![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CELLULAR AND MOLECULAR
Department of Pharmaceutical/Medicinal Chemistry II, Institute of Pharmacy, University of Regensburg, Regensburg, Germany (H.P., P.G., A.K., S.D., A.B.); and Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Regensburg, Germany (R.S.)
Received for publication
January 16, 2007
Accepted
February 28, 2007.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionConclusionsReferences |
|---|
, GsS, were expressed in Sf9 insect cells. In the membrane steady-state GTPase activity assay, cH2R-GsS but neither gpH2R-GsS nor rH2R-GsS showed the hallmarks of increased constitutive activity compared with hH2R-GsS, i.e., increased efficacies of partial agonists, increased potencies of agonists with the extent of potency increase being correlated with the corresponding efficacies at hH2R-GsS, increased inverse agonist efficacies, and decreased potencies of antagonists. Furthermore, in membranes expressing nonfused H2Rs without or together with mammalian GsS or H2R-Gs fusion proteins, the highest basal and GTP-dependent increases in adenylyl cyclase activity were observed for cH2R. An example of ligand selectivity is given by metiamide, acting as an inverse agonist at hH2R-GsS, gpH2R-GsS, and rH2R-GsS in the GTPase assay in contrast to being a weak partial agonist with decreased potency at cH2R-GsS. In conclusion, the cH2R exhibits increased constitutive activity compared with hH2R, gpH2R, and rH2R, and there is evidence for ligand-specific conformations in H2R species isoforms.
), human (Gantz et al., 1991a), rat (Ruat et al., 1991), and guinea pig (Traiffort et al., 1995) were cloned. The four H2R species isoforms are closely related to each other, as is reflected by an overall amino acid sequence identity of more than 80%. The highest conservation exists within the seven -helical transmembrane (TM) domains (sequence identity of more than 90%), whereas the N-terminal domain together with the extracellular end of TM1 and the C terminus are the least conserved regions (Fig. 1).
|
, GsS, such H2R-selective agonists are considerably more potent and efficacious at gpH2R-GsS than at hH2R-GsS (Kelley et al., 2001). By contrast, the small H2R agonists histamine (1, HA), dimaprit (2, DIM), amthamine (3, AMT), and betahistine (4, BET) are unselective between these species. Recently, a novel class of NG-acylated imidazolylpropylguanidines as represented by compounds 11 to 16 was developed (Ghorai, 2005; Xie et al., 2006a). Generally, by introduction of a carbonyl group adjacent to the guanidine moiety, the species selectivity of the agonists is preserved (Xie et al., 2006a). Comparison of the corresponding agonist efficacies in the GTPase assay and at stabilizing the high-affinity ternary complex of the H2R with nucleotide-free Gs indicate that N-[3-(1H-imidazol-4-yl)propyl]guanidines and their NG-acylated analogs stabilize different ligand-specific active conformations of hH2R and gpH2R (Kelley et al., 2001; Xie et al., 2006a). However, it is not known whether these differences also apply for other H2R species isoforms. Moreover, H2Rs are known to be constitutively active (Alewijnse et al., 1998), but the degree to which constitutive activity varies among several species isoforms remains elusive. To generate an expanded pharmacological profile of H2R species isoforms, in the present report, we compare human, guinea pig, rat, and canine H2Rs.
|
Sf9 cell membranes expressing H2R-GsS fusion proteins were used to measure steady-state GTPase activity. For this purpose, we studied several classes of H2R ligands (Fig. 2). HA (1) and related small H2R agonists DIM (2), AMT (3), and BET (4) similarly interact with the binding site of H2R. The amino group of HA forms an ionic interaction with Asp-98(3.32) in TM3, and the imidazolyl ring presumably interacts with Tyr-182(5.38) and Asp-186(5.42) in TM5 (Fig. 1). The guanidine-type H2R agonists impromidine (8, IMP), arpromidine (9, ARP), and BU-E-43 (10), as well as the NG-acylated derivatives 11 to 16 share a common N-[3-(1H-imidazol-4-yl)propyl)]guanidine moiety that mimics binding of HA and thus is crucial for agonistic activity (Dove et al., 2004). The 2-(5-methylimidazol-4-ylmethylthio)ethyl moiety of IMP and the 3-(4-fluorophenyl)-3-(2-pyridyl)propyl group of ARP are supposed to interact with a pocket formed by multiple residues in TM3, -6, and -7 (Kelley et al., 2001). The variable side chains of the ARP derivatives 10 to 16 consist of diverse mono- or diarylalkyl groups with different chain lengths between the aromatic ring system and the guanidine group. In compound 16 (Xie et al., 2006b), the aryl ring is replaced by a cyclohexyl moiety. Compound 13 is the pure (R)-enantiomer (eutomer). 2-Benzylhistamine (5) and suprahistaprodifen (6) represent H1R agonists with partial H2R agonism (Seifert et al., 2003). Burimamide (7) and metiamide (22) are neutral H2R antagonists, whereas cimetidine (17, CIM), ranitidine (18, RAN), famotidine (19, FAM), aminopotentidine (20, APT), and iodoaminopotentidine (21, IAPT) act as inverse agonists (Hill et al., 1997; Dove et al., 2004).
