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CELLULAR AND MOLECULAR

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

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands (H.D.L., A.J., R.A.B., E.H., I.J.P.d.E., R.L.); and Boehringer Ingelheim Austria GmbH, Vienna, Austria (E.H.)

Received for publication April 24, 2008
Accepted July 16, 2008.

	Abstract

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Using the natural variation in histamine H₄ receptor protein sequence, we tried to identify amino acids involved in the binding of H₄ receptor agonists. To this end, we constructed a variety of chimeric human-mouse H₄ receptor proteins to localize the domain responsible for the observed pharmacological differences between human and mouse H₄ receptors in the binding of H₄ receptor agonists, such as histamine, clozapine, and VUF 8430 [S-(2-guanidylethyl)-isothiourea]. After identification of a domain between the top of transmembrane domain 4 and the top of transmembrane domain 5 as being responsible for the differences in agonist affinity between human and mouse H₄Rs, detailed site-directed mutagenesis studies were performed. These studies identified Phe¹⁶⁹ in the second extracellular loop as the single amino acid responsible for the differences in agonist affinity between the human and mouse H₄Rs. Phe¹⁶⁹ is part of a Phe-Phe motif, which is also present in the recently crystallized β₂-adrenergic receptor. These results point to an important role of the second extracellular loop in the agonist binding to the H₄ receptor and provide a molecular explanation for the species difference between human and mouse H₄ receptors.

The histamine H₄ receptor (H₄R) is the latest member of the histamine receptor family and was first reported in 2000 as a direct consequence of the efforts to elucidate the human genome sequence (Oda et al., 2000). The H₄R is mainly expressed in a variety of cells of the immune system, and activation of the H₄R results in chemotaxis of eosinophils, mast cells, and monocyte-derived dendritic cells, and modulation of chemical mediator production (Hofstra et al., 2003; Ling et al., 2004; Gutzmer et al., 2005; Dunford et al., 2006). Current experimental evidence suggests that the H₄R is a potential drug target for inflammatory diseases, such as allergic asthma, rheumatoid arthritis, and pruritis (de Esch et al., 2005; Thurmond et al., 2008).

The H₄R protein is a G_i-protein-coupled receptor of 390 amino acids, possessing all the hallmarks of the family A or rhodopsin-like family of GPCRs (de Esch et al., 2005). Like all aminergic receptors, the H₄R possesses an aspartic acid residue in transmembrane domain (TM) 3, which has been implicated in the binding of a variety of aminergic ligands to their respective GPCR proteins (Shi and Javitch, 2002). Moreover, in TM5, a glutamic acid residue is conserved with the related H₃R protein, and this amino acid is most likely responsible for the relative high affinity of both receptors for their agonist histamine. Mutagenesis and computational studies indeed confirm the involvement of those two amino acids (Shin et al., 2002; Uveges et al., 2002; Jongejan et al., 2008).

It has been reported that histamine and clozapine have a lower affinity for the mouse H₄R in comparison with the human H₄R (Liu et al., 2001). The existence of such pharmacological differences is not surprising given that the homology between the protein sequences of human and mouse H₄Rs is relatively low (67%) for species orthologs. However, the previously identified aspartic acid and glutamic acid residues in TM3 and 5, respectively, are conserved in both species (Liu et al., 2001), indicating that additional residues are involved in the agonist binding to the H₄R protein. By using the natural variation in H₄R protein sequence, we tried to identify additional amino acids involved in agonist binding to the H₄R. We constructed a variety of chimeric human-mouse H₄R proteins to localize domains responsible for the observed pharmacological differences. After identification of a domain between the top of TM4 and the top of TM5 as being responsible for the observed differences in agonist affinity between human and mouse H₄R, our chimeric approach was followed by site-directed mutagenesis to identify amino acid residues involved in the agonist binding to the H₄R.

	Materials and Methods

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Materials. Dulbecco's modified Eagle medium (DMEM), penicillin, and streptomycin were purchased from Invitrogen Life Technologies (Merelbeke, Belgium). Fetal bovine serum was from Integro BV (The Netherlands), and cell culture plastic wares were obtained from Greiner Bio-one (Wemmel, Belgium). Tris base was from AplliChem (Darmstadt, Germany), whereas linear 25-kDa polyethyleneimine (PEI) was from Polysciences (Warrington, PA). Histamine dihydrochloride, clozapine, and 750-kDa PEI were purchased from Sigma-Aldrich (St. Louis, MO), whereas VUF 8430 (Lim et al., 2006) and JNJ 7777120 (Thurmond et al., 2004) were synthesized at the Department of Medicinal Chemistry (Vrije Universiteit, Amsterdam, The Netherlands). [³H]Histamine (18.10 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Oligonucleotides primers for PCR were synthesized by Isogen Bioscience (Maarsen, The Netherlands). Endonuclease restriction enzymes, T₄ DNA ligase, and Pfu DNA polymerase were obtained from MBI Fermentas (St. Leon-Rot, Germany).

