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CELLULAR AND MOLECULAR

Mutations of Cys-17 and Ala-271 in the Human Histamine H₂ Receptor Determine the Species Selectivity of Guanidine-Type Agonists and Increase Constitutive Activity

Departments of Pharmaceutical/Medicinal Chemistry II (H.P., P.G., A.K., S.D., A.B.) and Pharmacology and Toxicology (R.S.), Institute of Pharmacy, University of Regensburg, Regensburg, Germany

Received January 25, 2007; accepted March 2, 2007.

	Abstract

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In a steady-state GTPase activity assay, N-[3-(1H-imidazol-4-yl)propyl)]guanidines and N^G-acylated derivatives are more potent and efficacious at fusion proteins of guinea pig (gpH₂R-G_sS) than human (hH₂R-G_sS) histamine H₂ receptor, coupled to the short splice variant of G_s, G_sS. Whereas Ala-271 (hH₂R) and Asp-271 (gpH₂R) in transmembrane domain 7 were identified to determine the potency differences of guanidine-type agonists, the molecular basis for the efficacy differences remains to be elucidated. A homology model of the gpH₂R suggested that an H-bond between Tyr-17 and Asp-271 stabilizes an active receptor conformation of the gpH₂R. In the present study, we generated a mutant hH₂R-G_sS with Cys-17 Tyr-17/Ala-271 Asp-271 exchanges (hH₂RgpH₂R) that exhibited an enhanced level of constitutive GTPase activity and adenylyl cyclase activity compared with wild-type hH₂R-G_sS and gpH₂R-G_sS. Potencies and efficacies of guanidines and N^G-acylguanidines were increased at this mutant receptor compared with hH₂R-G_sS, but they were still lower than at gpH₂R-G_sS, suggesting that aside from Tyr-17 and Asp-271 additional amino acids contribute to the distinct pharmacological profiles of both species isoforms. Another hH₂R-G_sS mutant with a Cys-17 Tyr-17 exchange showed inefficient coupling to G_sS as revealed by reduced agonist-stimulated GTPase and basal adenylyl cyclase activities. Collectively, our present pharmacological study confirms the existence of an H-bond between Tyr-17 and Asp-271 favoring the stabilization of an active receptor conformation. Distinct potencies and efficacies of agonists and inverse agonists further support the concept of ligand-specific conformations in wild-type and mutant H₂R-G_sS fusion proteins.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Structures of H2R agonists and inverse agonists. 1 to 3, small H2R agonists; 4, H1R agonist with partial agonism at the H2R; 5 to 7, guanidine-type H2R agonists; 8 to 12, NG-acylated N-[3-(1H-imidazol-4-yl)propyl]guanidines with agonistic H2R activity; 13, (R)-N-[3-(2-amino-4-methylthiazol-5-yl)propyl]-N'-(3-phenylbutanoyl)guanidine, an H2R agonist; and 14 to 18, H2R inverse agonists.

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A three-dimensional homology model of the gpH₂R suggested that the nonconserved Asp-271 in transmembrane domain (TM) 7 confers high potency to the guanidines, which was subsequently confirmed by an Ala-271 Asp-271 mutation in hH₂R-G_sS (hH₂R-A271D-G_sS) (Kelley et al., 2001). However, the efficacies of guanidines at this mutant and at hH₂R/gpH₂R chimeras were lower than at gpH₂R, demonstrating that guanidine efficacy depends on additional or other interactions. As a rationale, an interhelical H-bond between Tyr-17 in TM1 and Asp-271 was predicted from the model, stabilizing an active guanidine-bound conformation only in gpH₂R but not in hH₂R (containing Cys-17 and Ala-271) (Kelley et al., 2001).

To test this hypothesis, we generated an hH₂R-G_sS mutant with a Cys-17Tyr-17 exchange and a double mutant with Cys-17Tyr-17 and Ala-271Asp-271 exchanges in the sequence of hH₂R. Sf9 cell membranes expressing mutant and wild-type H₂R-G_sS were used to measure steady-state GTPase activity, because this system was previously shown to be reliable and very sensitive to analyze ligand potencies and efficacies (Seifert et al., 1999; Milligan, 2000). due to the defined 1:1 stoichiometry of receptor and G_s in fusion proteins, ligand potencies and efficacies in the steady-state GTPase assay are independent of the expression levels, allowing for the comparison of various membrane preparations with different expression levels. We also assessed AC activity in Sf9 membranes as a sensitive readout to compare distinct levels of constitutive activity of mutant and wild-type H₂R-G_sS fusion proteins. Figure 1 shows the structures of H₂R agonists examined in the present study. Impromidine (IMP; 5), arpromidine (ARP; 6), and BU-E-43 (7) are representatives of N-[3-(1H-imidazol-4-yl)propyl]guanidines. Their N^G-acylated derivatives contain diverse diarylpropanoyl (9 and 11), 3-(hetero)arylbutanoyl (8 and 10), and 3-(cyclohexylbutanoyl) (12) groups. Compound 13 contains a 2-amino-4-methylthiazol-5-yl group and exhibits enhanced selectivity relative to the H₃R (Ghorai, 2005). Compounds 10 and 13 are the pure (R)-enantiomers. In addition, the inverse agonists cimetidine (CIM; 14), ranitidine (RAN; 15), famotidine (FAM; 16), aminopotentidine (APT; 17), and iodoaminopotentidine (IAPT; 18) were studied (Hill et al., 1997; Dove et al., 2004).

