indicated that histamine affects at least two distinct receptors, namely the H1R and histamine H2 receptor (H2R) (5). Unambiguous evidence for the existence of two different receptors arose from the development of the first (H2R-) selective antagonists burimamide and metiamide by Sir James Black and colleagues (6). These compounds prevented the histamine-induced release of HCl in the stomach, but did not affect the responses supposed to be mediated by the H1R. Consequently, in the following years a plethora of selective drugs was developed against either H1R or H2R to relief either allergic symptoms or inhibit abundant gastric acid release in the stomach, respectively. These H1R (antihistamines) and H2R antagonists became worldwide blockbuster drugs that are now readily available from the local drugstore without a prescription. The sedative effects of the first generation antihistamines revealed the presence of H1R receptors in the brain. To prevent drowsiness as side effect, a second generation of H1R antihistamines was developed, that could not pass the blood brain barrier. At the same time, brain-penetrating antihistamines were marketed and even newly developed as sleep aid.

In the eighties, a new histamine receptor that could not be inhibited according to the known pharmacology of the H1R or H2R antagonists was identified in rat brain (7). This new receptor was classified as the histamine H3 receptor (H3R) and is now known to be involved in the regulation of e.g. sleep/wake cycle and cognitive processes, by presynaptically inhibiting the release of histamine, but also other neurotransmitters like dopamine, noradrenaline, acetylcholine and serotonin. The H3R is seen as a potential target in central nervous system (CNS) disorders such as Alzheimer's disease, schizophrenia and attention deficit hyperactivity disorder (ADHD) (8, 9).

In addition to the "classical" techniques such as ex vivo organ-based studies to pharmacologically define histamine-induced effects, new molecular biology techniques became available in the late twentieth century, eventually leading to the sequencing of complete genomes. Thanks to these developments, histamine receptors can now be recombinantly expressed in convenient host cell lines and biochemically and pharmacologically characterized. Moreover, using site-directed mutagenesis the role of specific amino acids in protein function can be studied. In 1991, both H1R- and H2R-encoding genes were readily cloned, but it lasted until 1999 before the gene of the H3R was identified (10). The histamine receptor proteins are all classified as G protein-coupled receptors (GPCRs), which are characterized by seven transmembrane (TM) alpha-helices. In a quest for new GPCRs that show homology to the H3R gene, a deduced sequence was found that shares considerable amino acid similarity. Several groups cloned this new gene at the same time, and the corresponding protein was named histamine H4 receptor (H4R) (11-16).

In this essay we will give a broad overview of past en present H4R research, as well as highlight some of the emerging developments in the H4R field.

3. H4R GENE

The protein encoded by the human histamine H4R gene (i.e. HRH4) shows very low (~19%) amino acid sequence homology to the histamine H1 and H2 receptors and shares ~37% identity with the H3R. The HRH4 is 16.98kb in size and is mapped by radiation hybrid experiments on chromosome 18q11.2 as a single copy (17). Increased copy numbers of HRH4 are associated with the occurrence of the autoimmune disorder systemic lupus erythematosis (SLE), arthritis, and proteinuria, due to elevated H4R expression levels (18). On the other hand, lower copy numbers are associated with decreased proteinuria (18).

Transcription of HRH4 results in a mRNA molecule of 3.7kb. The open reading frame is 1173bp in size and encodes 390 amino acids. The HRH4 contains two introns of 7867 bp and >17500 bp, dividing the actual coding region into three exons (amino acids 1-65, 66-119 and 120-390) (fig. 1). The HRH3 and HRH4 share a similar intron/exon distribution (19), suggesting that they have evolved from a common ancestral gene. In contrast, HRH1 and HRH2 are intron-less in their coding region (20, 21). Four alternative splicing variants of HRH4 have been identified so far, namely H4R (302), H4R (67) (fig. 2) and H4bR and H4cR. The H4R (302) splice variant contains TM1, part of TM2 and TM5-7, whereas H4R (67) is truncated due to a frame shift and only contains TM1 and the first half of TM2 (22). Both these HRH4 splice variants are predominantly located intracellularly and are not able to activate signaling pathways. However, these splice variants act as dominant negative partners for the full length H4R (see 5.1). The splice variants H4bR and H4cR were cloned from human spleen cDNA by Merck (patent WO 03/020907 A2). The H4bR is identically spliced as H4R (67), whereas H4cR contains exon 2 and recognizes an alternative acceptor site in exon 3, which causes a 33 amino acid deletion. In contrast to H4R (302) and H4R (67), both splice variants are reported in a patent to bind (3H)-histamine and to activate downstream pathways like cAMP inhibition, Ca2+ release and MAPK activation (22), but this has so far not been confirmed.

The promoter region of HRH4 contains several binding motifs for transcription factors (TF), such as nuclear factor kappa B, nuclear factor to interleukin 6, interferon regulatory factor and interferon-stimulated response element (17), but lacks TATA or CAAT box sequences. This suggests that HRH4 expression could be induced by inflammatory factors like interferon, TNFa or IL-6.

