Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Histamine is an endogenous, biogenic amine that is known to mediate numerous physiological processes. Since its first description in 1910 (Barger and Dale, 1910), the list of activities ascribed to histamine has steadily grown to include activities in inflammation, gastric acid secretion, and neurotransmission. The diversity of physiology affected by histamine has led to the progressive development of antihistaminergic compounds that saw utility first as pharmacological tools and ultimately as therapeutic agents. The continued expansion of this pharmacological "toolbox" allowed detailed pharmacological studies to be carried out that revealed the existence of three types of histamine receptor: H₁, H₂, and H₃. In addition, several studies have described physiological responses to histamine whose pharmacology does not clearly correspond to H₁, H₂, or H₃ receptors. The H₁, H₂, and H₃ receptor subtypes have subsequently been confirmed by molecular cloning of cDNAs encoding G protein-coupled receptors (GPCRs) that exhibit corresponding pharmacological properties (Gantz et al., 1991; Yamashita et al., 1991; Lovenberg et al., 1999); however, the molecular identity of other pharmacologically defined sites has remained elusive.

Recent efforts to determine the complete sequence of the human genome, both by sequencing of expressed-sequence tags (ESTs) and large scale genomic sequencing, have uncovered many novel members of the GPCR superfamily. Using the sequence of receptors for known biologically active substances, it has been possible to discover new members of receptor subfamilies that were not previously detected by pharmacological methods (Sibley and Monsma, 1992; Kroeze and Roth, 1998). With the addition of these novel receptor subtypes, many new insights into the function of the respective ligand/receptor systems have been gained.

The current study describes the discovery and characterization of a novel GPCR, designated SP9144, which appears to be a member of the histamine receptor family. SP9144 is most similar to the recently cloned H₃ histamine receptor but is preferentially expressed in leukocytes. During the preparation of this manuscript, Oda et al. (2000) described the cloning of a novel histamine receptor that is essentially identical in sequence, expression pattern, and pharmacology to SP9144. The identification and further characterization of this receptor will likely provide new insights into the role of histamine in the modulation of immunological function.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. Human mRNA was obtained from Clontech (Palo Alto, CA). cDNA synthesis kits and all cell culture and transfection reagents were obtained from Life Technologies (Gaithersburg, MD). The vector pcDNA3.1 was obtained from Invitrogen (Carlsbad, CA). Reagents for DNA sequencing and quantitative PCR were from PE-Biosystems (Foster City, CA). The sources of compounds were Schering-Plough Department of Chemical Research for burimamide, R()--methylhistamine, thioperamide, dimaprit, chlorpheniramine, and cimetidine; Smith, Kline, and French Laboratories (Philadelphia, PA) for impromidine; Sigma/Research Biochemicals International (St. Louis, MO) for histamine, imetit, clobenpropit, N-methylhistamine, S(+)--methylhistamine, probenecid, and Fluo 3-AM. [³H]Histamine (15 Ci/mmol) was from DuPont NEN (Boston, MA).

Molecular Cloning. The amino acid sequences of known GPCRs were used to conduct a BLAST search of nucleotide databases. The search identified a 200-base pair nucleotide sequence as being a putative GPCR, with homology to the sixth transmembrane domain of the 5HT_1B receptor. The corresponding cDNA clone (designated SP9144) was obtained and sequenced further to reveal the sixth and seventh transmembrane domains.

Transfection of Cells. HEK-293 cells were seeded 24 h before transfection in DMEM with 10% fetal bovine serum, then transiently transfected overnight using LipofectAMINE 2000 (Life Technologies). Where indicated, cells were cotransfected with SP9144 (1 µg/10 mm²), chimeric G protein -subunits (Conklin et al., 1993; Coward et al., 1999), and G₁₆, either individually or as mixtures (as 10% of total DNA used for transfection). The chimeric G subunits used consist of G_q with the five C-terminal amino acids replaced by those of G_i1 (which are identical in G_i2), G_i3, G_o, and G_z (referred to as G_q/i1,2, G_q/i3, G_q/o, and G_q/z). G protein mix 1 included equal amounts of G₁₆ and G_q/o; G protein mix 2 included equal amounts of G_q/z, G_q/i3, and G_q/i1,2. Cells were used for experiments 48 h following transfection.

