Molecular and Cellular Analysis of the Histamine H₄-Receptor

Background: Histamine plays an important role as local mediator and neurotransmitter and exerts its effects through H₁-, H₂-, H₃- and H₄-receptors (H_xRs) [1]. H_1-3Rs are all well served with potent and selective agonists and antagonists [2]. The H₄R was cloned independently by seven groups [3-9]. The H₄R is predominantly expressed in white blood cells, specifically T-lymphocytes, mast cells and eosinophils, and activation of the H₄R stimulates those cells [10-13]. Based on its expression pattern, the H₄R is assumed to play an important role in the pathogenesis of bronchial asthma and autoimmune diseases [14]. The H₄R couples to pertussis toxin-sensitive G-proteins of the G_i-family to mediate adenylyl cyclase inhibition and activation of phospholipase C, the latter effect probably being mediated by Gβγ-subunits [1,11].

Structurally, the H₄R is most closely related to the H₃R [1]. Therefore, it is not surprising that many H₃R ligands also display substantial affinity for the H₄R [1]. In addition, the H₄R does bind some H₂R ligands [6]. Recently, a potent and selective H₄R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ 7777120)) was introduced [15]. In fact, this compound exhibits potent anti-inflammatory effects in in vivo animal models [16]. The development of potent and selective H₄R ligands is complicated by the fact that H₄R isoforms from various species exhibit substantial differences in their pharmacological properties [17]. Species-specific pharmacology is a general feature of H_xRs [18-20].

Unresolved questions: Although the H₄R has attracted great attention during the past years [1,14], there are still several important unresolved issues. First, characterization of the H₄R with H₁R ligands is rudimentary, and the available data are controversial [1]. Second, there are some indications that the H₄R also binds H₂R ligands [6], but the large libraries of H₂R agonists and antagonists [21] were not yet studied systematically at the H₄R. Third, the atypical anti-psychotic drug clozapine is a partial H₄R agonist [3], but it is unclear whether the H₄R is involved in the clozapine-induced agranulocytosis [22]. Fourth, the structural basis for the pharmacological differences among H₄R species isoforms is unknown [17]. Fifth, in human HL-60 promyelocytes, a H_xR with unique pharmacological properties was identified [23,24], but it is unclear whether this H_xR represents the H₄R or another as yet unidentified H_xR subtype.

Long-term project goals: The long-term goals of the project are to learn about the (patho)physiological role of the H₄R and to understand, at the molecular level, the interaction of the H₄R with agonists and antagonists. The achievement of the latter goal is the prerequisite for the future design of potent and selective H₄R agonists and antagonists. The availability of such ligands will then, in turn, facilitate further analysis of the (patho)physiological role of the H₄R and may ultimately result in the development of novel therapeutic agents.

Methodological approach: Recombinant H₄R species isoforms will be co-expressed with different G-proteins in Sf9 insect cells [25]. The structure/activity relationships of agonists and antagonists will be determined in steady-state GTPase and [³⁵S]GTPγS binding experiments [20,26]. Moreover, H₄R isoforms will be analyzed in receptor binding studies using the agonist [³H]histamine and the antagonist [³H]JNJ 7777120 as radioligands [3,15]. In collaboration with Prof. S. Dove from the Graduate Training Program, molecular modeling studies on the ligand-binding site of the H₄R will be performed [19]. Those studies will be complemented by site-directed mutagenesis of the H₄R isoforms [20]. Based on the aggregate results of those studies, novel H₄R ligands will be designed in collaboration with Profs. A. Buschauer and S. Elz from the Graduate Training Program. Moreover, functional experiments, for example measurements of chemotaxis [11], intracellular Ca²⁺ concentrations and inositol phosphate accumulation [23], will be conducted with various human myeloid cell lines.

Specific aims: The first specific aim of the project is to provide a systematic and comprehensive pharmacological analysis of all available H_xR agonists and antagonists at various H₄R species isoforms expressed in Sf9 cells coupled to various G-proteins. These studies will fill important gaps in our knowledge on the pharmacological properties of the H₄R and hopefully reconcile discrepancies in the literature [1]. In this context, we will specifically address the hypothesis that ligand-specific H₄R conformations exist that result in ligand-dependent coupling of the H₄R to various G-proteins and differences in the antagonist profile, depending on which agonist is studied [26]. Those studies will provide us with a detailed characterization of the H₄R pharmacophore and establish the basis for future mutagenesis and molecular modeling studies. Mutagenesis and modeling studies will further refine our knowledge on the H₄R ligand binding site. Based on the available data, we expect that the H₄R is promiscuous and interacts with both H₁- and H₂R ligands, but in a different order of affinity than the H₁R and H₂R. Those studies will, therefore, provide us with new lead strategies for the future design of H₄R ligands.

The second specific aim of the project is to analyze the H₄R in human cell lines. As starting point, we will analyze the pharmacological profile of the H₄R in purified human eosinophils measuring chemotaxis and intracellular Ca²⁺ concentration. Subsequently, we aim at identifying a suitable human cell culture model for the H₄R. The availability of such a system would be most valuable for elucidating the (patho)physiological function of the H₄R since studies with purified eosinophils are difficult to perform. We will focus our attention on a specific eosinophilic HL-60 subclone [27] and the human mast cell line HMC-1 [28]. In those cell lines, we will detect H₄R transcripts via reverse transcriptase-PCR and search for novel H_xR isoforms by PCR with degenerate primers. The H₄R will be analyzed in the chemotaxis, intracellular Ca²⁺ and inositol phosphate accumulation assays. By combining molecular biology and pharmacological techniques, we will specifically address the question whether the previously identified H_xR in HL-60 cells with unique pharmacological properties [23,24] represents, indeed, the H₄R. In order to address the issue whether clozapine-induced agranulocytosis is mediated by the H₄R, we will examine the effect of the H₄R on apoptotic pathways in myeloid cell lines.

References

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