Numerous single nucleotide polymorphisms (SNPs) have been identified in the HRH4. Most SNPs are located in non-coding regions with 37 SNPs in intron 1, 45 in intron 2 and 21 in the 3' untranslated region. However, four SNPs were identified in the coding region, resulting in Val4.48Ala, Arg5.70His, Cys6.16Ser or a frame shift at Leu7.75. The first two changes give rise to the differences between the HRH4 sequences as published by Oda et al (12) and other research groups. Recently, three SNPs (i.e. ss142022671, ss142022677 and ss142022679) were associated with atopic dermatitis (23). The SNP ss142022671 is located inside a consensus transcription factor binding motif in intron 1, resulting in increased transcription. On the other hand, the SNPs ss142022677 and ss142022679 are located in exon 3, resulting in the substitution of the Lys7.71-encoding sequence by a stopcodon or mutation of Lys7.71 to Ile, respectively. Moreover, when both these SNPs are present Lys7.71 is changed to Leu (23). Unfortunately, the effects of these SNPs on H4R pharmacology are not yet resolved.

Soon after the identification of the human HRH4, corresponding orthologs were cloned from mouse, rat, guinea pig, pig, monkey and dog (24, 25). The amino acid similarity between monkey and human H4R is the highest with 93%, but decreases when comparing with rodent or pig H4R proteins (65-72%, respectively) (fig. 2) (24, 26-28). Unraveling sequence differences between species is an interesting study for evolutionary scientists. However, it has proven to be a very useful tool to delineate ligand-binding sites as well (see 7.1).

4. H4R EXPRESSION PROFILE

The HRH4 is expressed in a variety of tissues such as bone marrow, spleen, peripheral blood, thymus, small intestine, colon, heart and lung, as revealed by quantitative polymerase chain reactions (qPCR), Northern blot analysis, microarray analysis or in situ hybridization (29-33). However, HRH4 seems to be predominantly expressed on cells of hematopoietic origin, e.g. eosinophils, mast cells, basophils, neutrophils, dendritic cells, monocytes and T cells (fig. 3). HRH4 mRNA of the splice variants could be detected in pre-monocytes and eosinophils (22). In addition, HRH4 expression was also detected in subsets of endocrine cells in the gastrointestinal tract (34), dermal fibroblasts and in the central nervous system (35). There is evidence of HRH4 expression in the mouse brain (14), but human data is still contradictory (13, 15, 17, 24, 36). Although the above-mentioned techniques detect HRH4 expression, they obviously do not give an indication regarding H4R protein levels.

A useful tool to detect H4R proteins on cells and tissues is a H4R specific antibody such as the polyclonal anti-H4R antibody developed against the last 17 amino acids of the C-tail (32). H4R proteins have been detected by antibody-based immunofluorescent staining in human monocyte-derived dendritic cells (MoDC) (29) as well as on primary Langerhans cells from murine and human skin samples (37). Moreover, it was observed that H4Rs are upregulated during the differentiation from monocytes into MoDC (38). More recently, this anti-hH4R receptor antibody was used to localize human and mouse H4R protein in the CNS (35). Histamine H4Rs are expressed in distinct deep laminae (particularly layer VI) in the human cortex and mouse thalamus, hippocampal CA4 stratum lucidum and layer IV of the cerebral cortex. In contrast, very low H4R expression was observed in the striatum and the remaining subfields of the hippocampus (35). The antibody data were confirmed by H4R-mediated electrophysiology responses in layer VI somatosensory cortex neurons in mice (35). In addition, H4R protein was also detected on mouse spinal cords motor neurons (39). On nerves of human nasal mucosa, H4R proteins were found to colocalize with both H1R and H3R (40). H1R, H2R, and H4R proteins are expressed in human intestinal tissue (41). Interestingly, both H1R and H4R expression was significantly decreased in colorectal tumors (41).

5. H4R PROTEIN STRUCTURE AND POST-TRANSLATIONAL MODIFICATIONS

Histamine receptors belong to the family of GPCRs. These receptor proteins consist of seven cell membrane-spanning alpha-helices that are connected by three extracellular loops (ELs) and three intracellular loops (ILs). The N-terminal tail is located extracellular and the C-tail intracellular (fig. 4). The histamine receptors have several conserved structural motifs that are common to the class A (rhodopsin-like) GPCRs, including the highly conserved residues Asn1.50, Asp2.50, Arg3.50, Trp4.50, Pro5.50, Pro6.50 and Pro7.50 (43). Like in most other GPCRs, (42) TM3 and the EL2 of H4R are presumably connected by a disulphide bridge between Cys3.25 and Cys45.50. The extracellular N-terminal tail contains two asparagines (Asn1.21 and Asn1.25) that are predicted to be involved in post-translational glycosylation, whereas the intracellular C-tail is possibly anchored to the cell membrane through palmitoylation of the Cys7.69 residue (fig. 4). In addition, like most GPCRs, H4R has an additional helix 8 that is located intracellularly and does not span the cell membrane.