View larger version (27K): [in this window] [in a new window]   Fig. 1. Snake plot of the human H4R protein. The residues of the human H4R identical with those of the mouse H4R are depicted in open circles. Differences between the two species homologs are indicated by the filled circles. The triangle pointing to the EL2 indicates the location of insertion of two residues in the mouse H4R, whereas the triangle pointing to the cytoplasmic tail shows the location of a deletion in the mouse H4R. The three arrows indicate the location of the used endonuclease restriction sites in the corresponding cDNA.

Site-directed mutagenesis, including the multiple residue alteration in the second extracellular loop (EL2), was performed with a fusion PCR method by introducing the mutation in oligonucleotide primers. To allow detection of expressed mutant receptor proteins, all mutant receptors were tagged with an N-terminal FLAG peptide (DYKDDDDK). This epitope tagging does not affect H₄R ligand binding (Shin et al., 2002). The identity of mutant cDNAs was verified by sequence analysis at ServiceXS (Leiden, The Netherlands).

Cell Culture and Transfection. HEK 293T cells were maintained in DMEM supplemented with 10% fetal bovine serum, 50 IU/ml penicillin, and 50 µg/ml streptomycin in a humidified 5% CO₂ atmosphere at 37°C. For transfection, approximately 4 million cells were seeded in a 10-cm dish and cultured overnight. For transfection of each dish of cells, a transfection mixture was prepared in 0.5 ml of serum-free DMEM containing 5 µg of receptor plasmid and 25 µl of 1 mg/ml 25-kDa linear PEI. The mixture was incubated for 5 to 10 min at room temperature before it was added to the HEK 293T cell monolayer loaded with 5 ml of fresh cell culture medium. Two days after transfection, the cells were detached from the plastic surface by adding 5 ml/dish of phosphate-buffered saline containing 1 mM EDTA. Transfected cells were collected as pellets by centrifugation for 3 min at 200g and stored at -20°C until use.

[³H]Histamine Binding. For radioligand binding studies, pellets of transfected cells were homogenized in H₄R binding buffer (100 mM Tris-HCl, pH 7.4). Saturation binding assay was performed using different concentrations of [³H]histamine (18.10 Ci/mmol), whereas nonspecific binding was determined by incubation in the presence of 3 to 10 µM JNJ 7777120 in a total assay volume of 200 µl. For displacement studies, cell homogenates were typically incubated with 10^-4 to 10^-11 M ligands (stock concentration was 10 mM in dimethyl sulfoxide) in the presence of approximately 7 or 20 nM [³H]histamine for the human H₄R-like or mouse H₄R-like receptors, respectively, in a total volume of 200 µl. The reaction mixtures were incubated for 1 h at room temperature (22°C) and harvested on 96-well glass fiber C plates that were pretreated with 0.3% 750-kDa PEI. Binding data were analyzed using Prism 4.0 (GraphPad Software Inc., San Diego, CA).

NFAT-Luciferase Reporter Gene Assay. Approximately 4 million resuspended HEK 293T cells were transfected with a mixture containing 5 µg of receptor plasmid, 2.5 µg of a pcDNA3.1-based plasmid construct that bears both an NFAT-luciferase reporter gene and a copy of G_qi5 gene (Conklin et al., 1993), and 35 µl of 1 mg/ml 25-kDa linear PEI. The transfected cells were immediately exposed to ligands in a white 96-well plate and incubated for 24 h. The luciferase activity was measured with a Victor² microplate reader (PerkinElmer Life and Analytical Sciences).

