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CELLULAR AND MOLECULAR

An 80-Amino Acid Deletion in the Third Intracellular Loop of a Naturally Occurring Human Histamine H₃ Isoform Confers Pharmacological Differences and Constitutive Activity

Leiden/Amsterdam Center for Drug Research, Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (G.B., P.v.M., R.A.B., R.L.); and Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois (K.M.K., T.R.M., J.L.B., B.R.E., D.G.W., M.I.S., M.D.C., A.A.H., T.A.E.)

Received for publication June 22, 2007
Accepted July 25, 2007.

	Abstract

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In this article, we pharmacologically characterized two naturally occurring human histamine H₃ receptor (hH₃R) isoforms, hH₃R(445) and hH₃R(365). These abundantly expressed splice variants differ by a deletion of 80 amino acids in the intracellular loop 3. In this report, we show that the hH₃R(365) is differentially expressed compared with the hH₃R(445) and has a higher affinity and potency for H₃R agonists and conversely a lower potency and affinity for H₃R inverse agonists. Furthermore, we show a higher constitutive signaling of the hH₃R(365) compared with the hH₃R(445) in both guanosine-5'-O-(3-[³⁵S]thio) triphosphate binding and cAMP assays, likely explaining the observed differences in hH₃R pharmacology of the two isoforms. Because H₃R ligands are beneficial in animal models of obesity, epilepsy, and cognitive diseases such as Alzheimer's disease and attention deficit hyperactivity disorder and currently entered clinical trails, these differences in H₃R pharmacology of these two isoforms are of great importance for a detailed understanding of the action of H₃R ligands.

For a good understanding of the biological effects of H₃R ligands, a detailed knowledge of the molecular pharmacology of the human H₃R (hH₃R) is indispensable. The molecular cloning of the hH₃R (Lovenberg et al., 1999) has revealed that the hH₃ gene is located on chromosome 20 (20q13.32-20q13.33) and contains three introns (Cogé et al., 2001), which give rise to a large number of hH₃R isoforms through alternative splicing (for review, see Hancock et al., 2003; Leurs et al., 2005). At present, there is no detailed knowledge on the pharmacological consequences of the various alternative splicing events. On the basis of the currently published information, one can conclude that the hH₃R(365) is one of the most abundantly expressed hH₃R isoforms next to the full-length hH₃R(445) (Cogé et al., 2001; Wellendorph et al., 2002; Esbenshade et al., 2006). The hH₃R(365) isoform lacks 80 amino acids in the third intracellular loop (IL3) and has been described to be nonfunctional in a cAMP, guanosine-5'-O-(3-[³⁵S]thio) triphosphate ([³⁵S]GTPS) binding, and Ca²⁺ mobilization assay (Cogé et al., 2001) but to be functional in an R-SAT reporter assay (Wellendorph et al., 2002). For other G-protein-coupled receptors (GPCRs), the IL3 has been shown to dictate G-protein specificity (Burstein et al., 1996; Senogles et al., 2004) and bind -arrestins (Gelber et al., 1999) and calmodulin (Turner et al., 2004). Furthermore, the carboxyl terminus of the IL3 has been shown to play a role in constitutive activity of GPCRs (Chakir et al., 2003). In view of the relative high abundance of the hH₃R(365) isoform and the known importance of the IL3 loop in GPCR-mediated signal transduction, we pharmacologically characterized the hH₃R(445) and the hH₃R(365) isoforms in full detail in this study.

Materials and Methods
Materials. Dulbecco's modified Eagle's medium, trypsin-EDTA, penicillin, L-glutamine, and streptomycin were from Invitrogen (Breda, The Netherlands), and fetal calf serum was from Integro (Zaandam, The Netherlands). Culture dishes were from Costar (Haarlemermeer, The Netherlands). cAMP was obtained from Sigma-Aldrich Chemie B.V. (Zwijndrecht, The Netherlands). All H₃R ligands were (re-) synthesized at the Vrije Universiteit Amsterdam or at Abbott Laboratories. G-418 was obtained from Duchefa Biochemie B.V. (Haarlem, The Netherlands). 3-Isobutyl-1-methylxanthine was obtained from Acros (Fischer Scientific, 's Hertogenbosch, The Netherlands). N-[methyl-³H]Histamine (83 Ci/mmol) was from PerkinElmer Life Sciences (Zaventem, Belgium). [³H]cAMP (40 Ci/mmol) was from Amersham (Hertogenbosch, The Netherlands). The following compounds were used: FUB322, A-304121, A-317920, A-320436, A-331440, A-349821, A-358239, and A-431404.

Cloning of the Histamine H₃ Receptor Isoforms. The human histamine H₃ receptor gene was cloned using human thalamus poly-A RNA (Clontech, Palo Alto, CA) with RT-PCR methods using primers designed according to the published human histamine H₃ receptor gene sequences (GenBank accession no. AF140538 [GenBank] ) (Lovenberg et al., 1999). The cDNAs for the human full-length [hH₃R(445)] and a shorter histamine H₃ receptor isoform [hH₃R(365)], with an 80-amino acid deletion from the third intracellular loop, were cloned into the pCIneo expression vector.