5.1. H4R assembly in the cell membrane

In the era of the first two histamine receptors it was generally believed that GPCRs function as monomeric entities. However the last two decades, a substantial amount of literature was published showing the existence of higher order structures consisting of two or more GPCRs (fig. 5). One of the most striking examples was given by two class C GPCRs, the GABAB1 and GABAB2 receptors. The GABAB proteins are obligatory heterodimers, not able to reach the cell surface or signal in the absence of one another (44-47). Although there is increasing evidence of GPCRs interacting with each other, the functional consequences of class A GPCR di- or oligomerization are less clear. Hence the quaternary organization for this class of GPCRs is not indisputably accepted (48).

All four histamine receptors form higher order assemblies (49-52). H4R dimers were detected in native cells (e.g. human phytohaemagglutinin (PHA) blasts and spleen lysates) using a selective anti-H4R antibody in co-immunoprecipitation experiments (32). However, co-immunoprecipitation experiments are disputable because of a-specific aggregation after disruption of the cellular environment. Therefore, supporting evidence for dimerization was obtained in transfected living cells (e.g. HEK293 and COS7 cells) that express H4R proteins at physiological relevant (~300 fmol/mg membrane protein) (32). Both homo- and heteromeric complexes were identified for the H4R using biophysical (bioluminescence & time-resolved fluorescence resonance energy transfer) techniques (32, 53). Moreover, H4R dimers are localized at the cell surface, as demonstrated by antibody-based time-resolved fluorescence resonance energy transfer (trFRET) measurement, that only allows detection of cell surface complexes.

H4R oligomers are formed constitutively and their formation is not modulated by (inverse) agonists or antagonists. H4R oligomerization does not require posttranslational N-glycosylation, although a possible role for glycosylation in stabilization of the complex was suggested, based on a decrease in the dimeric population upon deglycosylation (32). H1R-H4R heteromeric complexes are only detected at very high expression levels, and are most likely the consequence of random interactions.

The HRH4 splice variants H4R (67) and H4R (302) form complexes with the full length H4R, and retain the latter intracellularly in a dominant negative manner (22). Hypothetically, since the mRNA of these splice variants is differentially expressed in different cell types, it could well be that they have a role in the regulation of H4R expression at the cell surface (22).

6. H4R LIGANDS

Since the discovery of the H4R, numerous ligands have been identified that bind the receptor and affect downstream signaling pathways. Several efforts have been made to design and synthesize H4R selective (inverse) agonists and antagonists. Considering the amino acid similarity to the H3R, especially in the ligand binding pocket that is formed by the TM domain, it is not surprising that the majority of imidazol-containing H3R ligands have affinity for the H4R as well (54) (fig 6). Examples include R-a-methylhistamine (RAMH), immepip (both 40-fold selective for H3R), immetridine and methimepip (300 and 2000-fold H3R selective, respectively). Small changes in ligand structure result in great differences in histamine receptor subtype specificity. Even so remarkable is the change in efficacy for some compounds on the distinct histamine receptor subtypes. The H3R antagonist clobenpropit, for example, acts as a high affinity partial agonist at the H4R.

Besides H3R cross reactivity, some H4R ligands are able to bind H1R or H2R (fig. 6) (55). The majority of these shared ligands contain the characteristic imidazole heterocycle, with the exception of clozapine (analogues) (56). Interestingly, clozapine binds promiscuously to several GPCRs, but only acts as an agonist on H4R (36).

6.1. H4R agonists

Optimization of the dibenzodiazepine clozapine resulted in the rigid structure VUF6884, a H1R antagonist and H4R agonist (57). Another non-imidazole H4R agonist is the dimaprit analog VUF8430 (54, 58). The latter is a full agonist with high affinity for the H4R and 30-fold selectivity over the H3R. In contrast to the H3R, H4R agonists are thus not limited to imidazole containing structures. Ligand optimization studies and evaluation of other histamine receptor compounds resulted in more selective H4R agonists. OUP-16 (59) and 4-methylhistamine (54), display respectively 40-fold and 100-fold H4R selectivity over the other histamine receptor subtypes.

In more recent years new classes of H4R agonists were discovered, each with their advantages and disadvantages. The acylguanidine agonists were developed as H3R/H4R interacting compounds and can be useful to study H4R pharmacology in absence of H3R (e.g. in some immune cells). Exchange of the acyl group with a cyano group resulting in the cyanoguanidines, improves H4R selectivity (60). UR-PI376 shows 30-fold binding selectivity over H3R, but shows a drop in potency on mouse H4Rs. An interesting aspect of this H4R agonist is that it is unable to activate any other histamine receptor subtype (61).

Recently, Johnson and Johnson (J&J) published their newest class of H4R agonists, the oxime analogues of JNJ 7777120 (see below) and JNJ 10191584 (VUF6002) (62). They show low affinity for the other histamine receptor subtypes and very promising, maintain their efficacy on H4R species orthologs (except for dog H4R) (62). Another interesting class of H4R agonists is the 2-arylbenzimidazoles. They were also developed by J&J and the best compound of this series has subnanomolar affinity for H4R. This compound is highly selective by showing more than 600-fold selectivity over the other histamine receptor subtypes, but is unfortunately less potent on the mouse H4R. Interestingly, minor changes in this 2-arylbenzimidazoles series lead to an efficacy shift from agonist into antagonists (63).