Homology Modeling. The human H₄R was modeled based on the crystal structure of β₂-adrenergic receptor (Protein Data Bank code 2RH1) (Cherezov et al., 2007), which lacks the N-terminal tail and contains T₄ ligase structure in the third intracellular loop (IL). The latter was removed in the model template. Sequence alignment, homology modeling, and energy minimization were all performed using default settings in Molecular Operating Environment, version 2007.09 (Chemical Computing Group, Inc., Montreal, QC, Canada). A large part of IL3 of the H₄R was removed to fit the length of the IL3 of the template. Alignment constraints were applied to avoid gaps in TM domains between Thr¹⁴⁶ and Gly¹²⁸, Leu¹⁶⁷, and Pro¹⁴⁹ of β₂-adrenergic receptor and H₄R, respectively. In EL2, constraints were put between Cys¹⁹¹ and Cys¹⁶⁴, Phe¹⁹³, and Phe¹⁶⁸, as well as Tyr¹⁷⁴ and Ser¹⁵⁶ of the β₂-adrenergic receptor and H₄R, respectively. This alignment was used to run homology modeling and resulted in models with a preserved disulfide bridge.

	Results

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The Human and Mouse H₄Rs Display Pharmacological Differences. To delineate the structural basis for the species differences between human and rodent H₄R proteins (Liu et al., 2001), we transiently expressed human and mouse H₄R in human embryonic kidney HEK 293T cells. Upon expression, both mouse and human H₄R are well expressed but display a clearly different affinity for [³H]histamine as determined by saturation binding analysis (Table 1; Supplemental Fig. 1). Whereas the human H₄R binds [³H]histamine with a K_d value of 9 nM, the mouse homolog binds [³H]histamine with an almost 9-fold lower affinity of 78 nM (Table 1). Similar results were obtained by displacement of [³H]histamine binding with unlabeled histamine (Table 1). Furthermore, displacement studies also revealed species differences for other H₄R agonists, such as clozapine and VUF 8430 (Table 1). Both compounds have a higher affinity for the human H₄R(pK_i values of 6.35 and 7.46, respectively) in comparison with the mouse H₄R(pK_i values of 5.49 and 6.75, respectively). For the H₄R antagonist JNJ 7777120, no difference in affinity for the two receptor proteins was noticed (Table 1). The results obtained for JNJ 7777120, histamine, and clozapine corroborate previous findings (Liu et al., 2001; Thurmond et al., 2004).

Generation and Characterization of Human-Mouse H₄R Chimeras. The pharmacological differences observed between human and mouse H₄Rs are not surprising in view of the relative low homology between the human and mouse H₄R protein sequences (67%). As can be seen in the snake-plot in Fig. 1, the residues that differ between the two species variants are distributed throughout the entire receptor protein. TM domains TM2, TM3, and TM7 and the first and second intracellular loops are relatively conserved, but in the other regions, substantial differences are observed, including the insertion/deletion of nonconserved stretches in the EL2 (DEGSE^159–163 in the human H₄R and NSTNTKD^159–165 in the mouse H₄R) and the C-terminal tail (HSR^384–386 in the human H₄R and NQ^386–387 in the mouse H₄R) (Fig. 1).

In view of the relatively large sequence variation, we adopted a chimeric receptor strategy to determine structural features responsible for the differences between the human and mouse H₄R in the binding of the H₄R agonists histamine, clozapine, and VUF 8430. Unique endonuclease restriction sites in the cDNAs, encoding the human and mouse H₄Rs (Fig. 1), allowed us to conveniently swap receptor domains at the bottom of TM3 (using ClaI), at the middle of TM5 (using EcoRI), and at the bottom of TM6 (using BstXI). In chimeras 1, 3, and 5, we gradually increased the extent of human H₄R protein sequence, whereas in chimeras 2, 4, and 6, reciprocal changes were introduced (Table 2). All six chimeric H₄R proteins were expressed upon transient expression in HEK 293T cells and bound histamine with moderate to high affinity. Exchanging the first part of the mouse H₄R protein (up to Arg¹¹² at the bottom of TM3) with the related human protein sequence as in chimera 1 resulted in a chimeric protein with moderate affinity for histamine (Fig. 2, pK_i = 7.20) and a pharmacological profile similar to the mouse H₄R (Table 2). Chimera 2, in which the first part of the human H₄R protein is replaced by the mouse protein sequence (Met¹-Arg¹¹²), retained a high affinity for histamine (Fig. 2, pK_i = 7.84), similar to value obtained for the human H₄R (Table 2). The same trend was observed for the other H₄R agonists, clozapine and VUF 8430 (Table 2), indicating that the TM domains 1 to 3 are not involved in the differential binding of the H₄R agonists.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Homologous displacement of [3H]histamine binding to chimeras 1, 3, and 5 (A), 2, 4, and 6 (B), 7 and 8 (C), and 9 and 10 (D) transiently expressed in HEK 293T cells. Data shown are representative of at least three independent experiments.