	Materials and Methods

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Materials. The generation of pGEM-3Z-SF-hH₂R-G_sS, pGEM-3Z-SF-hH₂R-A271D-G_sS, and pVL1392-SF-hH₂R-G_sS was described previously (Kelley et al., 2001). The generation of the baculoviruses encoding hH₂R-G_sS and gpH₂R-G_sS was described previously (Kelley et al., 2001; Houston et al., 2002). Compounds 8 and 11 and 13 (Ghorai, 2005; Xie et al., 2006a) and compound 12 (Xie et al., 2006b) were prepared as described. IMP was synthesized as described previously (Durant et al., 1978). ARP and BU-E-43 were synthesized as described previously (Buschauer, 1989). APT and IAPT were prepared as described previously (Hirschfeld et al., 1992). Suprahistaprodifen was synthesized as described previously (Elz et al., 2000). The structures of compounds were confirmed by elemental analysis (C, H, N), ¹H NMR, and mass spectrometry. Purity of compounds was >98% as determined by high-performance liquid chromatography or capillary electrophoresis. The anti-FLAG Ig (M1 monoclonal antibody) was from Sigma-Aldrich (St. Louis, MO), and the anti-His₆ Ig was from Clontech (Mountain View, CA). [-³²P]GTP was synthesized through phosphorylation of GDP by enzymatic conversion of L--glycerol phosphate to 3-phosphoglycerate following a procedure described previously (Walseth and Johnson, 1979). [³²P]P_i (8500 –9100 Ci/mmol orthophosphoric acid), [-³²P]ATP (800 Ci/mmol), and [³H]dihydroalprenolol (85–90 Ci/mmol) were from PerkinElmer Life and Analytical Sciences (Boston, MA). All unlabeled nucleotides, glycerol-3-phosphate dehydrogenase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase were from Roche Diagnostics (Indianapolis, IN). 3-Phosphoglycerate kinase, L--glycerol phosphate, HA, CIM, RAN, and FAM were from Sigma-Aldrich. AMT was from Tocris Cookson Inc. (Ballwin, MO). DIM was from Sigma/RBI (Natick, MA). All restriction enzymes and T4 DNA ligase were from New England Biolabs (Beverly, MA). Cloned Pfu DNA polymerase was from Stratagene (La Jolla, CA).

Construction of the cDNA for hH₂R-C17Y-G_sS. The Cys-17 Tyr-17 exchange in hH₂R was generated by sequential overlap-extension PCRs. With pGEM-3Z-SF-hH₂R-G_sS as template, PCR 1A was used to amplify a DNA fragment consisting of the cleavable signal peptide from influenza hemagglutinin (S), the FLAG epitope (F) recognized by the M1 monoclonal antibody, and the N-terminal portion of the hH₂R. The sense primer annealed with 18 base pairs of pGEM-3Z before the 5' end of SF. The antisense primer encoded the sequence 5'-GATCTTATATGCGGTAGAGTCTAGACAAAAGGAAGAGGCTG-3' to generate the Cys-17 Tyr-17 exchange and a new XbaI site (TCTAGA). In PCR 1B, the DNA sequence of the hH₂R, a hexahistidine tag, and the entire sequence of G_sS was amplified using pGEM-3Z-SF-hH₂R-G_sS as template. The sense primer encoded the sequence '-CTTTTGTCTAGACTCTACCGCATATAAGATCACCATCACCG-3' to generate the Cys-17 Tyr-17 exchange and the new XbaI site. The antisense primer annealed with the cDNA encoding the five C-terminal amino acids of G_sS, the stop codon, and an XbaI site. In PCR 2, the products of PCR 1A and 1B annealed in the region encoding the newly created Cys-17 Tyr-17 exchange and the new XbaI site. Here, the sense primer of PCR 1A and the antisense primer of PCR 1B were used. In that way, the complete cDNA for the hH₂R-C17Y-G_sS fusion protein was amplified. The product of PCR 2 was digested with SacI and KpnI and cloned into pGEM-3Z-SF-hH₂R-G_sS digested with SacI and KpnI. pGEM-3Z-SF-hH₂R-C17Y-G_sS was digested with SacI and EcoN I and cloned into the baculovirus transfer vector pVL1392-SF-hH₂R-G_sS digested with SacI and EcoNI. PCR-generated DNA sequences were confirmed by extensive restriction enzyme analysis and enzymatic sequencing.

Construction of the cDNA for hH₂R-C17Y-A271D-G_sS. To generate the DNA for fusion proteins with two amino acid exchanges Cys-17 Tyr-17 and Ala-271 Asp-271, pGEM-3Z-SF-hH₂R-A271D-G_sS was digested with KpnI and BglII and cloned into pGEM-3Z-SF-hH₂R-C17Y-G_sS digested with KpnI and BglII. pGEM-3Z-SF-hH₂R-C17Y-A271D-G_sS was digested with NcoI and BglII and cloned into the baculovirus transfer vector pVL1392-SF-hH₂R-G_sS digested with NcoI and BglII.