Analysis of H₃R Isoform Expression. Tissue expression of H₃R isoforms was analyzed by RT-PCR. Human mRNA (1 µg; Clontech, Mountain View, CA) was reverse-transcribed using SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA) and amplified for 35 cycles of 95°C for 45 s, 60°C for 1.5 min, 72°C for 2 min, and a final extension at 72°C for 7 min using 1 unit of Platinum Taq High Fidelity DNA polymerase (Invitrogen). The 5' forward and the 3' reverse primers used were F754 (nucleotides 754–778) and R1406 (nucleotides 1383–1406) (Cogé et al., 2001). The PCR products were subcloned into pCR Blunt II-TOPO cloning vector (Invitrogen) and sequenced. PCR products were analyzed using agarose gels stained with ethidium bromide.

Cell Culture. The H₃R isoforms were stably expressed in rat C6 glioma cells and were maintained at 37°C in a humidified 5% CO₂, 95% air atmosphere in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin in the presence of 400 µg/ml G-418.

Radioligand Binding. Cells were scraped from their dishes, centrifuged (3 min, 1000 rpm), and the pellets were stored at–20°C until use. Before use, the pellets were dissolved in 50 mM Na₂HPO₄, pH 7.4 (for N-[methyl-³H]histamine and [³H]A-349821) or in 25 mM Tris, 145 mM NaCl, and 5 mM MgCl₂ (for [¹²⁵I]iodophenpropit) and homogenized for 2 s (40 W, Labsonic 1510). The cell homogenates (10–20 µg) were incubated for 60 min at 25°C with or without competing ligands in a total volume of 200 µl. For competition-binding experiments, 0.6 nM N-[methyl-³H]histamine (83.0 Ci/mmol) or 2.5 nM [¹²⁵I]iodophenpropit was used. Saturation experiments were perform with various concentrations of N-[methyl-³H]histamine (0.1–20 nM), [¹²⁵I]iodophenpropit (0.5–50 nM), or [³H]A-349821 (0.01–1.5 nM), and nonspecific binding was defined by 100 µM thioperamide. The incubation was terminated by rapid filtration over polyethylenimine (0.3%)-pretreated Unifilter GF/C filter plates with two subsequent washes with ice-cold 50 mM Tris-HCl, pH 7.4. Radioactivity retained on the filter was determined by liquid scintillation counting on the Microbeta Trilux with 25 µlof Microscint "O."

GTPS Binding Assay. Human embryonic kidney cell membranes expressing the human H₃R were prepared by homogenization in cold buffer containing 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM MgCl₂, and protease inhibitors. The homogenate was centrifuged two times at 40,000g for 20 min at 4°C, and the resulting pellet was resuspended in buffer containing 50 mM Tris HCl, pH 7.4, 5 mM EDTA, and 10 mM MgCl₂. Glycerol and bovine serum albumin were added to a final concentration of 10% glycerol and 1% bovine serum albumin before freezing the membranes. The inverse agonist activity of H₃R antagonists was determined as described previously (Krueger et al., 2005). In brief, membranes were diluted in GTPS assay buffer (25 mM HEPES, 2.5 mM MgCl₂, and 75 mM NaCl, pH 7.4), and 10 µg of membrane protein was incubated in a 96-well deep-well block in the presence of 5.0 µM unlabeled GDP, approximately 0.5 nM [³⁵S]GTPS, and various concentrations of H₃R antagonists. Samples were subsequently incubated at 37°C for 20 min. For assays to determine antagonist activity, (R)--methylhistamine (30 nM) was added in addition to the assay components described above, and the samples were incubated at 37°C for 5 min. The assays were terminated by the addition of cold buffer (50 mM Tris-HCl, 75 mM NaCl, and 2.5 mM MgCl₂, pH 7.6) and subsequent harvesting by vacuum filtration onto a Packard Unifilter 96-well GF/B plate (PerkinElmer Life Sciences). After extensive washing, the plates were dried, Microscint 20 was added to the samples, and the amount of bound [³⁵S]GTPS was determined utilizing the Topcount (PerkinElmer Life Sciences, Boston, MA). The percentage of [³⁵S]GTPS bound in each sample was calculated as a percentage of that bound to control samples incubated in the absence of histamine H₃R ligands. Triplicate determinations were obtained at each concentration, and the data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA) to obtain EC₅₀ or IC₅₀ values and Hill slopes. pK_b values were calculated using the generalized Cheng-Prusoff equation (Cheng and Prusoff, 1973; Leff and Dougall, 1993). The mean ± S.E.M. of at least three independent experiments is reported.