Intracellular Calcium ([Ca²⁺]_i) Mobilization Assay. Cells were harvested 24 h post-transfection without trypsin and seeded at 2.5 × 10⁵ cells/well in DMEM with 10% fetal bovine serum in poly(D-lysine)-treated 96-well clear bottom black plates (Becton Dickinson, Franklin Lakes, NJ). Experimental compounds were diluted in Hanks' balanced salt solution, 20 mM HEPES, 2.5 mM probenecid, 1% bovine serum albumin (wash buffer). Forty-eight hours post-transfection, cells were loaded for 1.5 h with 2 µM Fluo 3-AM (F-6142; Sigma), 2.5 mM probenecid, and 20 mM HEPES in DMEM with 10% fetal calf serum. Cells were washed extensively with wash buffer to remove excess dye and evaluated for ligand-induced [Ca²⁺]_i release using the fluorometric imaging plate reader (FLIPR) (Molecular Devices, Sunnyvale, CA). Results are given as the relative change in fluorescence from the initial reading and measured over a 3-min period following addition of compound.

Membrane Preparation. HEK-293 cells transfected with SP9144 as described above were harvested by incubating in 5 mM EDTA/phosphate-buffered saline followed by repeated pipetting. The cells were centrifuged for 5 min at 1000g. The EDTA/PBS was decanted, and an equal volume of ice-cold 50 mM Tris-HCl, pH 7.5, was added and cells were broken up with a Polytron homogenizer (PT-10 tip, setting 5, 30 s). Nuclei and unbroken cells were sedimented at 1,000g for 10 min and then the supernatant was centrifuged at 50,000g for 10 min. The supernatant was decanted, the pellet was resuspended by Polytron homogenization, a sample was taken for BCA protein assay (Pierce, Rockford, IL), and the tissue was again centrifuged at 50,000g. Pellets were stored frozen at 20°C.

Radioligand Binding. For saturation binding, increasing concentrations of [³H]histamine (30-60 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) were incubated without and with 10⁵ M histamine in triplicate with 40 to 60 µg of membrane protein in a total volume of 200 µl of 50 mM Tris-HCl, pH 7.5, for 1 h at 30°C. The bound radioactivity was separated by filtration through Unifilter-96 GF/B filters (Packard, Meriden, CT) pretreated with 0.1% polyethyleneimine (Sigma). The filters were washed eight times with 400 µl of ice-cold 50 mM Tris-HCl (pH 7.5), and radioactivity retained on the filters was quantitated by liquid scintillation counting in a Topcount (Packard) at 34% efficiency. For competition binding assays, five concentrations of compounds were incubated in triplicate with 18 nM [³H]histamine and 70 µg of membrane protein under the conditions as described above. Samples were filtered through Whatman (Clifton, NJ) GF/B filters and washed three times with 2 ml of cold Tris buffer. Filters were dried in a microwave oven, impregnated with Meltilex wax scintillant (PerkinElmer-Wallac Inc., Gaithersburg, MD), and counted at 45% efficiency. Binding data were analyzed by nonlinear least-squares curve-fitting to appropriate models with Prism software (GraphPad, San Diego, CA), and K_i values were calculated from IC₅₀ values according to Cheng and Prusoff (1973).

cAMP Assay. Cells were transfected as previously described and assayed 48 h post-transfection. Cells that were subjected to pertussis toxin pretreatment were incubated overnight before the assay with pertussis toxin (100 ng/ml) in full serum media. On the day of the assay, cells were harvested in 2 mM EDTA/PBS and resuspended to a final concentration of 5 × 10⁶ cells/ml in cold (4°C) adenylate cyclase buffer (AC buffer) (250 mM sucrose, 75 mM Tris-HCl, 12.5 mM MgCl₂, 1.5 mM EDTA, pH 7.4) to which ascorbic acid (10 mg/50 ml) and dithiothreitol (31 mg/50 ml) were added fresh daily. The phosphodiesterase inhibitor Ro 20-1724 (4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone) was added at a final concentration of 100 µM, and the cells were incubated for either 15 min (room temperature) or 30 min (on ice). Drugs were prepared at 2× final concentrations in AC buffer ± forskolin (10 µM for Chinese hamster ovary cells; 100 nM for the HEK-293 cells). For the assay, 50 µl of drug solution was added to 50 µl of cell suspension in a 1 ml × 96-well assay block, incubated at 37°C in an incubator-shaker for 15 min, boiled for 3 min, and then cooled on ice. The cell lysates were then assayed for total cyclic AMP using the NEN cyclic AMP Flashplate Assay (New England Nuclear Life Science Products, Inc., Boston, MA) according to the manufacturer's protocol. Total cAMP produced for each condition was determined as follows: %B/Bo for each sample = (average net counts for sample or standard × 100)/average net counts of zero standard. A standard curve was generated by plotting the %B/Bo for each standard versus log[pmol of cAMP]. The concentration of cAMP for each sample could be interpolated from the standard curve. Results are expressed as femtomoles of cAMP/well.