When a larger part of the human H₄R sequence was used to replace the mouse sequence, as in chimera 3 (Met¹-Glu¹⁸² in the middle of TM5) and chimera 5 (Met¹-Ser³⁰⁴ in the bottom of TM6), the affinity of histamine increased to pK_i values of 7.98 (chimera 3) and 8.00 (chimera 5). These values are not significantly different from the value obtained for the binding of histamine to the human H₄R (Fig. 2; Table 2). The reciprocal counterparts, chimeras 4 and 6, showed opposite results. Both chimeric receptors lost affinity for histamine upon the introduction of larger stretches of the mouse H₄R sequence (Fig. 2; Table 2). The change in affinity of chimeras 3, 4, 5, and 6 is not only observed for histamine, similar changes in affinity were found for clozapine and VUF 8430 (Table 2).

The pharmacological properties of the chimeric human-mouse H₄R proteins 1 to 6 altogether suggest that the domain determining the differences in binding of the tested H₄ agonists is located between Arg¹¹² at the bottom of TM3 and Glu¹⁸² in the middle of TM5. To investigate this hypothesis, we constructed chimeras 7 and 8 in which just the middle parts of the receptor proteins (bottom TM3 up to the middle of TM5 using the ClaI and EcoRI DNA restriction sites; Fig. 1) were exchanged. As can be seen in Table 3 and Fig. 2, the affinity of all three agonists is dependent on the nature of the middle part of the H₄R protein sequence. Chimera 7, which possesses a small part of the human protein sequence (Arg¹¹² up to Glu¹⁸²) in a large mouse H₄R backbone, shows a human H₄R pharmacology with respect to the three tested agonists (Table 3). The findings with the reciprocal chimera 8 are completely in line with these results (Table 3; Fig. 2). Introduction of the mouse protein sequence between the bottom of TM3 and the top of TM5 (Arg¹¹² up to Glu¹⁸⁴) in the human H₄R backbone results in a chimeric protein with mouse H₄R pharmacology.

To further refine our search to the H₄R domain responsible for the human-mouse species difference, we created chimera 9 and chimera 10, which both have only small amino acid stretches exchanged within TM domains 4 and 5 (Table 3). Chimera 9 displays similar affinity for histamine (Fig. 2D), clozapine, and VUF 8430 as found for the human H₄R (Table 3), whereas the binding properties of chimera 10 closely resemble the binding profile of the mouse H₄R (Fig. 2; Table 3). These data clearly pinpoint to the receptor domain between the extracellular half of TM4 and extracellular half of TM5 (Val¹⁴¹ up to Glu¹⁸²) as a structural determinant for high affinity agonist binding to the H₄R.

Phenylalanine 169 in the EL2 As Determining Structural Element. As can be seen in Table 4, in the domain between the extracellular halves of TM4 and TM5, eight single amino acid residues are different, and a stretch of six amino acid residues (DEGSE^159–163, located adjacent to the conserved cysteine residue in the EL2) in the human H₄Ris replaced by the eight amino acid stretch NSTNTKD^159–165 of the mouse protein sequence. Therefore, nine new mutants of the human H₄R were constructed, including V141I, V153A, E155D, DEGSE^159–163NSTNTKD^159–165, F169V, S170T, A176T, S179M, and F180L (Table 4). All human H₄Rs mutants were well expressed after transient transfection of HEK 293T cells, and all proteins bound [³H]histamine in a saturable manner (Table 4). From the nine tested H₄Rmutants, the human H₄R F169V was the only receptor protein with a shift in agonist binding profile. Replacement of phenylalanine 169 in the human H₄R protein with a valine residue, as found in the mouse and rat H₄Rs, resulted in a clear change in binding profile (Table 4). The agonist affinities for the human H₄R F169V mutant are significantly different from the wild-type human H₄R. The observed pK_i values for the H₄R F169V of 7.12 (histamine), 5.52 (clozapine), and 7.05 (VUF8430) are similar to those observed for the mouse H₄R (Table 4). Furthermore, the F169V mutant behaves similarly to the mouse H₄R in response to histamine in a G_qi5-driven NFAT-luciferase reporter gene assay (Supplemental Fig. 2), with pEC₅₀ values of 5.86 ± 0.09 and 5.82 ± 0.03, respectively, which are lower than the potency at the human H₄R (pEC₅₀ = 6.29 ± 0.05). Despite the gaps between pK_i and pEC₅₀ values, the order of histamine potency at these receptors is maintained.