6.2. H4R Antagonists/inverse agonists

One of the first identified antagonists (later discovered to be inverse agonist) was thioperamide, but this compound is equiactive on both H3R and H4R receptors (64-67). The first H4R-selective (non-imidazole) neutral antagonist JNJ 7777120 (>1000 fold selective over other histamine receptor subtypes) was discovered by J&J following a high throughput screen. This compound has equal affinity for human, mouse and rat H4Rs (68, 69) initially making it a valuable compound to extend the in vitro pharmacology to in vivo studies. Intriguingly, the more research is performed on this compound, the more "active" it becomes. It was reported that in a steady-state GTPase assay JNJ 7777120 acts as a partial inverse agonist on the human H4R. In the same assay, however, it behaves as a partial agonist on mouse, rat and dog H4Rs (70). Very recently JNJ 7777120 was identified as partial biased agonist, able to recruit beta-arrestin in a G protein-independent manner (71) (see 8.4). Hence, the usefulness of this compound as reference neutral H4R antagonist is currently under debate. JNJ 7777120 has a poor half-life (2h) upon oral administration to rats (72, 73), which should be taken into account when using it for in vivo H4R targeting.

Another class of H4R antagonists that has gained enormous interest from industry is the aminopyrimidines. According to filed patents, Bayer Healthcare AG, Palau Pharma, Pfizer and J&J all have their current research programs based on these structures. Also Abbott Laboratories followed this series of pyrimidines, eventually leading to the anti-inflammatory A-987306 (Ki H4R = 5.8 nM) with very good pharmacokinetic properties and an in vivo half-life up to 3.7 hours (74).

The new class of quinoxalinone H4R ligands was found in a fragment-based drug discovery project at the VU University Amsterdam as well as at J&J, where they originated from 5-HT3 ligand discovery projects (75). The VU University Amsterdam developed these quinoxalines by combining clozapine and JNJ 7777120 in a pharmacophore model. These compounds were shown to have anti-inflammatory properties in a carrageenan-induced paw-edema model in rats (76). Subsequent scaffold hopping resulted in the discovery of inverse agonists quinazolines (56). Interestingly those structures can be extended with a sulfonamide group (resulting in the inverse agonists quinazoline sulfonamides) with a variety of substituents without loosing affinity for the H4R (77). H4R ligands and their clinical applications have been extensively discussed in recent reviews by Smits et al. (78) and Engelhardt et al. (79).

6.3. H4R radioligands

The agonists histamine and UR-PI294 (61) can be readily labeled with tritium and used as radioligand in ligand/receptor binding studies. Since histamine and UR-PI294 interact with high affinity to other histamine receptor subtypes (i.e. H3Rs), only 4-methylhistamine is a H4R-selective radioligand.

The only available H4R antagonist radioligand is (3H)-JNJ 7777120 (69), which is particularly useful when studying mutant H4Rs that are unable to bind histamine.

7. ELUCIDATION OF LIGAND BINDING MODES IN H4R

By relating distinct pharmacology of H4R orthologs to their sequence divergence, information can be obtained on ligand binding modes (fig. 7) and receptor activation. To this end, chimeras between H4R of human and other species have been constructed and evaluated, subsequently followed by site-directed mutagenesis to pinpoint the specific amino acid (s) involved in ligand interaction.

7.1. Species differences

Recently, an extensive study was performed in which H4Rs of several species were characterized (28). These H4R orthologs were heterologously expressed and binding affinities of several well-known ligands were determined. Histamine and 4-methylhistamine show an equal trend when comparing their H4R binding affinities between species. VUF8430 (58) shows a different pattern with a decreased affinity for pig, dog, and guinea pig H4R, in comparison to other H4R orthologs. Clozapine loses affinity for the pig, dog, mouse and rat, compared to the human H4R. Interestingly, monkey and guinea pig show higher affinity for this tricyclic compound (28). Clobenpropit has lower affinity for pig and dog H4R, but similar affinities for the other tested H4R species variants, whereas the inverse agonist thioperamide has equipotent affinity for all H4R orthologs. Both antagonists JNJ 7777120 and VUF6002 show significant lower affinity for monkey, pig, dog and guinea pig receptors as compared to the other tested species.

Most of the tested ligands show significant differences in affinity at the different species variants (24). Therefore, careful in vitro pharmacological characterization of H4R orthologs is of major importance as several animal models are used to study H4R (ligands) function in vivo. Considering the H4R differences between animals, caution should be taken in selecting appropriate species to evaluate specific ligands. JNJ 7777120 is currently the most commonly used reference compound to block H4Rs in animal models (55). However, recent developments request re-evaluation whether JNJ 7777120 should still be used as reference antagonist (see sections 6.2 and 8.4).