MAP Kinase Assay. Cells were transfected as described above. Twenty-four hours post-transfection, cells were harvested and reseeded at a density of 1 × 10⁶ cells/well in six-well dishes. Full serum media was replaced 5 to 8 h after seeding with 0.5% serum media overnight. Cells that were subjected to pertussis toxin pretreatment were incubated overnight before the assay with pertussis toxin at 100 ng/ml in 0.5% serum media. One hour before the drug challenge, cells were placed in media without serum to reduce background MAP kinase activation. Drug was then added at the appropriate concentration and incubated for 5 min at 37°C. Cells were then washed once with cold PBS and lysed in 100 µl of cold lysis buffer [150 mM NaCl, 50 mM Tris pH 8.0, 5 mM EDTA pH 8.0, 10 mM NaF, 10 mM dibasic sodium pyrophosphate, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate (RIPA)] containing one Complete protease inhibitor cocktail tablet/50 ml (Roche Molecular Biochemicals, Indianapolis, IN). Cell lysates were collected in microfuge tubes and spun at 13,000g for 15 min at 4°C to pellet cellular debris. The protein concentration of the lysates was determined using the BCA protein assay. Twenty micrograms of protein was added to an equal volume of 2× SDS polyacrylamide gel electrophoresis sample buffer and boiled for 5 min, then separated on a 10% Tris-glycine polyacrylamide gel (Novex, Carlsbad, CA). Proteins in the gel were transferred to a nitrocellulose membrane in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) using a semidry transfer apparatus (Bio-Rad, Hercules, CA). Membranes were incubated in blocking solution [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% (v/v) Tween 20 (TTBS)] containing 5% (w/v) milk for 1 h or more at room temperature. Membranes were rinsed three times with TTBS then developed using the PhosphoPlus p44/42 MAP Kinase (Thr202/Tyr204) Antibody Kit (Cell Signaling Technology, Inc., Beverly, MA) according to the manufacturer's instructions.

[³⁵S]GTPS Binding Assay. A scintillation proximity assay was used for [³⁵S]GTPS binding assays. For each assay point, 2 to 3 µg of membranes, prepared essentially as previously described (excluding phosphatase inhibitors) (Hipkin et al., 2000), were preincubated for 15 min at room temperature with 300 µg of wheat germ agglutinin-coated scintillation proximity assay beads (WGA-SPA; Amersham, Arlington Heights, IL) in SPA binding buffer (50 mM HEPES, 1 mM CaCl₂, 5 mM MgCl₂, 50 mM NaCl, 0.002% NaN₃) containing 0.1% bovine serum albumin (Factor V, lipid free) and 3.75 µM GDP (Sigma). The beads and membranes were transferred to a 96-well Isoplate (Wallac, Gaithersburg, MD) and incubated for 60 min at room temperature with 50 pM [³⁵S]GTPS (tetraethylammonium salt, specific activity = 1250 Ci/mmol; NEN) in the absence or presence of histamine and/or thioperamide. Membrane-bound [³⁵S]GTPS was measured by scintillation proximity assay using a 1450 Microbeta Plus liquid scintillation counter (Wallac).

Messenger RNA Expression Analysis. Expression of SP9144 mRNA was examined using dot blots and Northern blots obtained from a commercial source (Clontech). Hybridization to blots was carried out using a PCR-generated DNA fragment encompassing 400 base pairs at the 3'-end. The DNA fragments were random-prime labeled with [³²P]dCTP, and the blots were hybridized for 14 h in ExpressHyb (Clontech) containing ~2 million cpm/ml of radiolabeled probe. The following day the blots were washed and exposed to Kodak Biomax MS film (Eastman Kodak, Rochester, NY for 3 days at 70°C. The films were analyzed for relative expression levels using the MCID M4 image analysis system (Imaging Research, Ontario, Canada).