Generation of Recombinant Baculoviruses, Cell Culture, and Membrane Preparation. Recombinant baculoviruses encoding hH₂R-C17Y-G_sS and hH₂R-C17Y-A271D-G_sS were generated in Sf9 cells using the BaculoGOLD transfection kit (BD Biosciences PharMingen, San Diego, CA) according to the manufacturer's instructions. After initial transfection, high-titer virus stocks were generated by two sequential virus amplifications. Sf9 cells were cultured in 250-ml disposable Erlenmeyer flasks at 28°C under rotation at 125 rpm in SF 900 II medium (Invitrogen, Carlsbad, CA) supplemented with 5% (v/v) fetal calf serum (Cambrex Bio Science Walkersville Inc., Walkersville, MD) and 0.1 mg/ml gentamicin (Cambrex Bio Science Walkersville Inc.). Cells were maintained at a density of 0.5 to 6.0 x 10⁶ cells/ml. For infection, cells were sedimented by centrifugation and suspended in fresh medium. Cells were seeded at 3.0 x 10⁶ cells/ml and infected with a 1:100 dilution of high-titer baculovirus stocks encoding H₂R-G_sS fusion proteins. Cells were cultured for 48 h before membrane preparation. Sf9 membranes were prepared as described previously (Seifert et al., 1998a), using 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml benzamidine, and 10 µg/ml leupeptin as protease inhibitors. Membranes were suspended in binding buffer (12.5 mM MgCl₂, 1 mM EDTA, and 75 mM Tris-HCl, pH 7.4) and stored at –80°C until use.

SDS-PAGE and Immunoblot Analysis. Membrane proteins were separated on SDS polyacrylamide gels containing 12% (w/v) acrylamide. Proteins were transferred onto Immobilon-P membranes (Millipore Corporation, Bedford, MA) and reacted with M1 antibody, or anti-His₆ Ig (1:1000 each). Protein bands were visualized by enhanced chemoluminescence (Pierce Chemical, Rockford, IL) using sheep anti-mouse IgG, coupled to peroxidase.

Steady-State GTPase Activity Assay. Membranes were thawed, sedimented, and resuspended in 10 mM Tris-HCl, pH 7.4. Assay tubes contained Sf9 membranes expressing H₂R-G_sS fusion proteins (10 µg of protein/tube), 1.0 mM MgCl₂, 0.1 mM EDTA, 0.1 mM ATP, 100 nM GTP, 0.1 mM adenylyl imidodiphosphate, 5 mM creatine phosphate, 40 µg of creatine kinase, and 0.2% (w/v) bovine serum albumin in 50 mM Tris-HCl, pH 7.4, and H₂R ligands at various concentrations. Reaction mixtures (80 µl) were incubated for 2 min at 25°C before the addition of 20 µl of [-³²P]GTP (0.1 µCi/tube). All stock and work dilutions of [-³²P]GTP were prepared in 20 mM Tris-HCl, pH 7.4. Reactions were conducted for 20 min at 25°C. Preliminary studies under basal conditions and with HA, IMP, and ARP showed that under these conditions, GTP hydrolysis was linear. Reactions were terminated by the addition of 900 µl of slurry consisting of 5% (w/v) activated charcoal and 50 mM NaH₂PO₄, pH 2.0. Charcoal absorbs nucleotides but not P_i. Charcoal-quenched reaction mixtures were centrifuged for 7 min at room temperature at 15,000g. Six hundred microliters of the supernatant fluid of reaction mixtures was removed, and ³²P_i was determined by liquid scintillation counting. Enzyme activities were corrected for spontaneous degradation of [-³²P]GTP. Spontaneous [-³²P]GTP degradation was determined in tubes containing all of the above-described components plus a very high concentration of unlabeled GTP (1 mM) that, by competition with [-³²P]GTP, prevents [-³²P]GTP hydrolysis by enzymatic activities present in Sf9 membranes. Spontaneous [-³²P]GTP degradation was <1% of the total amount of radioactivity added using 20 mM Tris-HCl, pH 7.4, as solvent for [-³²P]GTP. The experimental conditions chosen ensured that not more than 10% of the total amount of [-³²P]GTP added was converted to ³²P_i.

AC Activity Assay. AC activity in Sf9 membranes was determined as described previously (Houston et al., 2002). In brief, membranes were thawed and sedimented by a 15-min centrifugation at 4°C and 15,000g to remove residual endogenous guanine nucleotides as far as possible, and they were subsequently resuspended in binding buffer. Tubes contained Sf9 membranes expressing H₂R-G_sS fusion proteins (20 µg of protein/tube), additionally 5 mM MgCl₂, 0.4 mM EDTA, and 30 mM Tris-HCl, pH 7.4. Assay tubes containing membranes and various additions in a total volume of 30 µl were incubated for 3 min at 37°C before starting reactions by the addition of 20 µl of reaction mixture containing (final) [-³²P]ATP (0.3 µCi/tube) plus 40 µM unlabeled ATP, 2.7 mM mono(cyclohexyl)ammonium phosphoenolpyruvate, 0.125 IU of pyruvate kinase, 1 IU of myokinase, and 0.1 mM cAMP. Reactions were conducted for 20 min at 37°C. Reactions were terminated by the addition of 20 µl of 2.2 N HCl. Denatured protein was sedimented by a 3-min centrifugation at 25°C and 15,000g. Sixty-five microliters of the supernatant fluid was applied onto disposable columns filled with 1.3 g of neutral alumina (A-1522, super I, WN-6; Sigma-Aldrich). [³²P]cAMP was separated from [-³²P]ATP by elution of [³²P]cAMP with 4 ml of 0.1 M ammonium acetate, pH 7.0. Recovery of [³²P]cAMP was 80%. Blank values were routinely 0.01% of the total amount of [-³²P]ATP added. [³²P]cAMP was determined by liquid scintillation counting. The experimental conditions chosen ensured that not more than 1 to 3% of the total amount of [-³²P]ATP added was converted to [³²P]cAMP.