Previous studies showed that the determination of adenylyl cyclase (AC) activity in Sf9 cell membranes is a very sensitive system to elucidate differences in the constitutive activities of GPCRs (Seifert et al., 1998b). Therefore, we also assessed AC activity in membranes expressing nonfused H2Rs (coupling to endogenous Gs-like G proteins), in membranes coexpressing H2R and mammalian GsS, and in membranes expressing H2R-GsS fusion proteins.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
). The cDNA for the cH2R was kindly provided by Dr. I. Gantz (University of Michigan, Medical School and Ann Arbor VA Medical Center, Ann Arbor, MI) (Gantz et al., 1991b). The generation of the baculoviruses encoding hH2R, gpH2R, hH2R-GsS, and gpH2R-GsS was described previously (Kelley et al., 2001; Houston et al., 2002). The baculoviruses encoding GsS were kindly provided by Drs. R. Sunahara and A. G. Gilman (Department of Pharmacology, University of Southwestern Medical Center, Dallas, TX). The generation of pGEM-3Z-SF-
1AR-GsS and pVL1392-SF-1AR-GsS was described previously (Wenzel-Seifert et al., 2002). Compounds 11 through 15 (Ghorai, 2005; Xie et al., 2006a) and compound 16 (Xie et al., 2006b) were prepared as described previously (Ghorai, 2005). The preparation of IMP, ARP, BU-E-43, APT, and IAPT was described previously (Dove et al., 2004). Suprahistaprodifen and 2-benzylhistamine were synthesized as described previously (Elz et al., 2000; Seifert et al., 2003). The structures of compounds were confirmed by 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). The anti-Gs Ig (C-terminal) was from Santa Cruz Biotechnology (Santa Cruz, CA), and the anti-His6 Ig was from Clonetech (Mountain View, CA). [
-32P]GTP (6000 Ci/mmol), [-32P]ATP (800 Ci/mmol), and [3H]dihydroalprenolol (85–90 Ci/mmol) were from PerkinElmer Life and Analytical Sciences (Boston, MA). All unlabeled nucleotides were from Roche Diagnostics (Indianapolis, IN). HA, BET, CIM, RAN, and FAM were from Sigma-Aldrich. AMT was from Tocris Cookson Inc. (Ballwin, MO). DIM was from Sigma/RBI (Natick, MA). Burimamide and metiamide were from Dr. W. Schunack (Free University of Berlin, Berlin, Germany). All restriction enzymes, T4 DNA ligase, and calf intestinal phosphatase were from New England Biolabs (Beverly, MA). Cloned Pfu DNA polymerase was from Stratagene (La Jolla, CA).