Measurement of cAMP. C6 cells stably expressing the H₃R were washed once with DMEM supplemented with 25 mM HEPES (pH 7.4 at 37°C) and preincubated in the same medium for 30 min at 37°C. Thereafter, 5 x 10³ cells/50 µl was added per well to a 96-well plate containing the respective ligands in 50 µl of DMEM supplemented with 0.3 mM 3-isobutyl-1-methylxanthine and 1 µM forskolin (final concentration, 0.5 µM). After 10 min, incubations were terminated by the addition of 20 µl of 0.3 mg/ml saponin to each well to lyse the cells. Subsequently, cAMP levels were determined with a competitive protein kinase A (PKA) binding assay, as described. Protein kinase A-containing fraction was isolated from bovine adrenal glands, as described previously (Wieland et al., 2001). In brief, a PKA-containing fraction was isolated from bovine adrenal glands, which were homogenized in 10 volumes of 100 mM Tris-HCl, 250 mM NaCl, 10 mM EDTA, 0.25 M sucrose, and 0.1% 2-mercaptoethanol (pH 7.4 at 4°C) and centrifuged for 60 min at 30.000g at 4°C. The supernatant, containing PKA, was carefully recovered and frozen in 1-ml aliquots at–80°C. Before use, the PKA was diluted 12-fold in ice-cold phosphate-buffered saline (PBS) and kept on ice. To each well, 20 µl of 0.6 nM [³H]cAMP in PBS (48.0 Ci/mmol) and 60 µlof PKA in PBS was added. After 30 min, the reaction was terminated by filtration over Unifilter GF/B filter plates with two subsequent washes with 200 µl of ice-cold 50 mM Tris-HCl, pH 7.4. Retained radioactivity was determined by liquid scintillation counting on the Microbeta Trilux with 50 µl of Microscint "O." The amount of cAMP in each sample was calculated with GraphPad Prism version 4.01 for Windows (GraphPad Software), using a generated standard cAMP curve (0.1 mM–10 pM).

Statistical Analysis. Statistical analyses were performed using GraphPad Prism version 4.01 for Windows (GraphPad Software). Differences among means were evaluated by analysis of variance, followed by the Dunnett's post test. For all analyses, the null hypothesis was rejected at the 0.05 level.

Results
Differential Expression in the Central Nervous System. The relative expression of the hH₃R(445) and hH₃R(365) isoforms was assessed by RT-PCR in several areas of the human CNS. For the hH₃R(445), high expression was found in the cerebellum and the caudate, and moderate expression was found in the hypothalamus, cerebrum, and thalamus (Fig. 1). Low expression was found in all other tested regions, and very low expression was found in the spinal cord. In most of the tested regions, the hH₃R(365) was found to be approximately 1.4-fold higher expressed than the hH₃R(445). The biggest differences in expression for the two isoforms was found in the regions where the hH₃R(445) was higher expressed than the hH₃R(365), such as the caudate (3.5-fold), corpus callosum (2.8-fold), and spinal cord (2.2-fold).

View larger version (42K): [in this window] [in a new window]   Fig. 1. Expression of the hH3R(445) and the hH3R(365) isoforms identified in the human central nervous system by RT-PCR. A and B, mRNA of human brain tissues was amplified by RT-PCR using primers flanking the intronic region. PCR products were analyzed using agarose gels stained with ethidium bromide. The lengths of amplicons were estimated by molecular mass markers. B, human GAPDH amplification was used as an internal standard.

Saturation Analysis Reveals Distinct Populations for the hH₃R Isoforms. The hH₃R isoforms (445 and 365) stably transfected into rat glioma C6 cells were characterized by saturation analysis with the agonist N-[methyl-³H]histamine and with the inverse agonists [¹²⁵I]iodophenpropit (Jansen et al., 1994) and [³H]A-349821 (Witte et al., 2006). Saturation binding experiments with N-[methyl-³H]histamine revealed a single high-affinity binding site for both isoforms with a similar maximal number of binding sites [hH₃R(445), B_max = 630 ± 100 fmol/mg protein, n = 7; and hH₃R(365), B_max = 670 ± 40 fmol/mg protein, n = 4]. Yet, for the radioligand N-[methyl-³H]histamine, a significantly lower affinity (p < 0.001) was measured for the hH₃R(445) compared with the hH₃R(365) [K_d values of 0.81 ± 0.07 nM (n = 7) and 0.33 ± 0.04 nM (n = 5) were obtained for hH₃R(445) and hH₃R(365), respectively; see Fig. 2, A and D]. Both hH₃R isoforms exhibited similar affinities for [¹²⁵I]iodophenpropit [hH₃R(445), K_d = 3.2 ± 2 nM; and hH₃R(365), K_d = 3.1 ± 2 nM], but a significant difference (p < 0.05) was found for the maximal number of binding sites [hH₃R(445), B_max = 2000 ± 500 fmol/mg protein, n = 3; and hH₃R(365), B_max = 830 ± 20 fmol/mg protein, n = 3] (Fig. 2, A and D). As observed for [¹²⁵I]iodophenpropit, the affinity values for the H₃R inverse agonist [³H]A-349821 did not differ between the two isoforms [hH₃R(445), K_d = 0.081 ± 0.01 nM, n = 4; and hH₃R(365), K_d = 0.10 ± 0.02 nM, n = 4], but a significant difference was again found for the maximal number of binding sites [hH₃R(445), B_max = 1800 ± 100 fmol/mg protein, n = 4; and hH₃R(365), B_max = 780 ± 100 fmol/mg protein, n = 4] (Fig. 2, C and D).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Typical saturation curves for N-[methyl-3H]histamine (A), [125I]iodophenpropit (B), and [3H]A-349821 (C) on membranes of C6 cells expressing either the hH3(445) () or the hH3(365) (). Nonspecific binding was determined in the presence of 100 µM thioperamide. D, maximal specific binding (Bmax) determined with N-[methyl-3H]histamine, [125I]iodophenpropit, and [3H]A-349821 at hH3(445)- and hH3(365)-expressing membranes. *, p < 0.05; **, p < 0.01 versus hH3R(365). E, relative effects of coincubation of 0.1 mM GDP on the maximal specific binding of membranes of C6 cells expressing the hH3R(445) or the hH3R(365) as determined with N-[methyl-3H]histamine or [3H]A-349821. For each condition, the control samples without 0.1 mM GDP are set to one, indicated by the dashed line. *, p < 0.05 versus hH3R(365). F, effects of various agents [pertussis toxin (PTX), GDP, and GTPS] that uncouple the receptor from the G-protein on the specific binding in a homologous competition experiment. *, p < 0.05 versus control. Results represent the mean ± S.E.M of a typical experiment (A–C) or the mean ± S.E.M. of at least three independent experiments performed in triplicate (D–F).