Chimeras between human and pig H4Rs, followed by site-directed mutagenesis, identified residues at positions 45.55, 4.57, 5.39 and 5.43 responsible for the observed species differences in ligand affinity (28). Residue 5.39 was shown to be also responsible for the differences in ligand binding between human and monkey (28), whereas Phe45.55 in EL2 (fig 4) of the human H4R was identified to be responsible for the increase in agonist affinity compared to mouse H4R (80).

7.2. Binding pockets in H4R

H4R ligands bind the receptor in the common binding pocket located in the cavities formed by the TM helices. This binding pocket consists of two subpockets, which are designated i and ii (81). Pocket i is located between TM domains 2, 3 and 7, whereas pocket ii is located between TMs 3, 4, 5 and 6. Besides these general pockets, two hydrophobic subpockets exist in pocket ii. The first hydrophobic subpocket is located between TM3, TM4, TM5 and TM6 in the vicinity of Trp6.48. The second hydrophobic subpocket is situated between TM3, TM5, TM6 and EL2 (and EL3) towards the extracellular side of the receptor (82). Compound classes such as the aminopyrimidines, quinoxalines and quinazolines are proposed to occupy these subpockets with their hydrophobic moieties (82).

7.3. Binding mode of histamine

Although the similarity with the other histamine receptor family members is relatively low (55), earlier identified key residues in ligand binding to the H1R-H3R, are also present in the H4R (83). The amine group of histamine interacts with Asp3.32 (83, 84) in subpocket i of H4Rs and the protonated nitrogen of the basic imidazole ring is forming a hydrogen bond with Glu5.46 in subpocket ii (fig. 7) (83, 85). Interestingly, both in H3R and H4R a Glu residue is present at position 5.46, compared to Asn in H1R and Thr in H2R. The importance of Glu5.46 is also shown by the inability or decrease in binding of (3H)-histamine to H4Rs in which Glu5.46 is substituted with Ala or Gln, respectively (83, 84). It is hypothesized that the presence of this negatively charged residue is the reason for increased binding affinity of histamine to H3R and H4R as compared to H1R and H2R (83). Asn4.57 and Ser6.52 were also suggested to be important residues in histamine-induced activation of H4R (84), but do not play a direct critical role in histamine binding (28). The non-imidazole small molecule agonist VUF8430 is believed to bind in a similar binding mode as histamine (83). In contrast, for the tricyclic agonist clozapine the interaction with residue Glu5.46 is less pronounced, as illustrated by its ability to bind the mutated H4R-Glu5.46 (83, 86, 87).

7.4. Binding mode JNJ 7777120

Although JNJ 7777120 binds in the same binding cavity as histamine, the actual binding mode is proposed to be different. This is supported by the fact that (3H)-histamine binding is almost lost in an E4.56Q mutant, but (3H)-JNJ 7777120 is still able to interact with this mutant receptor. The positively charged piperidine nitrogen atom is believed to form a hydrogen bond with Asp3.32 (83). The indole nitrogen atom donates a hydrogen bond to the carboxylate group of Glu5.46. Considering the drop in affinity for JNJ 7777120 on the L5.39V mutant compared to a increase in affinity for clozapine for the same H4R mutant, the chlorinated aromatic ring of JNJ 7777120 is believed to be positioned in the vicinity of this residue (fig. 7) (86).

The binding mode of a variety of H4R ligands has recently been extensively reviewed by Istyastono et al. (86).

8. SIGNALING OF H4R

Although H4R signaling has been studied in cells endogenously expressing H4Rs, determination of the exact mechanisms of H4R functioning as well as ligand characterization is predominantly performed in H4R transfected cells. In HEK293T cells, transfected with H4R and a cAMP-response element (CRE) reporter gene construct, H4R activation inhibits forskolin-induced cAMP production by adenylyl cyclase (AC) and subsequent CRE-driven gene transcription in a pertussis toxin (PTX)-sensitive manner, indicating the involvement of Galphai/o proteins (14, 16, 36). Interestingly, no changes in cAMP levels were observed in H4R-expressing HEK293 cells in response to histamine, even though other Galphai-mediated responses (mitogen-activated protein kinase (MAPK) phosphorylation and PTX sensitivity) could be measured (15). In addition, in the presence of chimeric Galphaq/i1/2, Galphaq/i3 or Galpha16 proteins H4R stimulation resulted in increased calcium mobilization in transfected CHO, COS-7 and HEK293 cells (15).

Furthermore, H4R stimulation resulted in an increase in (35S)-GTPgammaS binding in SK-N-MC and HEK293 cells. Interestingly, increased basal levels are observed in this assay when comparing H4R expressing cells with control cells. Thioperamide inhibits this basal signaling. The same phenomenon is seen in Sf9 insect cells co-expressing mammalian Galphai2 and Gbeta1gamma2 proteins (88). This indicates that H4Rs are constitutively active (i.e. signal in absence of a ligand) and thioperamide is acting as inverse agonist (54). Constitutive activity has been demonstrated for other histamine receptor family members (89-92) but in the steady state GTPase assay, the H4R shows the highest amount of constitutive activity compared with H1R-H3R (88).