5

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Using a set of biogenic amine receptor sequences, public and proprietary EST databases were searched by BLAST (Altschul et al., 1990) to look for novel members of this GPCR family. One proprietary EST was identified that exhibited significant homology with transmembrane region seven of biogenic amine receptors. Subsequently, a matching fragment of nucleotide sequence from chromosome 18 was identified in GenBank (accession number AC007922). From this genomic sequence, a virtual transcript exhibiting high homology to the cloned H₃ histamine receptor (Lovenberg et al., 1999) was identified. This sequence was used to design PCR primers to the predicted amino and carboxy termini that amplified a 1173-base pair fragment from eosinophil cDNA. Sequence analysis of the cloned fragment confirmed its identity to the predicted transcript. Hydrophobicity analysis of the predicted 390-amino acid protein indicated the presence of seven hydrophobic, putative transmembrane regions, a feature common to G protein-coupled receptors. BLAST analysis with this protein sequence revealed homology to known GPCRs with the highest degree of similarity to the H₃ histamine receptor. Furthermore, a phylogenetic analysis (Wisconsin Package, Genetics Computer Group, Madison, WI) with the amino acid sequences of all known human biogenic amine receptors and SP9144 showed that SP9144 clustered with the H₃ histamine receptor (results not shown). Sequence alignment analysis using the Clustal V method (Higgins et al., 1992) (Fig. 1) showed 43% identity overall between SP9144 and the H₃ histamine receptor and 58% within the predicted transmembrane regions. The identity to the H₁ and H₂ receptors was 29 and 31%, respectively. These analyses suggest that the protein encoded by the SP9144 open reading frame could be a novel receptor for histamine.

View larger version (69K): [in this window] [in a new window]   Fig. 1.   Alignment of amino acid sequences of histamine receptors. The deduced amino acid sequence of SP9144 has been aligned with the reported amino acid sequences of the human H1, H2, and H3 receptors using the Clustal V method. Residues matching SP9144 are boxed, and the predicted transmembrane regions are indicated by bars. The nucleotide sequence associated with the amino acid sequence reported here has been deposited in the GenBank database with accession number AF329449.

To identify the ligand for SP9144, the cDNA was transiently transfected into HEK-293 cells with and without chimeric G protein -subunits representing G_q with the five C-terminal amino acids replaced with those of the -subunits G_i1,2, G_i3, G_o, or G_z. Coexpression of a GPCR and the chimeric G protein mix allows receptors not normally coupled to G_q to be assayed by monitoring mobilization of [Ca²⁺]_i (Conklin et al., 1993; Coward et al., 1999). The mobilization of [Ca²⁺]_i due to agonist addition was monitored in a FLIPR. HEK-293 cells transfected with SP9144 and chimeric G proteins were exposed to a variety of potential agonist molecules, including histamine, dopamine, serotonin, epinephrine, and norepinephrine, all at 10 µM final concentration. Of these agonists, only histamine mobilized [Ca²⁺]_i in SP9144-transfected cells (Fig. 2A). Furthermore, [Ca²⁺]_i mobilization in response to histamine was only observed when SP9144 was cotransfected with a mixtures of chimeric G proteins (G mix 1 or G mix 2, Fig. 2B). To determine the G protein specificity of this receptor, individual chimeric G proteins were cotransfected with SP9144. Under these conditions, histamine-induced [Ca²⁺]_i mobilization was observed only when SP9144 and G_q/i1,2, G_q/i3, or G₁₆ were cotransfected. No response was observed with G proteins alone or when SP9144 was cotransfected with G_q/o or G_q/z (Fig. 2B).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A, mobilization of [Ca2+]i by biogenic amines in HEK-293 cells expressing SP9144 and chimeric G proteins. HEK-293 cells cotransfected with SP9144 and the chimeric G proteins Gq/i1,2, Gq/i3, and Gq/z were loaded with the Ca2+-sensitive dye Fluo 3-AM, and the change in fluorescence in response to addition of various biogenic amines (10 µM final concentration) was monitored in a FLIPR as described under Experimental Procedures. The data are expressed as arbitrary fluorescence units normalized to the initial baseline level. B, mobilization of [Ca2+]i in response to 10 µM histamine in HEK-293 cells cotransfected with empty pcDNA3.1 (solid bars) or SP9144 (gray bars) and the indicated chimeric G proteins. Data are the maximal (±S.E.M., n = 3) fluorescence values observed after stimulation with 10 µM histamine.