Miscellaneous. Protein concentrations were determined using the DC protein assay kit (Bio-Rad, Hercules, CA). [³H]Dihydroalprenolol saturation binding was performed as described previously (Seifert et al., 1998a). All analyses of experimental data were performed with the Prism 4 program (GraphPad Software Inc., San Diego, CA). K_B values were calculated using the Cheng and Prusoff (1973) equation. Expression levels of recombinant proteins were determined using the GS-710 calibrated imaging densitometer and the software tool Quantity One version 4.0.3 (Bio-Rad).

	Results

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Immunological Detection of Recombinant Proteins in Sf9 Cell Membranes. In Sf9 cells hH₂R-C17Y-G_sS and hH₂R-C17Y-A271D-G_sS were well expressed (Fig. 2, A and B). Monomeric nonfused H₂R expressed in Sf9 cells migrates as an 33-kDa band in SDS-PAGE (Fukushima et al., 1997; Houston et al., 2002), and the apparent molecular mass of G_sS is 45 kDa (Graziano et al., 1989). SDS-PAGE analysis of membranes expressing hH₂R-C17Y-A271D-G_sS yielded intense bands at 80 kDa, recognized by both the anti-FLAG and the anti-His₆ antibodies, that coincide with the expected apparent molecular masses of H₂R-G_sS monomers (Kelley et al., 2001; Houston et al., 2002). Both bands seemed somewhat diffuse, representing different glycosylation forms of the proteins. With the anti-His₆ antibody, an additional doublet band was detected at 45 kDa not recognized by the anti-FLAG antibody, which is presumably due to a lack of epitope exposure. By contrast, SDS-PAGE of membranes expressing hH₂R-C17Y-G_sS yielded strong and diffuse bands at 40 kDa and lacked the expected bands at 80 kDa. These bands could either represent atypically migrating glycosylated forms of H₂R-G_sS monomers or degraded proteins. Because the anti-FLAG Ig recognizes the N terminus and the anti-His₆ Ig the C terminus of the H₂R, it can be concluded that for either case the complete amino acid sequence of hH₂R-C17Y was expressed. Additional diffuse bands at 110 kDa may correspond to GPCR dimers or higher oligomers and were also observed in wild-type hH₂R-G_sS fusion proteins (Kelley et al., 2001). Comparison with the peak intensities of calibrated Sf9 membranes expressing the ₂-adrenergic receptor (AR) at 7.5 pmol mg^–1 (as determined by [³H]dihydroalprenolol saturation binding) revealed approximately similar expression levels of 2 pmol mg^–1 for hH₂R-C17Y-G_sS and hH₂R-C17Y-A271D-G_sS.

View larger version (52K): [in this window] [in a new window]   Fig. 2. Immunological detection of recombinant proteins in Sf9 cells. Sf9 membranes expressing various proteins were prepared, separated by SDS-PAGE on gels containing 12% (w/v) acrylamide, transferred onto Immobilon P membranes, and probed with the respective Ig indicated on top of each panel. In each lane, 10 µg of membrane protein was loaded onto the gel. Numbers on the left of membranes designate masses of marker proteins in kilodaltons.