8.1. Calcium mobilization

Histamine induces calcium release from intracellular calcium stores in both mast cells and eosinophils (65), which was antagonized by thioperamide indicating a role for H4Rs (64). Direct proof of H4R-mediated calcium mobilization came from experiments with H4R knock out mice, as mast cells isolated from these mice failed to mobilize calcium in response to 30 mM histamine (65). Upon histamine binding, H4R couples to PTX-sensitive Galphai/o proteins which subsequently can activate phospholipase C (PLC) via their Gbetagamma subunits. PLC hydrolyzes phosphatidylinositol 4,5-biphosphate (PIP2) to diacylglycerol and inositol 1,4,5-triphosphate (InsP3). InsP3 can bind to its receptor on the endoplasmatic reticulum (ER) membrane, thereby stimulating the release of calcium, which can on its turn induce chemotaxis towards histamine (65) (fig. 8).

8.2. Migration

More than 25 years before the identification of H4R, Clark and colleagues already observed that low concentrations of histamine induced chemotaxis of eosinophils (93). Soon after the introduction of the newest member of the histamine receptor family a role for H4R in this eosinophil migration was discovered (94). More recently, the chemotactic properties of isolated human eosinophils were tested in the presence of several H1R/H3R agonists, but only histamine could provoke a response. Interestingly, only H3R/H4R antagonists were able to block this effect. Since eosinophils do not express H3R, the H4R is responsible for the histamine-induced migration of eosinophils (94-96).

The actin cytoskeleton determines both cell morphology and cell movement. The real "moving" is occurring because the G actin monomers are rearranged to form F actin polymers. An imaging approach was used to show that stimulation of H4R-expressing eosinophils induces maximal actin polymerizaton to F actin already after 5-10 seconds (64, 96). Histamine-induced actin polymerization was inhibited by JNJ 7777120 and H3R/H4R inverse agonist thioperamide, confirming an exclusive role for H4Rs (96). H4R agonists also induced F-actin polymerization via H4Rs in MoDC as shown by the observation that clobenpropit-induced polymerization could be blocked by JNJ 7777120 (38).

In addition, H4R agonists induce a rapid PTX-sensitive shape change in eosinophils, which was blocked by H4R antagonists, but not by antagonists of other histamine receptors (64). In comparison to the chemokine eotaxin (CCL11)-induced eosinophil migration, histamine was found to be a relative weak chemotactic factor in a whole blood migration assay (95). Of concern is the observation that histamine failed to stimulate guinea-pig eosinophils in a variety of these shape change or chemotaxis assays (64). This again emphasizes the importance of understanding the species differences in H4R pharmacology.

Histamine stimulation of the H4R also induces mast cell migration in vitro. Investigation of the signaling components proofed that both Galphai/o and PLC are involved in the downstream signaling events. Histamine inhalation led to an increase in the total number of mast cells and sub-epithelial mast cells in trachea of mice, which could be inhibited by JNJ 7777120 (69), thereby ruling out H1R and H2R-mediated effects.

In an ex vivo migration assay, skin dendritic cells isolated from mice and guinea pig were able to the enhance chemotaxis upon stimulation with histamine or H3R/H4R ligand clobenpropit. These observations were confirmed in in vitro migration experiments with bone marrow derived dendritic cells from human and mice (29). This histamine/clobenpropit-induced effect was fully inhibited by JNJ 7777120. However, the histamine-induced chemotaxis could also be fully inhibited by H1R antagonist diphenhydramine (29), suggesting crosstalk between these two histamine receptor subtypes.

In a human epidermis and murine in vivo assay it was shown that Langerhans cell migration from the epidermis was increased upon H4R activation. In addition, a downregulation of the production of CCL2 was observed (37).

More recently the effect of histamine on migration of human fetal lung fibroblasts to human plasma fibronectin (HFn) in vitro was examined (97). Histamine did not induce migration of these cells, but appeared to potentiate the migration to Hfn in a bell shaped relation. Addition of JNJ 7777120 inhibited this effect, as did PTX, indicating an H4R/Galphai/o-mediated process (fig. 8) (97).

8.3. Modulation of protein expression and physiological effects regulated by H4R

Leukocyte chemoattractants affect the function and cell surface expression of adhesion molecules, which play an important role in the interaction of leukocytes with the microvascular endothelium. Histamine is also able to upregulate adhesion molecules (e.g. CD11b/CD18 and CD54) in eosinophils. This upregulation occurs already within 10 min after stimulation (95). Upregulation of CD11b could be inhibited by preincubation with thioperamide (64) indicating a role for the H4R. In addition, pre-stimulation with histamine increased the amount of eosinophils to migrate towards CCL11, which could also be inhibited by thioperamide (64).