The initial pharmacological characterization of SP9144 was carried out by examining responses to the histamine derivatives R()--methylhistamine, S(+)--methylhistamine, and N-methylhistamine. Analysis of the potency of histamine and these derivatives (Fig. 3A) revealed a rank order of histamine > N-methylhistamine > R()--methylhistamine S(+)--methylhistamine (Table 1). Furthermore, both N-methylhistamine and R()--methylhistamine behaved as full agonists in this assay. S(+)--Methylhistamine was found to be completely inactive up to 10 µM (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   A, dose-response analysis of histamine and analogs at SP9144 coexpressed with chimeric G protein. HEK-293 cells were transfected with SP9144 or empty pcDNA3.1 and the chimeric G protein Gq/i1,2. Forty-eight hours later, the mobilization of [Ca2+]i in response to the indicated compounds was determined using the FLIPR. Data are presented as the percentage of maximal fluorescence for a given compound. Maximal fluorescence values for each compound were approximately 4000 units. Each data point is the mean ± S.E.M. of three independent experiments performed in triplicate. B, dose-response analysis of H3 receptor antagonists in HEK-293 cells coexpressing SP9144 and Gq/i1,2. Data shown are expressed as percentage of maximal histamine fluorescence (approximately 4000 units). Each data point is the mean ± S.E.M. of three independent experiments performed in triplicate. C, thioperamide inhibition of histamine-induced [Ca2+]i mobilization in HEK-293 cells transiently transfected with SP9144 and Gq/i1,2. Cells were incubated for 5 min with increasing concentrations of thioperamide before the addition of 10 µM histamine. Data are expressed as percentage of histamine response in the absence of thioperamide and are the mean ± S.E.M. of three independent experiments conducted in triplicate.

View this table: [in this window] [in a new window]   TABLE 1 Pharmacological values for histaminergic compounds at SP9144 Values for binding were determined from competition binding experiments against [3H]histamine using membranes from HEK-293 cells stably transfected with SP9144 performed three times in triplicate. Values for [Ca2+]i mobilization are derived from analysis of dose-response curves carried out on HEK-293 cells transiently transfected with SP9144 and Gq/i1,2 carried out three times in triplicate.

The pharmacology of SP9144 was further characterized by comparison to that of the known histamine receptors using compounds selective for each histamine receptor subtype. Neither the H₁ antagonist chlorpheniramine nor the H₂ antagonist cimetidine had any effect on histamine-induced [Ca²⁺]_i mobilization in SP9144/G_q/i1,2-cotransfected cells (Fig. 3C). In contrast, a number of compounds known to interact selectively with the H₃ histamine receptor were also active at SP9144. Thus, as described above, the H₃-selective agonist R()--methylhistamine was able to induce [Ca²⁺]_i mobilization in SP9144/G_q/i1,2-cotransfected cells, although with significantly less potency than that reported for the H₃ receptor (Lovenberg et al., 1999). Imetit, another H₃-selective agonist, also exhibited agonism at SP9144, albeit with reduced efficacy as compared with histamine (Fig. 3B). Several H₃-selective antagonists were also active at SP9144; however, unlike their action at the H₃ receptor, they exhibited partial agonist activity at SP9144 (Fig. 3B). The rank order of potency for these compounds in the [Ca²⁺]_i assay was clobenpropit > imetit > impromidine > burimamide. In contrast, the H₃-selective antagonist thioperamide was also an antagonist at SP9144 in this assay (Fig. 3C). The EC₅₀, efficacy, and K_i values for these compounds are summarized in Table 1.