Construction of the cDNAs for rH2R and rH2R-GsS. The cDNAs encoding for the proteins were generated by sequential overlap-extension PCRs. With pGEM-3Z-SF-gpH2R-GsS 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 start codon of the rH2R. The sense primer annealed with 18 bp of pGEM-3Z before the 5' end of SF. The antisense primer annealed with 15 bp of the 3' end of SF and with ATG. In PCR 1B, the cDNA encoding the rH2R followed by a hexahistidine tag in 3' position was generated. The hexahistidine tag was included to allow future purification and to provide additional protection against proteolysis (Seifert et al., 1998a). The sense primer consisted of 15 bp of the 3' end of SF and the first 22 bp of the 5' end of the rH2R. The antisense primer consisted of 18 bp of the C terminus of the rH2R, the hexahistidine tag, the stop codon, and an XbaI site. The cDNA for the rH2R was extracted from pcDNA-rH2R after restriction digestion with HindIII and BglII and was used as template. In PCR 2, the products of PCR 1A and PCR 1B annealed in the region encoding SF and ATG. Here, the sense primer of PCR 1A and the antisense primer of PCR 1B were used. In that way, a fragment encoding SF, the rH2R, the hexahistidine tag, the stop codon, and an XbaI site was obtained. This fragment was digested with SacI and XbaI and cloned into pGEM-3Z-SF-hH2R digested with SacI and XbaI to yield pGEM-3Z-SF-rH2R. pGEM-3Z-SF-rH2R was digested with SacI and XbaI and cloned into the baculovirus transfer vector pVL1392-SF-hH2R digested with SacI and XbaI. With pGEM-3Z-SF-rH2R as template, the sense primer of PCR 1A, and an antisense primer encoding six histidines, in PCR 3A a fragment encoding SF, the cDNA for the rH2R, and the hexahistidine tag was generated. In PCR 3B, a fragment encoding the hexahistidine tag, the cDNA of GsS, the stop codon, and an XbaI site was generated. Here, the sense primer annealed with the hexahistidine tag and the start codon of GsS, and the antisense primer annealed with the cDNA encoding the five C-terminal amino acids of GsS, the stop codon, and an XbaI site. pGEM-3Z-SF-gpH2R-GsS was used as template. In PCR 4, the products of PCRs 3A and 3B annealed in the hexahistidine region, and the sense primer of PCR 1A and the antisense primer of PCR 3B were used. In that way, the complete cDNA for the rH2R-GsS fusion protein, consisting of SF, the cDNA for the rH2R, the hexahistidine tag, and the cDNA of GsS was amplified. The product of PCR 4 was digested with SacI and BglII and cloned into pGEM-3Z-SF-1AR-GsS digested with SacI and BglII. In addition, the PCR 4 product was digested with SacI and BglII and directly cloned into pVL1392-SF-1AR-GsS that was digested with SacI and BglII and treated with calf intestinal phosphatase to yield the baculovirus transfer vector pVL1392-SF-rH2R-GsS.
Construction of the cDNAs for cH2R and cH2R-GsS. The strategy for the generation of the cDNAs for the epitope-tagged cH2R and cH2R-GsS was analogous to the strategy for the generation of the cDNAs for rH2R and rH2R-GsS. With pGEM-3Z-SF-gpH2R-GsS as template, in PCR 1A the SF region and the start codon of the cH2R were amplified. The sense primer annealed with 18 bp of pGEM-3Z before the 5' end of SF, and the antisense primer annealed with 15 bp of the 3' end of SF and with ATG. In PCR 1B, the cDNA encoding the sequence for the cH2R followed by the hexahistidine tag in 3' position was generated. The sense primer consisted of 15 bp of the 3' end of SF and the first 21 bp of the 5' end of cH2R. The antisense primer consisted of 18 bp of the C terminus of the cH2R, the hexahistidine tag, the stop codon, and an XbaI site. The cDNA for the cH2R was extracted from CMVneo-cH2R after digestion with BglII and was used as template. In PCR 2, the products of PCR 1A and PCR 1B annealed in the region encoding SF and ATG. Here, the sense primer of PCR 1A and the antisense primer of PCR 1B were used. In that way, a fragment encoding SF, the cH2R, the hexahistidine tag, the stop codon, and an XbaI site was obtained. This fragment was digested with SacI and XbaI and cloned into pGEM-3Z-SF-hH2R digested with SacI and XbaI to yield pGEM-3Z-SF-cH2R. pGEM-3Z-SF-cH2R was digested with SacI and XbaI and cloned into the baculovirus transfer vector pVL1392-SF-hH2Rdigested with SacI and XbaI. PCR 3 was used to generate a fragment encoding the C terminus of the cH2R, the hexahistidine tag, and GsS. The sense primer encoded the last 10 amino acids of the C terminus of the cH2R, the hexahistidine tag, and the start codon of GsS, and the antisense primer encoded the five C-terminal amino acids of GsS, the stop codon, and an XbaI site. Here, pGEM-3Z-SF-hH2R-GsS was used as template. This fragment was digested with XhoI and XbaI and cloned into pGEM-3Z-SF-cH2R digested with XhoI and XbaI to yield pGEM-3Z-SF-cH2R-GsS. pGEM-3Z-SF-cH2R-GsS was digested with SacI and BglII and cloned into pVL1392-SF-1AR-GsS that was digested with SacI and BglII and treated with calf intestinal phosphatase to yield the baculovirus transfer vector pVL1392-SF-cH2R-GsS.