	Discussion

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Soon after the cloning of the human H₄R (Oda et al., 2000), rat, mouse, and guinea pig orthologs were reported as well (Liu et al., 2001). Mouse H₄R shows a substantial lower affinity for H₄R agonists, like histamine, VUF 8430 (Lim et al., 2006), and clozapine, whereas the H₄R antagonist JNJ 7777120 does not distinguish between the human and mouse H₄R (Thurmond et al., 2004; this study). Considering the relative low homology between the human and mouse H₄R protein sequences (67%), the observed pharmacological differences between human and mouse H₄Rs are not surprising. Despite the fact that such species differences might hamper preclinical evaluation of H₄R ligands, such a natural genetic variation also offers an opportunity to investigate receptor-ligand interactions.

In this study, we employed a chimeric receptor approach to localize receptor domains that could be responsible for the observed pharmacological differences between the human and mouse H₄Rs. Such an approach has been proven to be quite successful for a number of other GPCRs (Yin et al., 2004). All chimeric human/mouse H₄R proteins were expressed in HEK 293T cells at detectable levels as measured by [³H]histamine radioligand binding. The ability of the chimeras to act as functional receptors is most probably due to the reasonable homology between the human and mouse H₄Rs within the TM domains. A systematic analysis of a set of 10 chimeric H₄Rs enabled us to localize the receptor domain responsible for the difference in agonist binding to a region that spans from the extracellular half of TM4 to the extracellular half of TM5, including the EL2 (Table 3).

To identify specific amino acid residues between the extracellular half of TM4 and the extracellular half of TM5 responsible for the pharmacological differences, we employed site-directed mutagenesis of the human H₄R at all divergent positions. Our detailed mutagenesis studies identified Phe¹⁶⁹ in EL2 as the single amino acid responsible for the differences in affinity between the human and mouse H₄Rs. Mutation of Phe¹⁶⁹ of the human H₄R into the corresponding residue of the mouse H₄R, Val¹⁷¹, results in the mutant F169V human H₄R protein, which binds H₄R agonists like the mouse H₄R does. Val¹⁷¹ is conserved between mouse and rat H₄R, whereas the Phe¹⁶⁹ found in the human H₄Ris conserved in the monkey receptor. As expected on the basis of our mutagenesis results, the rat H₄R indeed shows a low affinity for histamine (Liu et al., 2001), whereas the monkey H₄R resembles the human H₄R in this respect (Oda et al., 2005).

The EL2 has been suggested to be involved in the binding of diverse types of ligands, such as aminergic receptor ligands (Laurila et al., 2007; Scarselli et al., 2007), nicotinic acid (Tunaru et al., 2005), leukotriene B₄ (Basu et al., 2007), or vasopressin (Conner et al., 2007). The involvement of EL2 in the binding pocket of aminergic GPCRs has originally been suggested for the dopamine D₂ receptor on the basis of detailed substituted-cysteine accessibility analysis (Shi and Javitch, 2004). Residues Ile¹⁸⁴ and Asn¹⁸⁶, which are, respectively, located +2 and +4 relative to the highly conserved Cys¹⁸² in EL2, were proposed to be directly involved in the binding pocket the D₂ receptor. These studies were supported by the observation that, in the X-ray crystal structure of bovine rhodopsin, EL2 forms a β hairpin located on top of the seven transmembrane pore protruding into the ligand binding cavity (Palczewski et al., 2000) and, therefore, may become part of the ligand binding pocket.

