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The histamine H(4) receptor subtype (H(4)R) belongs to the class 1 of G protein-coupled receptors and is involved in inflammatory and immunological processes. The aim of this study was to elucidate the importance of extracellular regions for the large species differences between human (h) and canine (c) H(4)R. Therefore, chimeric receptors were generated by replacing corresponding domains of the hH(4)R with canine N-terminus (h(cNT)H(4)R) and three canine extracellular loops, respectively (h(cE1)H(4)R, h(cE2)H(4)R and h(cE3)H(4)R). Wild type and chimeric H(4) receptors were expressed in Sf9 insect cells and subsequently characterized in [(3)H]histamine-binding experiments and in steady-state GTPase activity assays, where standard H(4)R ligands histamine, 5-methylhistamine, thioperamide, 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) and clozapine were examined. The exchange of N-terminus or first extracellular loop did not influence hH(4)R pharmacology. The effect of altered second extracellular loop (E2-loop) and third extracellular loop (E3-loop) was rather ligand specific than agonist/inverse agonist specific. At h(cE3)H(4)R, the potency of histamine and 5-methylhistamine significantly decreased. The efficacy of the inverse agonist thioperamide was strongly reduced at h(cE2)H(4)R and h(cE3)H(4)R. Surprisingly, JNJ7777120 as weak inverse agonist at hH(4)R exhibited partial agonistic activity at h(cE2)H(4)R and h(cE3)H(4)R. Molecular dynamic simulations suggest that the E2- and E3-loops are independently of each other involved in the partial/inverse agonism of JNJ7777120 and that E2- as well as E3-loop do not directly interact with JNJ7777120 in the binding pocket. In conclusion, our study indicates an involvement of the E2- and E3-loops in H(4)R activation process after binding of some but not all examined ligands.

The histamine H4 receptor (H4R) is expressed on cells of the immune system including eosinophils, dendritic cells, and T cells and plays an important role in the pathogenesis of bronchial asthma, atopic dermatitis, and pruritus. Analysis of the H4R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H4R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H4R in comparison to human H4R expressed in Sf9 insect cells. Only hH4R and cH4R exhibited a sufficiently high [3H]histamine affinity for radioligand binding studies. Generally, cH4R exhibited lower ligand-affinities than hH4R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH4R than at other H4R species orthologs. Unlike the other H4R species orthologs, hH4R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H4R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH4R, rH4R, and mH4R, whereas at hH4R, JNJ7777120 was a partial inverse agonist. H4R agonists from the class of NG-acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H4R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H4R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H4R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of “the prototypical H4R antagonist” JNJ7777120 as H4R antagonism.

Residues in the second extracellular loop (e2) play a role in ligand binding in certain aminergic G protein coupled receptors (GPCRs). N-[3-(1H-Imidazol-4-yl)propyl)]guanidines and N (G)-acylated derivatives are more efficacious and potent agonists at fusion proteins of the guinea pig histamine H(2) receptor and the short splice variant of G(salpha), G(salphaS) (gpH(2)R-G(salphaS)) than at the human isoform (hH(2)R-G(salphaS)). To elucidate the structural basis for this species-selectivity, we generated a mutant hH(2)R-G(salphaS) fusion protein with the four e2 residues differing in both species isoforms mutated into the gpH(2)R sequence, and a reverse mutant of the gpH(2)R-G(salphaS) with the corresponding mutations into the human species. In a steady-state GTPase activity assay, efficacies and potencies of guanidine-type agonists were similar at mutant and wild-type receptors indicating that e2 does not contribute to the species-selectivity. In several class 1 GPCRs, amino acids in the vicinity of a highly conserved cysteine in e2 participate in ligand binding. A three-dimensional homology model of the hH(2)R predicted Lys-173 and Lys-175, adjacent to Cys-174 in e2, to be in close proximity to the binding pocket of guanidine-type agonists. To elucidate the putative role of both residues for interactions with the agonists, two hH(2)R-G(salphaS) fusion proteins, with single-point mutations of Lys-173--Ala-173 and Lys-175--Ala-175 respectively, were generated. With these mutants, the efficacies and potencies of small and bulky H(2)R agonists did not significantly change. However, increases in GTPase activity upon agonist stimulation were reduced, suggesting an impact of both residues on the efficiency of receptor coupling to G(salphaS). In conclusion, none of the point mutations generated within this study substantially altered the efficacies and potencies of guanidine-type agonists relative to the wild-type receptors, suggesting that these residues do not directly face the H(2)R guanidine-binding pocket. Thus, agonist binding to residues in e2 is relevant for some but not all aminergic GPCRs.

The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca(2+)]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca(2+)]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.

The histamine H₄ receptor (H₄R) is expressed on cells of the immune system including eosinophils, dendritic cells, and T cells and plays an important role in the pathogenesis of bronchial asthma, atopic dermatitis, and pruritus. Analysis of the H₄R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H₄R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H₄R in comparison to human H₄R expressed in Sf9 insect cells. Only hH₄R and cH₄R exhibited a sufficiently high [³H]histamine affinity for radioligand binding studies. Generally, cH₄R exhibited lower ligand-affinities than hH₄R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH₄R than at other H₄R species orthologs. Unlike the other H₄R species orthologs, hH₄R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H₄R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH₄R, rH₄R, and mH₄R, whereas at hH₄R, JNJ7777120 was a partial inverse agonist. H₄R agonists from the class of N ( G )-acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H₄R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H₄R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H₄R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of "the prototypical H₄R antagonist" JNJ7777120 as H₄R antagonism.

Formyl peptides activate superoxide anion (O2-) formation in human neutrophils and in HL-60 cells via pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins), and histamine (HA) mediates inhibition of O2- formation via H2-receptors. We have studied the effects of lipophilic arpromidine-derived guanidines, which are potent, full H2-receptor agonists in the guinea pig atrium, on O2- formation and on activation of G-proteins in HL-60 membranes and on purified G-proteins. We have also studied the effects of a HA trifluoromethyl-toluidide derivative (HTMT), a cationic-amphiphilic HA derivative which activates O2- formation in HL-60 cells through a mechanism which is independent of known HA receptor subtypes, on G-protein activation. Guanidines, at concentrations, up to 30 mumol/l inhibited and, at concentrations above 30 mumol/l, enhanced formyl peptide-induce O2- formation in neutrophils. In HL-60 cells, guanidines per se activated O2- formation. The stimulatory effects of guanidines on O2- formation were not inhibited by H1- or H2-receptor antagonists. In HL-60 membranes, guanidines and HTMT, activated high-affinity GTPase in a PTX-sensitive manner. These substances also increased GTP hydrolysis effected by transducin and Gi/G(o)-proteins. Our data suggest that lipophilic guanidines and HTMT may act as receptor-independent activators of PTX-sensitive G-proteins, resulting in stimulation of O2- formation.