Agonist and Inverse Agonist Effects on GTPase Activities in Sf9 Membranes Expressing hH₂R-G_sS, gpH₂R-G_sS, hH₂R-C17Y-G_sS, and hH₂R-C17Y-A271D-G_sS. The basal GTPase activity of hH₂R-G_sS amounted to 0.66 ± 0.09 pmol mg^–1 min^–1 (n = 10). Compared with it, the data were similar in membranes expressing gpH₂R-G_sS (0.69 ± 0.19 pmol mg^–1 min^–1; n = 8; p > 0.05) and hH₂R-C17Y-G_sS (0.78 ± 0.10 pmol mg^–1 min^–1; n = 9; p > 0.05), respectively, but significantly increased at hH₂R-C17Y-A271D-G_sS (1.67 ± 0.38 pmol mg^–1 min^–1; n = 9; p < 0.01). At the fusion proteins of both wild-type receptors and at hH₂R-C17Y-A271D-G_sS, stimulation with 100 µM HA yielded GTPase activities 400 to 600% of the basal levels. By contrast, at hH₂R-C17Y-G_sS, maximal HA GTPase activities amounted to just 140% of the basal signal, thereby providing an insufficiently low signal-to-noise ratio for detailed analysis of agonists (Fig. 3). Thus, for a comparative analysis of efficacies and potencies of compounds 1 to 13, only membranes expressing both wild-type receptors and the double mutant hH₂R-C17Y-A271D-G_sS were considered (Table 1). The efficacies of the small agonists DIM (2) and AMT (3) were slightly increased at gpH₂R-G_sS and hH₂R-C17Y-A271D-G_sS, relative to hH₂R-G_sS. The potencies of HA (1) (Fig. 3), DIM (2), and AMT (3) were increased at hH₂R-C17Y-A271D-G_sS compared with the wild-type receptors. The H₁R-selective agonist suprahistaprodifen (4) (Seifert et al., 2003) acted as a partial agonist with similar efficacies and potencies at hH₂R-G_sS, gpH₂R-G_sS, and hH₂R-C17Y-A271D-G_sS. In agreement with previous studies (Kelley et al., 2001; Xie et al., 2006a,b), N-[3-(1H-imidazol-4-yl)propyl]guanidines 5 to 7 and most of their N^G-acylated derivatives 8 to 13 were more efficacious and more potent at gpH₂R-G_sS than at hH₂R-G_sS. Except for IMP (5) being more efficacious at hH₂R-C17Y-A271D-G_sS, the efficacies of 5 to 7 were not significantly changed at the mutant receptor compared with wild-type hH₂R-G_sS. Compound 11 but not ARP (6) was more efficacious and both were more potent at hH₂R-C17Y-A271D-G_sS than at wild-type hH₂R-G_sS. Compounds 8 and 9 share a 2-thiazolyl moiety and were more potent at the double mutant compared with hH₂R-G_sS, although for compound 9 the difference was not significant. When the 2-thiazolyl group was replaced by a cyclohexyl group (12), the selectivity for the mutant was lost. Taken together, the small H₂R agonists 1 to 3 were considerably more potent at hH₂R-C17Y-A271D-G_sS than at the wild-type human and guinea pig H₂R-G_sS. Some guanidines and N^G-acylated guanidines displayed enhanced potencies at the mutant receptor compared with hH₂R-G_sS. However, these compounds were all less potent at hH₂R-C17Y-A271D-G_sS than at gpH₂R-G_sS, and the efficacies varied between the corresponding values at both wild-type receptors.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Concentration-dependent increase of GTPase activity by HA in membranes expressing hH2R-GsS (), gpH2R-GsS (), hH2R-C17Y-GsS (), and hH2R-C17Y-A271D-GsS (). GTPase activity in Sf9 membranes was determined as described under Materials and Methods. Reaction mixtures contained membranes (10 µg of protein/tube) expressing fusion proteins and HA at concentrations indicated on the abscissa. Data shown are the means ± S.E.M. of three independent experiments performed in duplicates. Data were analyzed by nonlinear regression and were best fitted to sigmoidal concentration-response curves.

View this table: [in this window] [in a new window]   TABLE 1 Agonist efficacies and potencies at hH2R-GsS, gpH2R-GsS, and hH2R-C17Y-A271D-GsS in the GTPase assay Steady-state GTPase activity in Sf9 membranes expressing hH2R-GsS, gpH2R-GsS, and hH2R-C17Y-A271D-GsS was determined as described under Materials and Methods. Reaction mixtures contained Sf9 membranes expressing fusion proteins and agonists at concentrations from 1 nM to 1 mM as appropriate to generate saturated concentration-response curves. Curves were analyzed by nonlinear regression and were best fitted to sigmoidal concentration-response curves. The maximal stimulatory effect of 100 µM HA amounted to 400 to 600% above basal. To calculate agonist efficacies, the maximum stimulatory effect of HA was set at 1.00, and the stimulatory effects of other agonists were referred to this value. Data shown are the means ± S.D. of three to six experiments performed in duplicate or triplicate. Efficacies and potencies, respectively, of ligands at hH2R-GsS were compared with the corresponding parameters at gpH2R-GsS, and hH2R-C17Y-A271D-GsS, respectively, using the t test. The control data for hH2R-GsS and gpH2R-GsS are identical with the control data for these constructs in Table 1 of Preuss et al. (2007).

At wild-type H₂R-G_sS and hH₂R-C17Y-A271D-G_sS CIM (14), RAN (15), FAM (16), APT (17), and IAPT (18) decreased GTPase activities below basal and thus acted as inverse agonists (Table 2). At hH₂R-C17Y-A271D-G_sS inverse agonist efficacies of 14 to 18 were significantly increased relative to wild-type receptors. Because inverse agonists stabilize an inactive receptor conformation (Milligan et al., 1995), the differences in inverse agonist efficacies reflect an increased level of constitutive activity of hH₂R-C17Y-A271D-G_sS relative to wild-type receptors. The magnitudes of constitutive activity measured critically depend on the relative stoichiometry of GPCR and G protein in the system (Kenakin, 2001). Physical tethering of H₂R with G_sS in the fusion proteins used provides a fixed 1:1 stoichiometry of both partners, allowing for a direct comparison of the efficacies in an expression-independent manner (Milligan, 2000). Compounds 16 and 18 were slightly more potent at hH₂R-C17Y-A271D-G_sS than at the wild-type receptors, whereas no significant differences in the K_B values were observed for 14, 15, and 17.