Recently, new structural information on the family of aminergic GPCRs has been obtained after the successful crystallization of the inactive conformation of the β₂ adrenergic receptor (β₂AR) (Cherezov et al., 2007; Rasmussen et al., 2007). The high-resolution crystal structure of the human β₂AR shows a highly intricate structure for the EL2 (Cherezov et al., 2007), which is clearly different from the EL2 in bovine rhodopsin. As found in the H₄R, the β₂AR contains a Phe-Phe motif in the EL2 (residues Phe¹⁹³-Phe¹⁹⁴). The crystal structure demonstrates the importance of Phe¹⁹³, as it protrudes deep into the binding pocket where it directly interacts with one of the aromatic rings of the 9H-carbazole group of the β₂AR antagonist carazolol (Cherezov et al., 2007; Rosenbaum et al., 2007). It is interesting that residue Phe¹⁹³ of the β₂AR aligns with Ile¹⁸⁴ of the D₂ receptor, which was shown to be involved in the binding pocket of D₂ receptor (Shi and Javitch, 2004). On the other hand, Phe¹⁹⁴ does not have any direct interactions with the ligand in the β₂AR crystal structure (Cherezov et al., 2007; Rosenbaum et al., 2007). The present study, however, clearly shows the importance of the analogous H₄R residue Phe¹⁶⁹ for agonist binding. In the β₂AR crystal structure, Phe¹⁹⁴ has a strong lipophilic interaction with Tyr¹⁸⁵, Ala¹⁸¹, and His¹⁷⁸ (Fig. 3A). We propose that these interactions are important to properly orient Phe¹⁹⁴ and hence position EL2 in the observed orientation. In agreement with the observations in the crystal structure of the β₂AR, our homology model of the H₄R clearly shows that the side chain of Phe¹⁶⁹ is also stabilized by lipophilic interactions, i.e., with residues Trp¹⁵⁷, Pro¹⁶⁶, Leu²⁴¹, Ser²⁴⁵, Pro²⁵⁰, and Tyr²⁵⁵ (Fig. 3B). A mutation to a smaller substituent, such as valine, the focus of the current study, would loosen the orienting interactions and increase the flexibility of the EL2 loop and concomitantly Phe¹⁶⁸. The concurrent increase in flexibility would result in a reduced binding affinity, which is in agreement with the observed binding differences between the human and mouse H₄Rs. Amino acids in this position, therefore, do not to directly interact with receptor-bound agonists but rather indirectly fine-tune the tightness of binding.

View larger version (48K): [in this window] [in a new window]   Fig. 3. Close-up of the EL2 as observed in the recent X-ray structure of the β2-adrenergic receptor (Cherezov et al., 2007) (A) and homology model of the human H4R (B). Phe193 points inwards into the GPCR binding pocket, whereas Phe194 interacts with various residues in the EL2 of the β2-adrenergic receptor, thereby stabilizing the conformation of the EL2 (Cherezov et al., 2007). Phe168 and Phe169 of the human H4R might play similar roles as Phe193 and Phe194 residues, respectively. The images were created with Molecular Operating Environment, version 2007.09 (Chemical Computing Group, Inc.). C, partial alignment of amino acid sequences of human β1–3 adrenergic, muscarinic M1–5, and histaminergic H1–4 receptors and bovine rhodopsin (BR). The sequences start at residue Trp4.50 and end at Phe5.47 (Ballesteros-Weinstein numbering system) (Ballesteros and Weinstein, 1995) and were aligned by ClustalW (Higgins et al., 1994). Residue Phe169 of the H4R is printed in boldface.

In summary, following the observation of species differences between the human and mouse H₄R for binding agonists, we identified phenylalanine 169 in the EL2 as the single amino acid responsible for the differences in agonist binding. Phenylalanine 169 is part of a Phe-Phe motif, which is also present in the recently crystallized β₂AR. These results point to an important role of the EL2 in the agonist binding to the H₄R and provide a molecular explanation for the species differences between human and rodent H₄Rs. Moreover, our findings also shed some light on the known differences in ligand binding in the family of muscarinic receptor subtypes.

	Acknowledgements

 
We thank Dr. Timothy Lovenberg (Johnson & Johnson Pharmaceutical Research & Development, LLC, La Jolla, CA) for the kind gift of the mouse H₄R cDNA and Payman Sadek for technical assistance.

	Footnotes

 
R.L. is recipient of a PIONIER award of the Technology Foundation (Stichting voor de Technische Wetenschapppen) of the Netherlands Foundation of Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.140343.

ABBREVIATIONS: GPCR, G-protein coupled receptor; β₂AR, β₂-adrenergic receptor; EL2, second extracellular loop; H₁R, histamine H₁ receptor; H₂R, histamine H₂ receptor; H₃R, histamine H₃ receptor; H₄R, histamine H₄ receptor; VUF 8430, S-(2-guanidylethyl)-isothiourea; TM, transmembrane; JNJ 7777120, 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; NFAT, nuclear factor of activated T cells; IL, interleukin.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Prof. Dr. Rob Leurs, Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. E-mail: r.leurs{at}few.vu.nl

	References

Top Abstract Materials and Methods Results Discussion References

 