A potentiation is also observed for CXCL12 chemotactic activity on the precursor mast cell population upon stimulation with histamine or supernatants from IgE-activated mast cells. Small interfering RNA (siRNA) was used to specifically block each histamine receptor subtype. This resulted in the identification of the H4R as the one responsible for the observed synergy. In addition, JNJ 7777120 was able to completely block this potentiation. Interestingly, CXCR4 (receptor for CXCL12) protein levels did not change. An explanation was put forward, in which the authors speculated on a shared signaling pathway between H4R and CXCR4 downstream of the Galphai/o protein (98).

Histamine suppresses polyIC-induced IL-12p70 production of MoDC via different pathways activated by H2R and H4R. H4R activates the MAPK pathway, resulting in the activation of AP-1 (fig. 8). Interestingly, this was independent of ERK1/2 phosphorylation. AP-1 induction by clobenpropit could be blocked by JNJ 7777120, demonstrating that AP-1 is indeed induced by H4R stimulation. The H2R-mediated IL-12p70 suppression involves cAMP production (38).

A recent study in peripheral blood mononuclear cell (PBMC) cultures from non-atopic human volunteers showed a possible role for the H4R in modulating signaling pathways via STAT1. It was already shown that H4R might play a role in lymphocyte signaling and in Th2 differentiation (69, 99, 100). JNJ 7777120 stimulated the production of STAT1alpha and its downstream phosphorylation in the non-atopic group. JNJ 7777120 could also enhance the binding of STAT1 to DNA. A model was proposed, in which histamine acts through the H4R on T cells, thereby inhibiting STAT1 activation and thus helps to drive the Th2 polarization by inhibiting IFN-gamma mediated events (101, 102).

Histamine-free histidine decarboxylase deficient mice (HDC-/-) show a functional deficit in invariant natural killer (iNKT) cells. This is clearly demonstrated by decreased IL-4 and IFN-gamma production. Addition of histamine induced a functional recovery that is mediated by the H4R, since JNJ 7777120 could prevent this. To unambiguously state that the H4R is involved, iNKT cells of H4R knock out mice were also tested. These cells generate lower amounts of circulating cytokines than WT mice, clearly showing a role for H4Rs in this process (103).

H4R proteins were recently shown to be involved in the production of interleukin 6 (IL-6) from mouse bone marrow-derived mast cells. Both histamine and H4R agonist JNJ 28610244 can induce the transient production of this cytokine. In turn, H4R antagonists could inhibit this effect. Additionally, H4R potentiates the prolonged lipopolysaccharide- (LPS) induced IL-6 production via MAPK (ERK) and Src/PI3Kgamma pathways, suggesting crosstalk between toll-like receptors (TLR) and H4R signaling pathways (104).

Murine and human progenitor cell populations express functional H4R. Upon activation of the receptor, the cells show a reduced growth factor-induced cell cycle progression. As a consequence, myeloid, erythroid and lymphoid colony formation is decreased, hence the cells show reduced proliferation. The H4R thus prevents induction of cell cycle genes, presumably via its cAMP and subsequent protein kinase A (PKA) pathway (fig. 8). A special role for the H4R was confirmed with H4R selective antagonists that restored cell cycle progression. Very interesting is the observation that the arrest of growth factor-induced G1/S phase transition (quiescence) protects the murine and human progenitor cells from the toxicity of the cell cycle dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy (103). This opens a new possible role for H4R targeting drugs (105).

The function of H4R in the stomach was explored in ulcer models in both rat and mice. JNJ 7777120 (10-30 mg/kg sc) reduced the indomethacin-induced gastric mucosal damage by approximately 70% in the conscious rat. In addition, JNJ 7777120 reduced indomethacin- and bethanechol-induced gastrolesive effects in conscious mice. H4R agonist VUF 10460 (4- (4-methylpiperazin-1-yl)-6-phenylpyrimidin-2-amine) reduced indomethacin-induced lesions in the rat, but not in mice (106).

8.4. Beta-arrestin recruitment to H4R / Biased agonism

In addition to G protein coupling, both histamine and 4-methylhistamine induce beta-arrestin recruitment to H4R as revealed in a protein-fragment complementation-based beta-arrestin recruitment assay (i.e. Tango assay Invitrogen) . Histamine-induced beta-arrestin recruitment to the H4R was recently confirmed by Rosethorne and Charlton in PathhunterTM U2OS-H4 / beta-arrestin cells (107). Interestingly, this recruitment was PTX insensitive, indicating a Galphai-independent mechanism. Surprisingly, the antagonist JNJ 7777120 was shown to behave as a partial agonist by inducing Galphai-independent beta-arrestin recruitment to the H4R (71).

Moreover, in the same cell line both histamine and JNJ 7777120 stimulate phosphorylation of ERK in time frames that are characteristic for G protein-mediated (seconds) or beta-arrestin-mediated (~20min) signaling, respectively (fig. 8) (71).