To confirm the pharmacology of SP9144, the binding of [³H]histamine was examined in wild-type and SP9144-transfected HEK-293 cells. Saturation binding analysis revealed the presence of high-affinity, saturable, specific [³H]histamine binding in cells transfected with SP9144 (Fig. 4A) but not in cells transfected with empty vector (data not shown). The K_d determined by Scatchard analysis was 15.3 (±3, n = 4) nM, with a B_max of 920 (±130, n = 4) fmol/mg of protein. The pharmacology of [³H]histamine binding to SP9144-transfected cells was further examined by inhibition of [³H]histamine binding by various histaminergic compounds. As indicated by the FLIPR assay, the H₁ antagonist chlorpheniramine did not inhibit [³H]histamine binding at concentrations 10⁵ M, whereas the H₂ antagonist cimetidine exhibited weak activity with an IC₅₀ of approximately 1 µM (data not shown). In contrast, compounds with H₃ receptor selectivity were able to potently inhibit [³H]histamine binding to SP9144 with the following rank order: imetit > clobenpropit > burimamide > thioperamide (Fig. 4C, Table 1).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   A, saturation binding of [3H]histamine to membranes from HEK-293 cells transiently transfected with SP9144. Inset, Scatchard transformation of saturation binding data. Data points shown are the mean of triplicate determinations and are representative of four independent experiments. B and C, inhibition of specific [3H]histamine binding by various histaminergic agonists (B) and antagonists (C). Data points shown are the mean of triplicate determinations and are representative of two independent experiments. Saturation and inhibition binding data were analyzed by nonlinear regression analysis using single-site binding models (Prism; GraphPad Software).

Since each of the known histamine receptors interact with different G proteins and second messenger systems, it was of interest to investigate the second messenger coupling of SP9144. The ability of the chimeric G_q/i proteins to interact with SP9144 suggested that agonist stimulation of SP9144 should lead to the inhibition of stimulated adenylyl cyclase activity via interaction with G_i. The ability of histamine to modulate cAMP levels was examined in HEK-293 cells stably transfected with FLAG-tagged SP9144 (without chimeric G proteins). Although these cells expressed functional SP9144 as verified by flow cytometry, [³H]histamine binding, and histamine-induced [Ca²⁺]_i mobilization (when transiently transfected with G_q/i1,2), exposure to histamine did not cause inhibition of forskolin-stimulated cAMP levels at concentrations up to 10 µM (Fig. 5). Interestingly, forskolin-stimulated cAMP levels were consistently lower in SP9144-transfected cells as compared with wild-type cells, and basal cAMP levels also tended to be lower (Fig. 5). Additional experiments using transiently transfected HEK-293 and Chinese hamster ovary cells yielded similar results (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effect of histamine on forskolin-stimulated cAMP levels in wild type and HEK-293 cells stably transfected with SP9144. The indicated cells were stimulated with 0.1 µM forskolin in the absence or presence of increasing concentrations of histamine as described under Experimental Procedures. Data are the average of triplicate determinations.

Given that SP9144 did not appear to modulate cAMP production, potential interaction with G proteins was determined by examining the stimulation of [³⁵S]GTPS binding following agonist stimulation of SP9144. As shown in Fig. 6A, histamine had no effect on [³⁵S]GTPS binding in wild-type HEK-293 cells, whereas in HEK-293 cells stably transfected with SP9144, histamine potently stimulated [³⁵S]GTPS binding, with an EC₅₀ of 9 (±5) nM. Interestingly, basal levels of bound [³⁵S]GTPS tended to be higher in SP9144-transfected cells as compared with wild types. As shown in Fig. 6B, preincubation with the H₃ antagonist thioperamide shifted the histamine dose-response curve to the right, although the maximal level of stimulation was not diminished. In addition, thioperamide decreased the basal level of [³⁵S]GTPS binding in the absence of histamine (Fig. 6C), thus indicating that thioperamide acts as an inverse agonist at SP9144.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   A, stimulation of [35S]GTPS binding by histamine in wild-type and SP9144 stably transfected HEK-293 cells. Results shown are the mean ± S.E.M. of triplicate determinations. B, effect of thioperamide on histamine stimulation of [35S]GTPS binding to membranes from HEK-293 cells stably transfected with SP9144. Membranes were incubated with increasing concentrations of histamine in the absence or presence of 10 µM thioperamide. C, comparison of the effects of histamine and thioperamide alone on [35S]GTPS binding to membranes of HEK-293 cells stably transfected with SP9144.