Ballesteros JA and Weinstein H (1995) Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci 25: 366-428.[CrossRef]
Basu S, Jala VR, Mathis S, Rajagopal ST, Del Prete A, Maturu P, Trent JO, and Haribabu B (2007) Critical role for polar residues in coupling leukotriene B₄ binding to signal transduction in BLT1. J Biol Chem 282: 10005-10017.[Abstract/Free Full Text]
Celanire S, Wijtmans M, Talaga P, Leurs R, and de Esch IJ (2005) Keynote review: histamine H₃ receptor antagonists reach out for the clinic. Drug Discov Today 10: 1613-1627.[CrossRef][Medline]
Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, et al. (2007) High-resolution crystal structure of an engineered human β₂-adrenergic G protein-coupled receptor. Science 318: 1258-1265.[Abstract/Free Full Text]
Conklin BR, Farfel Z, Lustig KD, Julius D, and Bourne HR (1993) Substitution of three amino acids switches receptor specificity of G_q alpha to that of G_i alpha. Nature 363: 274-276.[CrossRef][Medline]
Conner M, Hawtin SR, Simms J, Wootten D, Lawson Z, Conner AC, Parslow RA, and Wheatley M (2007) Systematic analysis of the entire second extracellular loop of the V(1a) vasopressin receptor: key residues, conserved throughout a G-protein-coupled receptor family, identified. J Biol Chem 282: 17405-17412.[Abstract/Free Full Text]
de Esch IJ, Thurmond RL, Jongejan A, and Leurs R (2005) The histamine H₄ receptor as a new therapeutic target for inflammation. Trends Pharmacol Sci 26: 462-469.[Medline]
de Graaf C, Foata N, Engkvist O, and Rognan D (2008) Molecular modeling of the second extracellular loop of G-protein coupled receptors and its implication on structure-based virtual screening. Proteins 71: 599-620.[Medline]
Dunford PJ, O'Donnell N, Riley JP, Williams KN, Karlsson L, and Thurmond RL (2006) The histamine H₄ receptor mediates allergic airway inflammation by regulating the activation of CD4⁺ T cells. J Immunol 176: 7062-7070.[Abstract/Free Full Text]
Ford DJ, Essex A, Spalding TA, Burstein ES, and Ellis J (2002) Homologous mutations near the junction of the sixth transmembrane domain and the third extracellular loop lead to constitutive activity and enhanced agonist affinity at all muscarinic receptor subtypes. J Pharmacol Exp Ther 300: 810-817.[Abstract/Free Full Text]
Goldman LA, Cutrone EC, Kotenko SV, Krause CD, and Langer JA (1996) Modifications of vectors pEF-BOS, pcDNA1 and pcDNA3 result in improved convenience and expression. Biotechniques 21: 1013-1015.[Medline]
Goodwin JA, Hulme EC, Langmead CJ, and Tehan BG (2007) Roof and floor of the muscarinic binding pocket: variations in the binding modes of orthosteric ligands. Mol Pharmacol 72: 1484-1496.[Abstract/Free Full Text]
Gutzmer R, Diestel C, Mommert S, Köther B, Stark H, Wittmann M, and Werfel T (2005) Histamine H₄ receptor stimulation suppresses IL-12p70 production and mediates chemotaxis in human monocyte-derived dendritic cells. J Immunol 174: 5224-5232.[Abstract/Free Full Text]
Hofstra CL, Desai PJ, Thurmond RL, and Fung-Leung WP (2003) Histamine H₄ receptor mediates chemotaxis and calcium mobilization of mast cells. J Pharmacol Exp Ther 305: 1212-1221.[Abstract/Free Full Text]
Hough LB (2001) Genomics meets histamine receptors: new subtypes, new receptors. Mol Pharmacol 59: 415-419.[Free Full Text]
Jongejan A, Lim HD, Smits R, de Esch IJ, Haaksma E, and Leurs R (2008) Delineation of agonist binding to the human histamine H₄ receptor using mutational analysis, homology modeling and ab initio calculations. J Chem Inf Model 48: 1455-1463.[CrossRef][Medline]
Laurila JM, Xhaard H, Ruuskanen JO, Rantanen MJ, Karlsson HK, Johnson MS, and Scheinin M (2007) The second extracellular loop of _2A-adrenoceptors contributes to the binding of yohimbine analogues. Br J Pharmacol 151: 1293-1304.[CrossRef][Medline]