These recent findings indicate that the H4R ligands may display distinct efficacies towards G protein-dependent and -independent pathways, with the reference antagonist JNJ 7777120 turning out to be a partial agonist with bias towards beta-arrestin driven pathways. Although this ligand-directed signaling is very exciting and opens the door for the development of pathway-selective compounds it also invites to re-assess the efficacies of known H4R ligands towards G-protein-independent pathways (70, 71, 107).

9. FINAL REMARKS

There is substantial evidence that H4R plays a role in inflammatory processes, based on their localization pattern on cells of hematopoietic origin and the medicinal in vivo studies so far. H4R inhibitors can be interesting drugs to counteract allergic reactions, hence the interest of the pharmaceutical industry. However, recently also other expression patterns were identified, such as the brain. In addition, a link between H4R and diseases as rheumatoid arthritis (108), colon (109, 110) and breast cancer (111) was postulated. This opens doors for even more possibilities than the initial anti-inflammatory properties.

New development programs for the search for novel H4R ligands are ongoing. In parallel with the computational design of ligands, and our increased understanding of receptor-ligand binding interactions the ultimate goal is to develop H4R-specific ligands with on forehand known efficacies and binding modes.

10. ACKNOWLEDGEMENTS

All authors are participating in the EU-KP7 COST program BM0806 (Histamine H4 receptor network). CdG is supported by VENI Grant 700.59.408 from the Netherlands Organization for Scientific Research.

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77. R. A. Smits, M. Adami, E. P. Istyastono, O. P. Zuiderveld, C. M. van Dam, F. J. de Kanter, A. Jongejan, G. Coruzzi, R. Leurs and I. J. de Esch: Synthesis and QSAR of quinazoline sulfonamides as highly potent human histamine H4 receptor inverse agonists. J Med Chem, 53 (6), 2390-400 (2010)
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80. H. D. Lim, A. Jongejan, R. A. Bakker, E. Haaksma, I. J. de Esch and R. Leurs: Phenylalanine 169 in the second extracellular loop of the human histamine H4 receptor is responsible for the difference in agonist binding between human and mouse H4 receptors. J Pharmacol Exp Ther, 327 (1), 88-96 (2008)
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83. A. Jongejan, H. D. Lim, R. A. Smits, I. J. de Esch, E. Haaksma and R. Leurs: Delineation of agonist binding to the human histamine H4 receptor using mutational analysis, homology modeling, and ab initio calculations. J Chem Inf Model, 48 (7), 1455-63 (2008)
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92. S. Morisset, A. Rouleau, X. Ligneau, F. Gbahou, J. Tardivel-Lacombe, H. Stark, W. Schunack, C. R. Ganellin, J. C. Schwartz and J. M. Arrang: High constitutive activity of native H3 receptors regulates histamine neurons in brain. Nature, 408 (6814), 860-4 (2000)
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102. B. Horr, H. Borck, R. Thurmond, S. Grosch and F. Diel: STAT1 phosphorylation and cleavage is regulated by the histamine (H4) receptor in human atopic and non-atopic lymphocytes. Int Immunopharmacol, 6 (10), 1577-85 (2006)
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103. M. C. Leite-de-Moraes, S. Diem, M. L. Michel, H. Ohtsu, R. L. Thurmond, E. Schneider and M. Dy: Cutting edge: histamine receptor H4 activation positively regulates in vivo IL-4 and IFN-gamma production by invariant NKT cells. J Immunol, 182 (3), 1233-6 (2009)

104. P. Desai and R. L. Thurmond: Histamine H (4) receptor activation enhances LPS-induced IL-6 production in mast cells via ERK and PI3K activation. Eur J Immunol, 41 (6), 1764-73 (2011)
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105. A. F. Petit-Bertron, F. Machavoine, M. P. Defresne, M. Gillard, P. Chatelain, P. Mistry, E. Schneider and M. Dy: H4 histamine receptors mediate cell cycle arrest in growth factor-induced murine and human hematopoietic progenitor cells. PLoS One, 4 (8), e6504 (2009)
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106. G. A. Coruzzi, M.' Pozzoli, C.; Smits, R.; De Esch, I.; Leurs, R.: Gastroprotective effects of histamine H4 receptor ligands in rodent ulcer models. Proceedings of the Britisch Pharmacological Society, 7 (4) (2010)

107. Y. Ikawa, M. Suzuki, S. Shiono, E. Ohki, H. Moriya, E. Negishi and K. Ueno: Histamine H4 receptor expression in human synovial cells obtained from patients suffering from rheumatoid arthritis. Biol Pharm Bull, 28 (10), 2016-8 (2005)
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109. C. Varga, K. Horvath, A. Berko, R. L. Thurmond, P. J. Dunford and B. J. Whittle: Inhibitory effects of histamine H4 receptor antagonists on experimental colitis in the rat. Eur J Pharmacol, 522 (1-3), 130-8 (2005)
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Key Words: Histamine H4 receptor, Histamine, GPCR, Inflammation, Dimerization, Review

Send correspondence to: Rob Leurs, VU University Amsterdam, Leiden, Amsterdam Center for Drug Research, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands, Tel:31205987579, Fax:31205987610, E-mail:r.leurs@vu.nl