To further investigate the intracellular signaling pathways affected by SP9144, the phosphorylation of MAP kinase in response to histamine was examined. In HEK-293/SP9144 cells, histamine potently stimulated phosphorylation of MAP kinase in a dose-dependent manner (Fig. 7), whereas only a slight response to histamine was observed in control cells. Pretreatment of the cells with pertussis toxin essentially abolished the ability of 10 µM histamine to stimulate SP9144-mediated MAP kinase phosphorylation (Fig. 7).

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Activation of MAP kinase following histamine stimulation of SP9144. HEK-293 cells, untransfected (HEK-293 wt) or stably transfected with SP9144, were incubated with increasing doses of histamine for 5 min, and the amount of phosphorylated MAP kinase was assessed by Western blot as described under Experimental Procedures. One set of cell samples was treated overnight with pertussis toxin (PTX) before stimulation with 10 µM histamine as indicated.

The potential physiological function of SP9144 was investigated by determining the distribution of SP9144 mRNA in human tissues by using both hybridization and quantitative reverse transcriptase-PCR. Using randomly primed ³²P-labeled probes specific for SP9144, a human multitissue dot blot was probed for SP9144 expression. After 3 days of exposure at 80°C, low-level expression was detected in several tissues including bone marrow, peripheral leukocytes, spleen, testis, small intestine, lymph node, heart, and kidney (Fig. 8A). Although expressed at low levels, SP9144 does appear to be preferentially distributed in tissues of immunological relevance. To examine the distribution of SP9144 mRNA in greater detail, quantitative PCR was used to examine SP9144 expression in a collection of cDNA libraries prepared from various lymphoid cells and tissues, as well as a collection of mRNA from various brain regions. Whereas no evidence of SP9144 expression was observed in any of the brain mRNA samples examined (data not shown), SP9144 was found to be selectively expressed in several types of immune cells (Fig. 8B), including T cells, dendritic cells (DC), monocytes, mast cells, neutrophils, and eosinophils. Furthermore, it was apparent that the expression of SP9144 in mononuclear cells is regulated upon cellular activation, depending on the specific cell type.

View larger version (68K): [in this window] [in a new window]   Fig. 8.   Distribution of SP9144 messenger RNA. A, multiple tissue mRNA dot blot hybridized with 32P-labeled probe to SP9144. Hybridization and washing were performed as described under Experimental Procedures. The tissues present on the blot are keyed to their position on the blot in the lower panel. B, quantitative PCR analysis of the distribution of SP9144 mRNA in specific tissues and lymphoid cells. cDNA libraries prepared from the indicated tissues and cell types were used as templates in PCR reactions with SP9144-specific primers as described under Experimental Procedures. Results are displayed as the ratio of target product to internal control product, and values above 105 are considered significant.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The current study describes the cloning and characterization of a novel receptor for histamine that is most similar to the recently described histamine H₃ receptor in primary sequence and pharmacology. The degree of homology between SP9144 and the H₃ receptor (43%) contrasts with the relatively low level of relatedness between the other histamine receptors. However, SP9144 differs from the H₃ receptor in several respects. For example, the potency of the agonists histamine and R()--methylhistamine is reversed at SP9144 in comparison with H₃, with histamine being more potent than R()--methylhistamine at SP9144, whereas R()--methylhistamine is more potent at the H₃ receptor (Lovenberg et al., 1999). Furthermore, several compounds generally recognized to be antagonists at the H₃ receptor, clobenpropit, burimamide, and impromidine, exhibit partial agonism at SP9144, while thioperamide exhibits antagonism at both receptors. These data indicate that while SP9144 is structurally similar to the H₃ receptor, it possesses a unique pharmacological profile.

The H₃ receptor has been shown to be a G_i-linked receptor that is able to inhibit forskolin-stimulated adenylyl cyclase activity (Lovenberg et al., 1999). While SP9144 shares several properties of G_i-linked receptors, such as stimulation of [³⁵S]GTPS binding, activation of MAP kinase phosphorylation, and pertussis toxin sensitivity, it has not been possible to demonstrate modulation of cAMP levels, even under conditions where other G_i-linked receptors are active. Despite the apparent preference for the G_q/i chimeric G proteins, the precise identification of the G protein species interacting with SP9144 will require further investigation.