View this table: [in this window] [in a new window]   TABLE 2 Potencies and inverse agonist efficacies of antagonists at hH2R-GsS, gpH2R-GsS, and hH2R-C17Y-A271D-GsS in the GTPase assay Steady-state GTPase activity in Sf9 membranes expressing hH2R-GsS, gpH2R-GsS, and hH2R-C17Y-A271D-GsS was determined as described under Materials and Methods. Reaction mixtures contained Sf9 membranes expressing fusion proteins, 1 µM HA as agonist and antagonists at concentrations from 1 nM to 1 mM as appropriate to generate saturated competition curves. Competition curves were analyzed by nonlinear regression. To determine the inverse agonist efficacies (Inv. Ago. Eff.), the effects of antagonists at fixed concentrations (10 µM RAN, FAM, APT, and IAPT; 100 µM CIM) on basal GTPase activity were assessed and referred to the stimulatory effect of 100 µM HA (=1.00). Data shown are the means ± S.D. of three experiments performed in duplicates. KB values and inverse agonist efficacies, respectively, of antagonists at hH2R-GsS were compared with the corresponding parameters at gpH2R-GsS and hH2R-C17Y-A271D-GsS, respectively, using the t test. The control data for hH2R-GsS and gpH2R-GsS are identical with the control data for these constructs in Table 2 of Preuss et al. (2007).

Regulation of AC Activities in Membranes Expressing hH₂R-G_sS, gpH₂R-G_sS, hH₂R-C17Y-G_sS, and hH₂R-C17Y-A271D-G_sS. AC activities were measured in Sf9 membranes expressing hH₂R-C17Y-G_sS and hH₂R-C17Y-A271D-G_sS, and they were compared with results at wild-type human and guinea pig H₂R-G_sS (Table 3). hH₂R-G_sS and gpH₂R-G_sS were similarly expressed in Sf9 cells (at 3 and 1 pmol mg^–1, respectively) and produced similar basal AC activities. By contrast, basal AC activities were increased 3-fold at hH₂R-C17Y-A271D-G_sS. At both mutant and both wild-type receptors, 10 µM GTP by itself increased AC activities above the basal level (Fig. 4), indicating constitutive activity of these receptors (Seifert et al., 1998a,b; Gille and Seifert, 2003). Accordingly, at all four H₂Rs, the inverse agonist IAPT (18) reduced this GTP-dependent AC activity. At hH₂R-G_sS and gpH₂R-G_sS, AC activity increases by 10 µM GTP achieved 73 and 77%, respectively, of the signal increases by 10 µM GTP plus 100 µM HA. Strikingly, at hH₂R-C17Y-A271D-G_sS, HA did not further enhance the GTP effect. Both higher basal AC activity and a strong stimulation by GTP caused exhaustion of the limiting pool of AC molecules in Sf9 cells and reflect an increased level of constitutive activity of hH₂R-C17Y-A271D-G_sS, compared with the wild-type fusion proteins. Similar reduced agonist-responsiveness due to high constitutive activity was shown for other aminergic GPCRs, e.g., ₂AR-G_s fusion proteins (Seifert et al., 1998a) and mutants of the 5-HT₄ receptor (Claeysen et al., 1999). At hH₂R-C17Y-G_sS much lower basal AC activities and a much smaller stimulatory effect of GTP were determined. In this case, GTP on its own caused only 26% of the effect with HA addition.

View this table: [in this window] [in a new window]   TABLE 3 AC activities in Sf9 membranes expressing hH2R-GsS, gpH2R-GsS, hH2R-C17Y-GsS, and hH2R-C17Y-A271D-GsS Basal AC activities and the effects of GTP and HA on AC activities in membranes expressing hH2R-C17Y-GsS and hH2R-C17Y-A271D-GsS were assessed and compared with the corresponding values at hH2R-GsS and gpH2R-GsS. AC activity in Sf9 membranes was determined as described under Materials and Methods. Reaction mixtures contained Sf9 membranes (20 µg protein/tube) expressing fusion proteins and distilled water (basal), 10 µM GTP, or 10 µM GTP plus 100 µM HA. Data shown are the means ± S.D. of three experiments performed in triplicates. To calculate the stimulatory effect of GTP (Rel. GTP Effect), the effect of 10 µM GTP was referred to the effect of 10 µM GTP plus 100 µM HA. The control data for hH2R-GsS and gpH2R-GsS are identical with the control data for these constructs in Table 3 of Preuss et al. (2007).

View larger version (12K): [in this window] [in a new window]   Fig. 4. Regulation of AC activities in Sf9 membranes expressing hH2R-GsS (A), gpH2R-GsS (B), hH2R-C17Y-A271D-GsS (C), and hH2R-C17Y-GsS (D). AC activity in Sf9 membranes was determined as described under Materials and Methods. Reaction mixtures contained Sf9 membranes expressing the proteins indicated on top of each panel and GTP at concentrations indicated at the abscissa. Reaction mixtures additionally contained H2O (), 100 µMHA (), or 10 µM IAPT (). Data shown are the means ± S.E.M. of one representative experiment performed in triplicates. The statistical analysis of AC activities is provided in Table 3. Data were analyzed by nonlinear regression and were best fitted to sigmoidal concentration-response curves. Please note the different scale of the ordinate in D. The control data for hH2R-GsS and gpH2R-GsS are identical with the control data for these constructs in Fig. 6 of Preuss et al. (2007).

At hH₂R-G_sS, gpH₂R-G_sS, and hH₂R-C17Y-A271D-G_sS, 100 µM HA reduced the basal AC activities in the absence of added GTP (Fig. 4, A–C). Similar effects were observed at ₂AR-G_sS fusion proteins (Seifert et al., 1998b), and they are due to dissociation of GDP from G_sS following agonist binding to the receptor without subsequent binding of GTP. G_s-GDP is more effective in activating AC than nucleotide-free G_s; therefore, AC activities were decreased.