Pharmacological Profile of Radioligand Binding to the hH₃R(445) and hH₃R(365) Isoforms. A series of imidazole (compounds 1–14 and 22–28) and nonimidazole (compounds 15–21) containing H₃R ligands were subsequently tested in a heterologous competitive binding assay with N-[methyl-³H]histamine and [¹²⁵I]iodophenpropit with membranes expressing either the hH₃R(445) or the hH₃R(365) (see Table 1). The competition binding curves for all compounds were best fitted according to a single binding site model and shown to have Hill slopes close to unity for both radioligands (Fig. 3, A–D). Equilibrium dissociation constants of the agonists correlate highly for competitive binding with either N-[methyl-³H]histamine (r² = 0.82) or [¹²⁵I]-iodophenpropit (r² = 0.85) (Fig. 3, E and F). Agonists exhibit a higher affinity for the hH₃R(365) compared with the hH₃R(445), on average 3.4- or 55-fold when determined with N-[methyl-³H]histamine or [¹²⁵I]iodophenpropit, respectively (Fig. 3E). Equilibrium dissociation constants of the inverse agonists correlate highly as well (r² = 0.95 for N-[methyl-³H]histamine and r² = 0.91 for [¹²⁵I]iodophenpropit) but display an opposite preference for the hH₃R isoforms and have a 2 to 3-fold higher affinity for the hH₃(445) isoform (Fig. 3F). In the correlation plots from the heterologous competitive binding assay with N-[methyl-³H]histamine or [¹²⁵I]iodophenpropit, some compounds are outside the 95% confidence interval of the linear regression for both the agonist and inverse agonist and therefore are statistical outliers (Fig. 3, E and F).

View this table: [in this window] [in a new window]   TABLE 1 Affinity values from competition experiments using N-[methyl-3H]histamine or [125I]iodophenpropit in membranes expressing hH3R(445) or hH3R(365) Affinities are expressed as mean pKi ± S.E.M. (n = 3-9).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Competition binding experiment curves and corresponding correlation plots. A to D, competition binding curves determined with 0.8 nM NMH or 2 nM IPP in the presence of various concentrations of H3R compounds in membranes of C6 cells stably expressing the hH3R(445) or the hH3R(365). Results represent the mean ± S.E.M of a typical experiment. E and F, affinity correlation plots for a series of selective H3R agonists (, compounds 1–14) and inverse agonists (, compounds 15–28) determined in radioligand competition binding experiments with either N-[methyl-3H]histamine (E) or [125I]iodophenpropit (F) on membranes expressing the hH3R(445) or the hH3R(365). A good agreement between binding affinities is observed when agonists and inverse agonists are considered as a different group. Compounds outside the 95% confidence interval for both groups are indicated as outliers (). Unity is indicated by the dashed line.

Pharmacological Profile of the hH₃R(445)- and hH₃R(365)-Induced [³⁵S]GTPS Binding. A series of compounds was also tested in a functional [³⁵S]GTPS binding assay with membranes expressing either the hH₃R(445) or the hH₃R(365) isoform (see Table 2). The dose-response curves for all compounds were best fitted according to a single binding site model and showed to have Hill slopes close to unity (Fig. 4, A–E). Potencies of the agonists correlated highly (r² = 0.96) and were found to be, on average, 4.6-fold more potent at the hH₃R(365) (Fig. 4F). However, the maximal increase in [³⁵S]GTPS binding for full agonists was approximately 80% lower at the hH₃(365) (1.2-fold increase of basal) compared with the hH₃R(445) (2.2-fold increase of basal) (Fig. 4, A–C).