The data presented in the present study also suggest that SP9144 exhibits a significant level of constitutive activity. This is indicated by the elevated basal levels of both [³⁵S]GTPS binding and MAP kinase phosphorylation seen in cells expressing SP9144 as compared with wild-type cells. In this regard it is interesting to note that in the [³⁵S]GTPS assay, thioperamide acts as an inverse agonist at SP9144. It is unlikely that the constitutive activity observed for SP9144 is due to over-expression of this receptor, as similar results are obtained from both transient transfections and from a stable cell line expressing relatively low levels of receptor. Furthermore, both the H₁ and H₂ histamine receptors (Alewijnse et al., 1998; Bakker et al., 2000), and more recently the H₃ receptor (Morisset et al., 2000), have been shown to exhibit constitutive activity. Interestingly, the study by Morriset et al. demonstrates that the constitutive activity of the H₃ histamine receptor exists in vivo and plays a role in regulation of histaminergic neurons in rodent brain. In the case of SP9144 however, it remains to be determined if in its native cellular environment it exhibits a similar degree of constitutive activity, and if so, what is the functional significance of such activity.

The tissue distribution of SP9144 and the H₃ receptor exhibit striking differences. Whereas the H₃ receptor is primarily expressed in the central nervous system, SP9144 is not found in the central nervous system and elsewhere has a very limited distribution, primarily in lymphoid tissues. In particular, SP9144 is expressed in T cells, DC, monocytes, mast cells, neutrophils, and eosinophils. Interestingly, it appears that SP9144 expression is either up-regulated or down-regulated upon activation and that this regulation may depend on the presence of IL-10 or IL-13. For example, activated monocytes expressed SP9144 only in the presence of neutralizing antibody to IL-10, and activated Th2 cells, which express IL-10 and IL-13, down-regulate SP9144 expression. Regulation of SP9144 expression in DC is also associated with IL-10/IL-13. Resting bone marrow-derived DC express SP9144; however this expression was dramatically decreased upon activation of these cells with phorbol-12-myristate-13-acetate and ionomycin. This strong stimulation has been shown to increase IL-13 expression in these cells (de Saint-Vis et al., 1998). The situation with monocyte-derived DC is more complex and depends not only on the type of stimulation used but also on maturation of the DC. A more complete understanding of SP9144 regulation in these cells will require more extensive studies in the future.

The expression of SP9144 in immune system cells is potentially significant since histamine has been shown to exhibit activity at various types of leukocytes. While many of these effects may be attributed to the expression of H₁ and/or H₂ receptors (Leino et al., 1993), the existence of at least one additional, novel histamine receptor has been postulated based on differential pharmacology in human eosinophils (Raible et al., 1994). The pharmacological profile of this eosinophil receptor is qualitatively similar to that found for SP9144 in the present study, although the potency of most compounds examined is lower in eosinophils as compared with heterologously expressed SP9144. Whether this discrepancy is due to the different cellular environments and signaling pathways or indicates the existence of yet another histamine receptor remains to be determined. During the preparation of this manuscript, Oda et al. (2000) reported the cloning and characterization of a novel histamine receptor that is essentially identical to SP9144. The sequence reported by Oda et al. (2000) (GenBank accession number AB0044934) differs from that of SP9144 by three nucleotides, resulting in three different amino acid residues (residue 138 in transmembrane region 4, residues 206 and 253 in intracellular loop 3). However, the nucleotide sequence determined for SP9144 exactly matches the reported chromosome 18 sequence (AC007922) at these positions. Thus, it remains to be determined if the alternate sequences reported by Oda et al. (2000) arise from sequencing errors or real variation in this receptor in the human population. In other respects, the mRNA expression pattern and pharmacology of SP9144 correlate well with those reported by Oda et al. (2000).

The data presented in the present study clearly indicate that SP9144 represents a fourth histamine receptor with a unique pharmacological profile and tissue distribution. Based on these unique properties, it is proposed that this receptor be referred to as the H₄ histamine receptor. It is anticipated that identification of SP9144 as an H₄ histamine receptor expressed in leukocytes will result in an improved understanding of the role of histamine in the modulation of immune function.