Lim HD, Smits RA, Bakker RA, van Dam CM, de Esch IJ, and Leurs R (2006) Discovery of S-(2-guanidylethyl)-isothiourea (VUF 8430) as a potent nonimidazole histamine H₄ receptor agonist. J Med Chem 49: 6650-6651.[CrossRef][Medline]
Ling P, Ngo K, Nguyen S, Thurmond RL, Edwards JP, Karlsson L, and Fung-Leung WP (2004) Histamine H₄ receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation. Br J Pharmacol 142: 161-171.[CrossRef][Medline]
Liu C, Wilson SJ, Kuei C, and Lovenberg TW (2001) Comparison of human, mouse, rat, and guinea pig histamine H₄ receptors reveals substantial pharmacological species variation. J Pharmacol Exp Ther 299: 121-130.[Abstract/Free Full Text]
Oda T, Morikawa N, Saito Y, Masuho Y, and Matsumoto S (2000) Molecular cloning and characterization of a novel type of histamine receptor preferentially expressed in leukocytes. J Biol Chem 275: 36781-36786.[Abstract/Free Full Text]
Oda T, Matsumoto S, Matsumoto M, Takasaki J, Kamohara M, Soga T, Hiyama H, Kobori M, and Katoh M (2005) Molecular cloning of monkey histamine H4 receptor. J Pharmacol Sci 98: 319-322.[CrossRef][Medline]
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, et al. (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289: 739-745.[Abstract/Free Full Text]
Parsons ME and Ganellin CR (2006) Histamine and its receptors. Br J Pharmacol 147: S127-S135.[Medline]
Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M, Ratnala VR, Sanishvili R, Fischetti RF, et al. (2007) Crystal structure of the human β₂ adrenergic G-protein-coupled receptor. Nature 450: 383-387.[CrossRef][Medline]
Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Yao XJ, Weis WI, Stevens RC, et al. (2007) GPCR engineering yields high-resolution structural insights into β₂-adrenergic receptor function. Science 318: 1266-1273.[Abstract/Free Full Text]
Scarselli M, Li B, Kim SK, and Wess J (2007) Multiple residues in the second extracellular loop are critical for M₃ muscarinic acetylcholine receptor activation. J Biol Chem 282: 7385-7396.[Abstract/Free Full Text]
Shi L and Javitch JA (2002) The binding site of aminergic G protein-coupled receptors: the transmembrane segments and second extracellular loop. Annu Rev Pharmacol Toxicol 42: 437-467.[CrossRef][Medline]
Shi L and Javitch JA (2004) The second extracellular loop of the dopamine D₂ receptor lines the binding-site crevice. Proc Natl Acad Sci U S A 101: 440-445.[Abstract/Free Full Text]
Shin N, Coates E, Murgolo NJ, Morse KL, Bayne M, Strader CD, and Monsma FJ Jr (2002) Molecular modeling and site-specific mutagenesis of the histamine-binding site of the histamine H₄ receptor. Mol Pharmacol 62: 38-47.[Abstract/Free Full Text]
Thompson JD, Higgins DG, and Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680.[Abstract/Free Full Text]
Thurmond RL, Desai PJ, Dunford PJ, Fung-Leung WP, Hofstra CL, Jiang W, Nguyen S, Riley JP, Sun S, Williams KN, et al. (2004) A potent and selective histamine H₄ receptor antagonist with anti-inflammatory properties. J Pharmacol Exp Ther 309: 404-413.[Abstract/Free Full Text]
Thurmond RL, Gelfand EW, and Dunford PJ (2008) The role of histamine H₁ and H₄ receptors in allergic inflammation: the search for new antihistamines. Nat Rev Drug Discov 7: 41-53.[CrossRef][Medline]
Tunaru S, Lättig J, Kero J, Krause G, and Offermanns S (2005) Characterization of determinants of ligand binding to the nicotinic acid receptor GPR109A (HM74A/PUMA-G). Mol Pharmacol 68: 1271-1280.[Abstract/Free Full Text]
Uveges AJ, Kowal D, Zhang Y, Spangler TB, Dunlop J, Semus S, and Jones PG (2002) The role of transmembrane helix 5 in agonist binding to the human H₃ receptor. J Pharmacol Exp Ther 301: 451-458.[Abstract/Free Full Text]
Yin D, Gavi S, Wang HY, and Malbon CC (2004) Probing receptor structure/function with chimeric G-protein-coupled receptors. Mol Pharmacol 65: 1323-1332.[Abstract/Free Full Text]

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