View this table: [in this window] [in a new window]   TABLE 2 Potency values from [35S]GTPS binding in membranes expressing hH3R(445) or hH3R(365) Potencies are expressed as mean pEC50 ± S.E.M. (n = 3-7). (R)--Methylhistamine, = 1 and ABT-239, = -1 by definition.

View larger version (23K): [in this window] [in a new window]   Fig. 4. [35S]GTPS binding curves and corresponding correlation plot. A to E, modulation of [35S]GTPS binding by increasing concentrations of H3R agonists and antagonists in membranes from C6 cells stably expressing the hH3R(445) or the hH3R(365). Data are expressed as a percentage of the basal [35S]GTPS binding in the absence of ligands (basal activities are isoform and time dependent: A–C, hH3R(445), 118 ± 3 fmol/mg; hH3R(365), 220 ± 25 fmol/mg; D and E, hH3R(445), 354 ± 45 fmol/mg, hH3R(365), 782 ± 72 fmol/mg). Results represent the mean ± S.E.M of a typical experiment. F, potency correlation plots for a series of selective H3R agonists (, 1–14) and inverse agonists (, 15–28) determined in a [35S]GTPS binding assay on membranes expressing the hH3R(445) or the hH3R(365). A good agreement between potencies is observed when agonists and inverse agonists are considered as a different group. Unity is indicated by the dashed line.

Potencies of the tested inverse agonist correlate highly as well (r² = 0.99) but were found to be, on average, 2.6-fold less potent at the hH₃R(365) (Fig. 4F). Interestingly, nonimidazole ligands, like A-349821, showed a significantly (p < 0.01) higher inhibition of basal [³⁵S]GTPS binding for the hH₃R(365) compared with the hH₃R(445) (Fig. 4, D and E).

Pharmacological Profile of the Forskolin-Induced cAMP Levels by the hH₃R(445) and the hH₃R(365). Histamine H₃R-specific compounds were also tested for their modulation of cAMP levels in forskolin (0.5 µM)-stimulated C6 cells expressing either the hH₃R(445) or the hH₃R(365) (see Table 3). All tested agonists and inverse agonists potencies correlate highly (r² = 0.91 and 0.88, respectively). Agonists are approximately 35-fold more potent at the hH₃R(365) (Fig. 5F). Similar to the [³⁵S]GTPS binding assay, full agonists are, however, more efficacious at the hH₃R(445) (80 and 44% inhibition of the forskolin-induced cAMP production, respectively; see Fig. 5, A–C). Conversely, inverse agonists are approximately 14-fold less potent at the hH₃R(445) but appear more efficacious (Fig. 5, D and E). Especially, nonimidazole compounds, like A-349821, showed increased efficacy at the hH₃R(365) (3.0-fold increase over basal) compared with the hH₃R(445) (1.5-fold increase over basal) (Fig. 5D).

View larger version (23K): [in this window] [in a new window]   Fig. 5. cAMP formation curves and corresponding correlation plot. A to E, modulation of 0.5 µM forskolin-stimulated cAMP formation by increasing concentrations of H3R ligands in C6 cells stably expressing the hH3R(445) or the hH3R(365). Data are expressed as a percentage of the basal cAMP levels in the absence of ligands. Results represent the mean ± S.E.M of a typical experiment. F, potency correlation plots for a series of selective H3R agonists (, compounds 1–14) and inverse agonists (, compounds 15–28) determined in a cAMP formation assay on C6 cells expressing the hH3R(445) or the hH3R(365). A good agreement between potencies is observed when agonists and inverse agonists are considered as a different group. Unity is indicated by the dashed line.

The hH₃R(365) Is More Constitutively Active Than the hH₃R(445). The observed differences in agonist/inverse agonist efficacies are good indications of substantial differences in the levels of constitutive activity of the two hH₃R isoforms. To investigate this issue, the amount of [³⁵S]GTPS formed over a time course of 10 min was measured on C6 cell membranes expressing either the hH₃R(445) or the hH₃R(365). The C6 parental cell line showed a gradual increase in the amount of [³⁵S]GTPS bound, and no modulation by the specific H₃R ligands (R)--methylhistamine and A-349821 was observed (Fig. 6A). Basal increase in [³⁵S]GTPS binding in C6 cells expressing the hH₃R(445) was comparable with the parental cell line, whereas for the hH₃R(365), the basal increase was significantly higher (Fig. 6, A–D). Stimulation of the hH₃R(445) with the H₃R agonist (R)--methylhistamine led to a steep increase in [³⁵S]GTPS binding over time, whereas the inverse agonist, A-349821, slightly inhibited the [³⁵S]GTPS binding (Fig. 6B). On membranes expressing the hH₃R(365), the increase in [³⁵S]GTPS binding induced by (R)--methylhistamine was moderate, whereas the effect of A-349821 was more pronounced compared with its effect at the hH₃R(445) (Fig. 6, A–D).