Generation of Recombinant Baculoviruses, Cell Culture, and Membrane Preparation. Recombinant baculoviruses encoding rH2R, cH2R, rH2R-GsS, and cH2R-GsS 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 H2Rs, GsS, and H2R-GsS 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 then transferred onto Immobilon-P transfer membranes (Millipore Corporation, Bedford, MA). Membranes were reacted with M1 antibody, anti-Gs Ig, or anti-His6 Ig (1:1000 each). Immunoreactive bands were visualized by enhanced chemoluminescence (Pierce Chemical, Rockford, IL) using sheep anti-mouse IgG (M1 and anti-His6 Ig) and donkey anti-rabbit IgG (anti-Gs Ig), respectively, 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-GsS 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 Pi. 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 32Pi 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 32Pi.
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 H2Rs (100 µg of protein/tube), H2Rs coexpressed with mammalian GsS (50 µg of protein/tube), or H2R-GsS 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 (Sigma A-1522, super I, WN-6). [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). KB values were calculated using the Cheng and Prusoff equation (1973) equation. Expression levels of recombinant proteins were determined using the Bio-Rad GS-710 calibrated imaging densitometer and the software tool Quantity One, version 4.0.3 (Bio-Rad).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
33 kDa (Gantz et al., 1991a,b; Fukushima et al., 1997). H2R species isoforms presumably exhibit similar glycosylation patterns, because the putative N-glycosylation sites for the H2R, Asn-4, and Asn-162 are fully conserved within their sequences (Fig. 1) (Fukushima et al., 1995). However, only rH2R and hH2R migrated as the expected bands for monomeric GPCRs (Fig. 3, A and B). Both bands occur as doublets, representing different glycosylation forms (Houston et al., 2002). Additional bands were detected at 70 kDa, presumably representing receptor dimers. A similar pattern of immunoreactive bands was previously observed for the hH2R (Houston et al., 2002). In contrast, both cH2R and gpH2R displayed strong doublet bands at 60 kDa that coincide with the expected bands of differentially glycosylated H2R dimers, whereas the bands for monomers were absent. Additional bands were detected at 150 kDa and above 250 kDa, possibly corresponding to H2R tetramers and higher oligomers, respectively. Dimerization and oligomerization of the cH2R has been described previously (Fukushima et al., 1997), but in those experiments, also receptor monomers were detected. Hence, gpH2R and cH2R possibly migrated atypically in SDS-PAGE, i.e., the bands at 60 and 150 kDa could correspond to monomers and dimers, respectively, and not to dimers and tetramers. With the anti-His6 Ig, in membranes expressing cH2R, an additional doublet band at 23 kDa was detected, and in rH2R- and hH2R membranes, an 27-kDa band was present. However, no such bands were detected in gpH2R membranes. The 23–27-kDa bands may represent differentially and atypically migrating H2R monomers not recognized by the M1 antibody because of a lack of epitope exposure. By analogy to formyl peptide receptors (Wenzel-Seifert and Seifert, 2003) differences in the C termini of H2R species isoforms may constitute the molecular basis for the species-selective migration pattern. Thus, H2R species isoforms are well expressed in Sf9 membranes, but due to their widely different migration, it is impossible to precisely assess their expression levels using 2AR-membranes calibrated with [3H]dihydroalprenolol saturation binding as standard (Kelley et al., 2001).
|
H2R-GsS fusion proteins of canine, rat, guinea pig, and human occurred as strong bands at 80 kDa (Fig. 3C). Because GsS has an apparent molecular mass of 45 kDa, these bands correspond to H2R-GsS monomers. Weaker bands were detected at 110 kDa, most probably representing differently glycosylated fusion proteins. With all species, additional bands at 250 kDa were detected, presumably representing H2R-GsS dimers or oligomers. cH2R-GsS was expressed at 5 pmol mg–1, rH2R-GsS at 4 pmol mg–1, gpH2R-GsS at pmol 1 mg–1, and hH2R-GsS at 3 pmol mg–1 using 2AR-membranes as standard. To account for the decreased expression level of gpH2R-GsS, in this case the amount of protein applied to the gel was adjusted to 40 µg.
Probing membranes expressing H2R-GsS species with the anti-Gs Ig yielded 80- and 250-kDa bands (Fig. 3D), which are consistent with those bands observed with the anti-FLAG Ig. Additional bands occurred at 45 kDa, representing atypically migrating or partially degraded fusion proteins. In all membranes coexpressing H2R species and GsS, the expected bands for GsS monomers were detected at 45 kDa. The expression levels of GsS in membranes coexpressing H2R and GsS were estimated using the 80-kDa peak intensities of H2R-GsS species as standard and were 2 pmol mg–1 in membranes coexpressing cH2R and GsS, 2 pmol mg–1 in membranes coexpressing rH2R and GsS, 1 pmol mg–1 in membranes coexpressing gpH2R and GsS, and 1 pmol mg–1 in membranes coexpressing hH2R and GsS.