	Discussion

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Impaired Coupling in Membranes Expressing hH₂R-C17Y-G_sS. In Sf9 cells expressing hH₂R-C17Y-G_sS, the anti-FLAG and the anti-His₆ antibodies recognized similarly migrating proteins in SDS-PAGE that did not coincide with the expected bands for monomeric H₂R-G_sS fusion proteins. Measurement of GTP hydrolysis at hH₂R-C17Y-G_sS yielded HA responses, but the increases in GTPase activity upon agonist stimulation were much lower than in the wild-type H₂R-G_sS species isoforms. Moreover, with this receptor mutant, substantial stimulatory effects of HA and inhibitory effects of IAPT on the GTP-dependent AC activity increases were observed, but the basal AC activities and the stimulatory effects of GTP were largely reduced relative to wild-type H₂R-G_sS species isoforms, and they were similar to the values typical for Sf9 membranes expressing nonfused H₂R species (Houston et al., 2002). These controversial results of hH₂R-C17Y-G_sS relative to hH₂R-G_sS and gpH₂R-G_sS imply the following conclusions: The proteins expressed in Sf9 cells made up the amino acid sequence for hH₂R-C17Y, and they were functional in the test systems used. However, coupling of hH₂R-C17Y to G_sS was much less efficient than is characteristic for GPCR-G_s fusion proteins (Seifert et al., 1999; Gille and Seifert, 2003). As a rationale, G_sS could be incorrectly expressed or degraded in Sf9 cells. Instead, hH₂R-C17Y possibly coupled to only a fraction of recombinant G_sS or to endogenous G_s-like G proteins with much lower efficiency.

Increased Constitutive Activity in Membranes Expressing hH₂R-C17Y-A271D-G_sS. In membranes expressing hH₂R-C17Y-A271D-G_sS, high-efficiency coupling was observed as GTPase activities were increased upon agonist stimulation similar to hH₂R-G_sS and gpH₂R-G_sS. Moreover, with this receptor mutant enhanced basal GTPase activities, increased potencies of the agonists as well as increased inverse agonist efficacies of antagonists were detected, representing the hallmarks of enhanced constitutive activity compared with the wild-type proteins (Lefkowitz et al., 1993). The determination of AC activity in Sf9 cell membranes has previously shown to be an alternative and sensitive system to quantify differences in the constitutive activities of GPCRs (Seifert et al., 1998a). In membranes expressing hH₂R-C17Y-A271D-G_sS the high basal AC activities and the strong AC activity increases upon stimulation with GTP additionally reflect high constitutive activity compared with hH₂R-G_sS, gpH₂R-G_sS, and hH₂R-C17Y-G_sS.

The discovery of increased constitutive activity at hH₂R-C17Y-A271D-G_sS further supports the concept of an H-bond between Tyr-17 in TM1 and Asp-271 in TM7 (Kelley et al., 2001) as basis for the distinct pharmacological properties of human and guinea pig H₂Rs. Our data suggest that this interhelical interaction stabilizes an active receptor conformation not only when agonists are bound but also when ligands are absent. However, gpH₂R-G_sS containing Tyr-17 and Asp-271 also was similarly constitutively active as hH₂R-G_sS which is presumably due to additional intramolecular interactions constraining the gpH₂R in an inactive conformation and thereby compensating for the activating function of both residues.

Of interest, the tertiary structure of the _1b-AR contains Lys-331 in TM7 corresponding to Ala-271 in hH₂R. Strikingly, _1b-AR mutants with Lys-331 exchanged by alanine or glutamate were more constitutively active than wild-type _1b-AR (Porter et al., 1996), suggesting a general role of an amino acid at this position for the activation mechanism of related GPCRs.

Species Selectivity of Guanidines and N^G-Acylguanidines at Wild-Type and Mutant H₂R-G_sS. The main intention of this study was to elucidate the impact of Cys-17/Tyr-17 and Ala-271/Asp-271 on the species selectivity of N-[3-(1H-imidazol-4-yl)propyl]guanidines and N^G-acylated imidazolypropylguanidines between hH₂R and gpH₂R. In our GTPase activity experiments, some of these agonists were more potent and more efficacious at hH₂R-C17Y-A271D-G_sS than at hH₂R-G_sS, and some compounds were not selective. Overall, the potencies and efficacies of the agonists were still higher at gpH₂R-G_sS than at hH₂R-C17Y-A271D-G_sS. The following conclusions can be drawn from these results.

First, both Tyr-17 and Asp-271 contribute to the enhanced potencies and efficacies of guanidines and N^G-acylguanidines at the gpH₂R. This investigation adds to a previous study at an hH₂R-A271D-G_sS mutant conferring high potency to guanidines without affecting the efficacies (Kelley et al., 2001). However, the pharmacological differences between hH₂R-C17Y-A271D-G_sS and gpH₂R-G_sS indicate that more than these two amino acids determine the species selectivity of agonists and will have to be identified in future mutagenesis studies.

Second, the concept of ligand-specific conformations in H₂R species (Kelley et al., 2001; Kenakin, 2003; Xie et al., 2006a) is further supported. The variable side chains of the compounds distinctly interact with wild-type and mutant H₂R-G_sS, which is represented by compounds 8 and 9 containing a 2-thiazolyl group and being more potent at hH₂R-C17Y-A271D-G_sS than at hH₂R-G_sS in contrast to compound 10 with a cyclohexyl group being similarly potent at both proteins. The 5-methyl-1H-imidazol-4-yl group in IMP (5) presumably directly interacts with Asp-271 (Kelley et al., 2001), yielding the high-potency increase of 4-fold at hH₂R-C17Y-A271D-G_sS versus hH₂R-G_sS.