View larger version (21K): [in this window] [in a new window]   Fig. 6. Basal and H3R ligand-mediated [35S]GTPS and cAMP signaling in C6 cells expressing the hH3R(445) or the hH3R(365). A to C, basal and ligand-mediated [35S]GTPS formed over a time course of 10 min in parental C6 cells or C6 cells expressing either the hH3R(445) or the hH3R(365). Total [35S]GTPS formed in 10 min (fmol/mg protein) in the parental C6 cells and expressing the hH3R(445) or the hH3R(365). D and E, basal and ligand [1 µM(R)--methylhistamine or 0.1 µM A-349821] H3R-mediated modulation of GTPS binding (fmol/mg protein/min) and forskolin (0.5 µM)-induced cAMP levels in C6 cells expressing either the hH3R (445) or the hH3R (365).

In addition, forskolin (0.5 µM)-induced cAMP levels were 2.3-fold (p < 0.001) lower in C6 cells expressing the hH₃R(365) compared with C6 cells expressing hH₃R(445) (Fig. 6E). The H₃R agonist (R)--methylhistamine inhibited the forskolin (0.5 µM) induced to a similar absolute level in both hH₃R(445)- and hH₃R(365)-expressing C6 cells. Likewise, stimulation of C6 cells expressing either the hH₃R(445) or the hH₃R(365) with the H₃R inverse agonist A-349821 increased the cAMP levels to a similar absolute level of cAMP.

Application of the Cubic Ternary Model to Explain the Observed Properties of the hH₃R(365) and hH₃R(445). We used the cubic ternary complex (CTC) model (Weiss et al., 1996a,b,c) to examine whether the observed differences in affinities and potencies, the maximal number of binding sites of agonist and inverse agonist and the effect of GDP on the maximal number of binding sites could all be explained by an increase in GPCR constitutive activity, represented in the CTC model by the equilibrium constant of the receptor (K_act; see Fig. 7A).

View larger version (25K): [in this window] [in a new window]   Fig. 7. Computer simulations according to the CTC model. A, CTC model with equilibrium association constants (adapted from Weiss et al., 1996a,b,c), in which Ka and Kg represent the association constants for a ligand (L) and G-protein (G), respectively; Kact is the equilibrium constant for the activation of the receptor (R); and represent the effect of ligand binding on activation and G-protein coupling of the receptor, respectively; represents the effect of receptor activation on G-protein coupling; and represents the extent to which the joint effect of any two (receptor activation, G-protein coupling, or ligand binding) varies the level of the third. B, theoretical maximal specific binding of an agonist (NMH) or inverse agonist (IPP) in the presence (Gtot = 1) or absence (Gtot = 0) of G-proteins. C and D, theoretical radioligand competition of an agonist (C) or inverse agonist (D) radioligand by an unlabeled agonist or inverse agonists on the hH3R(445) and hH3R(365). Curves were plotted as a percentage of the calculated Bmax. E, theoretical dose-response curve for the hH3R(445) and hH3R(365) for agonists and inverse agonists. All simulations were generated with the following parameters: for agonists, = 10 and = 1; for inverse agonists, = 0.1 and = 0.1; for the hH3R(445), Kact = 0.005 and Rtot = 1; and for the hH3R(365), Kact = 1 and Rtot = 0.3. The other parameters were kept constant ( = 10, = 10, Ka = KG = 109, Gtot = 1).

To examine the maximal number of binding site as found with the H₃R radioligands (Fig. 2A), the agonist radioligand N-[methyl-³H]histamine was assumed to bind the R_a and R_aG state of the receptor, to promote receptor activation ( > 1), and to facilitate G-protein coupling only for the receptor in its active form ( > 1). The inverse agonist radioligand [³H]A-349821 was assumed to bind the R_i and R_iG state of the receptor, to inhibit receptor activation ( < 1), and to facilitate G-protein coupling only for the receptor in its inactive form ( < 1). Furthermore, to examine whether the difference between the two isoforms could be due to a difference in K_act, the hH₃R(445) and the hH₃R(365) were assumed to have a K_act < 1 and K_act = 1, respectively. Hereby, the hH₃R(445) exists predominantly in the R_i conformation, and the hH₃R(365) has no preference for either the R_a of R_i conformation. Experimentally, we found an approximately 3-fold difference in maximal number of binding sites of [³H]A-349821 for the two hH₃R isoforms (Fig. 2D). To take this observation into account in the CTC model, the R_tot of the hH₃R(365) was assumed to be 3-fold lower compared with the hH₃R(445).

Subsequently, the maximal number of binding sites for the agonist, N-[methyl-³H]histamine (NMH), and inverse agonist, [¹²⁵I]iodophenpropit (IPP), were simulated in the presence (G_tot = 1) and absence (G_tot = 0) of G-proteins. This simulation revealed that only the maximal number of binding sites for the agonist, reflected by the receptor states LR_a and LR_aG, on the hH₃R(445) is affected by the removal of G-proteins (Fig. 7B). Furthermore, the maximal number of binding sites for the hH₃R(445) recognized by the inverse agonist, reflected by the receptor states LR_i and LR_iG, was found to be approximately three times higher, whereas for the hH₃R(365), agonist and inverse agonists give rise to the same maximal number of binding sites (Fig. 7B). To account for the differences in ligand affinity and potency found experimentally for the hH₃R(445) and the hH₃R(365), we calculated the apparent affinity constant when G-proteins are not limiting (K_Aapp) (Weiss et al., 1996b).

(1)

From this equation (eq. 1), it follows that for the hH₃R(445), the log(K_Aapp) for agonists is 10.2 and for inverse agonists, 10.0; in the case of the hH₃R(365), the log(K_Aapp) was increased for agonists to 11.0 and decreased for inverse agonists to 9.0. We defined a partial agonist to have an > 1 but lower than of a full agonist, e.g., the ligand is able to activate the receptor but to a lesser extent than a full agonist. The difference for partial agonists in the K_Aapp for the two isoforms decreases, and with an 1, the log(K_Aapp) = 10 for both the hH₃R(445) and the hH₃R(365).

Furthermore, we simulated radioligand competition and dose-response curves for the hH₃R(445) and hH₃R(365) in a G-protein-dependent manner (Monczor et al., 2003). For the competition curves, we assumed the radioligands and the competing ligands to have properties as described above. Subsequently, the concentration of a ligand (L) was varied, and the generated curves were plotted as percentage of the B_max for both hH₃R isoforms and radioligands (Fig. 7, C and D).

For simulation of dose-response curves, we assumed that both the LR_aG and R_aG give rise to a receptor response (as described by Chen et al., 2000). Subsequently, the following equation (eq. 2) was used to simulate the dose-response curves (Fig. 7E).

(2)

Furthermore, when [L] = 0, the above equation describes the basal signaling of the receptor, and this was found to be 19-fold increased for the hH₃R(365) compared with the hH₃R(445).

Discussion
The cloning of the hH₃R(445) by Lovenberg et al. (1999) has subsequently led to the discovery of a large number of hH₃R isoforms (Hancock et al., 2003; Leurs et al., 2005). In this study, we pharmacologically characterize the two most abundantly occurring hH₃R isoforms, the hH₃R(445) and the hH₃R(365) (Cogé et al., 2001; Wellendorph et al., 2002; Esbenshade et al., 2006). Using an RT-PCR approach, we confirmed the abundant expression of the hH₃R(445) and the hH₃R(365) isoforms and demonstrated differential isoform expression in various human brain areas. In most regions, the hH₃R(365) was slightly higher expressed. However, in brain regions with the highest hH₃R(445) expression levels, such as caudate, corpus callosum, and the spinal cord, the largest differences between these two isoforms were found.

The hH₃R(365) lacks 80 amino acids in the IL3 in comparison with the hH₃R(445) and was found to have different functionality. In particular, we show that the hH₃R(365) isoform stably expressed in C6 cells gives rise to a higher agonist-independent inhibition of forskolin-induced cAMP levels and to an increased basal [³⁵S]GTPS formation. These findings are consistent with an increased constitutive receptor activity of the hH₃R(365) compared with the hH₃R(445). Basal GPCR signaling highly depends on the receptor expression levels but is also regulated by the expression levels of its cellular signaling partners (Milligan, 2003). However, the observed increase in constitutive signaling of the hH₃R(365) cannot be explained by a higher level of receptor expression because it was found to have either the same (N-[methyl-³H]histamine) or lower ([³H]A-349821 or [¹²⁵I]iodophenpropit) number of radioligand binding sites as observed for the hH₃R(445). In addition, both isoforms were stably expressed using the same parental C6 cell line, and differences in cellular content, like G-protein expression, are therefore unlikely.

Previously, agonist-induced activation of the hH₃R(365) was not found in several signal transduction assays, like the G_i-mediated inhibition of cAMP formation, [³⁵S]GTPS binding, or Ca²⁺ mobilization (Cogé et al., 2001), but in another study, the hH₃R(365) could be activated by agonists in a reporter assay (Wellendorph et al., 2002). In the present study, we show that under our experimental conditions, H₃R agonists can activate the hH₃R(365) isoform, resulting in an increase in [³⁵S]GTPS binding and the expected inhibition of forskolin-induced cAMP. The apparent discrepancy between the results of Coge et al. and the present study might be due to the high constitutive activity of the hH₃R(365), which makes it difficult to measure agonist-mediated responses. Interestingly, the study of Coge et al. does not report on the functional effects of H₃R inverse agonists on the hH₃R(365).