GPCRs with enhanced constitutive activity exhibit an increased affinity for agonists with the affinity increase being correlated with the efficacy of the ligand (Samama et al., 1993). Accordingly, the parameter of constitutive activity not only affects elevated potencies of small H₂R agonists at hH₂R-C17Y-A271D-G_sS but also potency increases of the guanidines and N^G-acylguanidines. Different magnitudes of constitutive activity therefore add to the complexity of the system for the analysis of species-selective ligand/GPCR interactions. Moreover, inverse agonists are less potent at constitutively active than at quiescent GPCRs (Kenakin, 2001). Accordingly, 14 to 18 were expected to be less potent at the more constitutively active hH₂R-C17Y-A271D-G_sS than at hH₂R-G_sS. However, the potencies of inverse agonists were not decreased, and 16 and 18 were even more potent at the mutant receptor, assuming that not only guanidine-type agonists but also inverse agonists could stabilize ligand-specific conformations in H₂R species isoforms.

	Conclusions

Top Abstract Materials and Methods Results Discussion Conclusions References

 
In the present study, we demonstrate that an hH₂R-G_sS fusion protein with mutations of Cys-17 Tyr-17 in TM1 and Ala-271 Asp-271 in TM7 displayed enhanced constitutive activity compared with hH₂R-G_sS and gpH₂R-G_sS.We additionally showed that an interaction between Tyr-17 and Asp-271 in gpH₂R contributes to the species-selective action of N-[3-(1H-imidazol-4-yl)propyl]guanidines and their N^G-acylated derivatives. Distinct potencies and efficacies of agonists and inverse agonists further support the concept of ligand-specific conformations in wild-type and mutant H₂R-G_sS fusion proteins. A single point mutation of Cys-17 Tyr-17 was devoid of efficient GPCR-G protein coupling. By analogy, point mutations of Phe-153 Leu-153 or Ile-433 Val-433 in the hH₁R (hH₁RgpH₁R) resulted in functional inactivity, whereas a Phe-153 Leu-153/Ile-433 Val-433 double mutant was functionally active (Seifert et al., 2003). The reasons for the annihilating effects of the single point mutations hH₁R and hH₂R are not known, but they illustrate the limitations of site-directed mutagenesis experiments. The characterization of closely related wild-type GPCR species isoforms is, therefore, an important alternative approach to relate distinct pharmacological properties to relatively few molecular determinants.

Taken together, our mutational studies provide unique insight into the molecular mechanisms of H₂R functions and will help us to find potent and selective agonists for the hH₂R that may be useful as positive inotropic drugs for the treatment of severe congestive heart failure, as agents inducing cell differentiation in acute myelogenous leukemia, and as anti-inflammatory drugs.

	Acknowledgements

 
We thank Dr. E. Schneider (Department of Pharmacology and Toxicology, University of Regensburg) for helpful discussions and A. Seefeld and G. Wilberg for technical support. Thanks are also due to the reviewers of this article for constructive critique.

	Footnotes

 
This work was supported by the Research Training Program (Graduiertenkolleg) GRK 760 "Medicinal Chemistry: Molecular Recognition–Ligand-Receptor Interactions" of the Deutsche Forschungsgemeinschaft.

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

doi:10.1124/jpet.107.120519.

ABBREVIATIONS: H₂R, histamine H₂ receptor; GPCR, G protein-coupled receptor; HA, histamine; AC, adenylyl cyclase; G_s, -subunit of the G_s protein that mediates adenylyl cyclase activation; G_sS, short splice variant of the G_s protein G_s; gpH₂R, guinea pig histamine H₂ receptor; gpH₂R-G_sS, fusion protein of the guinea pig histamine H₂ receptor and the short splice variant of G_s; H₁R, histamine H₁ receptor; hH₂R, human histamine H₂ receptor; hH₂R-G_sS, fusion protein of the human histamine H₂ receptor and the short splice variant of G_s; hH₂R-C17Y-G_sS, fusion protein of the human histamine H₂ receptor bearing a Cys Tyr mutation at position 17 and the short splice variant of G_s; hH₂R-C17Y-A271D-G_sS, fusion protein of the human histamine H₂ receptor bearing a Cys Tyr mutation at position 17 and an Ala Asp mutation at position 271 and the short splice variant of G_s; DIM, dimaprit; AMT, amthamine; TM, transmembrane domain of a G protein-coupled receptor; IMP, impromidine; ARP, arpromidine; CIM, cimetidine; RAN, ranitidine; FAM, famotidine; APT, aminopotentidine; IAPT, iodoaminopotentidine; PCR, polymerase chain reaction; S, signal peptide from influenza hemagglutinin; F, FLAG epitope; PAGE, polyacrylamide gel electrophoresis; AR, adrenoceptor; ₂AR-G_s, fusion protein of the ₂-adrenoceptor and G_s.

¹ Current affiliation: Department of Chemistry, University of Nebraska, Lincoln, Nebraska.

Address correspondence to: Dr. Roland Seifert, Department of Pharmacology and Toxicology, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany. E-mail: roland.seifert{at}chemie.uni-regensburg.de

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