The hH₃R(365) displays higher potencies and affinities for agonists and likewise lower potencies and affinities for inverse agonists, consistent with an increase in constitutive activity. These findings are in line with previous observations with the rat H₃R_B and rat H₃R_C isoforms, which have a 32- or 48-amino acid deletion in the IL3 loop, respectively, and also display a slightly higher affinity and potency for agonists compared with the full-length rat H₃R_A isoform (Morisset et al., 2000; Drutel et al., 2001). Interestingly, partial agonists have a higher affinity and potency at the hH₃R(365) as well but to a less extent than full agonists. The observed statistical outliers in the binding affinity correlation plots, in fact, appear to be partial agonists with intrinsic activities between 0.0 and 0.4 or 0.0 and 0.7 in the correlation plots for N-[methyl-³H]histamine or [¹²⁵I]iodophenpropit, respectively (Fig. 3, E and F; Table 3). This is in good agreement with the CTC model simulations, where we found that the of a compound correlates with the difference in apparent affinity (K_Aapp) for the two isoforms, e.g., a decrease in leads to a smaller difference in K_Aapp for the two hH₃R isoforms. Interestingly, agents that prevent G-protein coupling were found to affect the maximal number of binding sites of N-[methyl-³H]histamine only for the hH₃R(445) but not for the hH₃R(365). In the present study, the binding of the inverse agonists was unaffected by a treatment that would uncouple the receptor from the G-protein. This seems in contrast with earlier studies (Witte et al., 2006) but is most likely due to increased ionic strength of the incubation buffers, which is known to shift the equilibrium of the receptor to the inactive conformation (Costa et al., 1990). To account for these observations, the CTC model was used to examine whether a higher K_act of a constitutively active receptor might be able to explain these observations. Indeed, the CTC model can account for the observed different maximal number of binding sites for the different radioligands and confirmed that the number of agonist-labeled sites was affected only at the hH₃R(445) when [G_tot] = 0. Furthermore, an increase of the K_act resulted in the experimentally observed increase of potency and affinity of agonists and decreased potency and affinity of inverse agonists at the hH₃R(365) compared with the hH₃R(445). The CTC model also accounted for the intermediate shift in affinity of partial agonists, showing that the preference over the hH₃R(365) is dependent on intrinsic activity of the compounds. We used the CTC model because other "simpler" models, such as the extended ternary complex model, could not explain the effect of GDP. In this model, the removal of the G-protein affects predominantly the receptor with the biggest K_act, which is not in accordance with our experimental observations.

In summary, our experimental and CTC modeling data indicate that the hH₃R(365) isoform mainly functions in a constitutive manner. These data indicate that the 80-amino acid stretch in the third intracellular loop either imparts a negative constraint on the GPCR activation directly or is involved in the interaction of intracellular proteins that inhibit H₃R activation. Future studies should address the role of the 80-amino acid stretch in the IL3 on H₃R receptor function in more detail. Because the H₃R is one of the few examples for which GPCR constitutive activity has been shown to be prominent under in vivo conditions (Morisset et al., 2000; Wieland et al., 2001), our present results suggest that this effect might be mainly mediated by the hH₃R(365) isoform. The nonimidazole compounds show a higher negative intrinsic activity at the hH₃R(365) compared with classical imidazole containing compounds like thioperamide. In combination with the potential differential expression in the CNS, this might lead to specific effects of these nonimidazole inverse agonists in brain areas where the hH₃R(365) is higher expressed than the hH₃R(445).

	Footnotes

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

doi:10.1124/jpet.107.127639.

ABBREVIATIONS: H₃R, histamine H₃ receptor; CNS, central nervous system; h, human; [³⁵S]GTPS, guanosine-5'-O-(3-[³⁵S]thio) triphosphate; IL3, intracellular loop 3; GPCR, G-protein-coupled receptor; [³H]A-349821, {4'-[3-((2R, 5R)-2,5-dimethyl-pyrrolidin-1-yl)-propoxy]-biphenyl-4-yl}-morpholin-4-yl-methanone; RT, reverse transcriptase; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; PKA, protein kinase A; PBS, phosphate-buffered saline; CTC, cubic ternary complex; NMH, N-[methyl-³H]histamine; IPP, [¹²⁵I]iodophenpropit; FUB322, 3-(1H-imidazol-4-yl) propyl-di(p-fluorophenyl)-methyl ether hydrochloride; A-304121, (R)-2-amino-1-{4-[3-(4-cyclopropanecarbonyl-phenoxy)-propyl]-piperazin-1-yl}-propan-1-one; A-317920, furan-2-carboxylic acid, ((R)-2-{4-[3-(4-cyclopropanecarbonyl-phenoxy)-propyl]-piperazin-1-yl}-1-methyl-2-oxo-ethyl)-amide; A-320436, furan-2-carboxylic acid, ((R)-2-{4-[3-(4'-cyano-biphenyl-4-yloxy)-propyl]-[1,4]diazepan-1-yl}-2-oxo-1-thiazol-4-ylmethyl-ethyl)-amide; A-331440, 4'-[3-((R)-3-dimethylamino-pyrrolidin-1-yl)-propoxy]-biphenyl-4-carbonitrile; A-349821, {4'-[3-((2R, 5R)-2,5-dimethyl-pyrrolidin-1-yl)-propoxy]-biphenyl-4-yl}-morpholin-4-yl-methanone; A-358239, 4-{2-[2-((R)-2-methyl-pyrrolidin-1-yl)-ethyl]-benzofuran-5-yl}-benzonitrile; A-431404, (4-fluoro-phenyl)-{2-[2-((R)-2-methyl-pyrrolidin-1-yl)-ethyl]-benzofuran-5-yl}-methanone.

¹ Current affiliation: Department of Metabolic Diseases, Boehringer Ingelheim Pharma GmbH and Co. KG, Biberach, Germany.

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

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