Efficacies and Potencies of Agonists at H2R-GsS Species Isoforms Derived from the GTPase Assay. Efficacies and potencies of compounds 1 to 22 at H2R-GsS fusion proteins of human, guinea pig, rat, and canine are summarized in Table 1. The small H2R agonists acted as full (1-3) or as nearly full (4) agonists at the four receptors with approximately similar efficacies. HA (1) and DIM (2) were equipotent at human, guinea pig, and rat H2R-GsS, they but showed lower EC50 values at cH2R-GsS. AMT (3) was slightly more potent at cH2R-GsS than at hH2R-GsS and gpH2R-GsS. At rH2R-GsS, the potency of AMT (3) was further decreased. BET (4) acted with increased potencies at gpH2R-GsS and cH2R-GsS, compared with hH2R-GsS and rH2R-GsS. In agreement with previous studies (Kelley et al., 2001; Xie et al., 2006a,b), N-[3-(1H-imidazol-4-yl)propyl]guanidines (8-10) and their NG-acylated analogs (11-16) were more potent and more efficacious at gpH2R-GsS than at hH2R-GsS (Table 1). At gpH2R-GsS, UR-PG222A (13) was more efficacious than HA (1). At hH2R-GsS and rH2R-GsS, the compounds exhibited similar efficacies and potencies. Only UR-PG214 (11) was slightly more potent at rH2R-GsS than at hH2R-GsS. Apart from ARP (9) and its NG-acylated analog UR-PG136 (15) that acted with similar efficacies at cH2R-GsS and hH2R-GsS, compounds 8 to 16 were more efficacious at cH2R-GsS than at hH2R-GsS. Compounds 8 to 16 were also more potent at cH2R-GsS than at hH2R-GsS. An exception of this rule was UR-PG123 (14) that exhibited the largest efficacy increase (4-fold) but was somewhat less potent at cH2R-GsS than at hH2R-GsS. In summary, small H2R agonists 1 to 4 acted with similar efficacies at all H2R-GsS species isoforms investigated, but they were more potent at cH2R-GsS compared with hH2R-GsS, gpH2R-GsS, and rH2R-GsS. Guanidines and NG-acylated guanidines 8 to 16 acted with increased efficacies and potencies at gpH2R-GsS and cH2R-GsS compared with hH2R-GsS, whereas no selectivity was observed between rH2R-GsS and hH2R-GsS.
|
Compounds 5 and 6 are representatives of H1R agonists with partial H2R agonism (Seifert et al., 2003). Both compounds were less efficacious at gpH2R-GsS than at hH2R-GsS and similarly efficacious at rH2R-GsS and hH2R-GsS (Table 1). In the GTPase assay at H2R-GsS fusion proteins, burimamide (7) was a weak partial agonist with similar efficacies at human, guinea pig, and rat species. Strikingly, compounds 5, 6, and 7 acted with significantly increased efficacies at cH2R-GsS compared with hH2R-GsS. Apart from 2-benzylhistamine (5) with 2-fold increased potency at gpH2R-GsS, the potencies of 5 to 7 did not significantly differ between the species investigated. Taken together, partial H2R agonists were considerably more efficacious at cH2R-GsS than at human, guinea pig, and rat H2R-GsS.
Potencies and Inverse Agonist Efficacies of Antagonists at H2R-GsS Species Isoforms Derived from the GTPase Assay. KB values and inverse agonist efficacies of the H2R antagonists CIM (17), RAN (18), FAM (19), APT (20), and IAPT (21) are listed in Table 2. The compounds decreased GTPase activities below basal values and thus acted as inverse agonists at all four species. At hH2R-GsS and gpH2R-GsS compounds 17 to 21 decreased the basal GTPase signal (0%) by 10% if the maximal stimulatory effect of 100 µM HA was set to 100%. At rH2R-GsS the inverse agonist efficacies of 17 to 21 were somewhat smaller. At cH2R-GsS all compounds except CIM (17) showed a significantly higher reduction of the basal GTPase activity by 20%. The KB values of 17 to 21 were similar at hH2R-GsS and gpH2R-GsS. At rH2R-GsS, 17 to 19 were less potent, and 20 and 21 were similarly potent compared with hH2R-GsS. By contrast, all compounds except FAM (19) were less potent at cH2R-GsS than at hH2R-GsS. Taken together, most of the H2R antagonists studied displayed increased inverse agonist efficacies and decreased potencies at cH2R-GsS compared with hH2R-GsS.
|
Constitutive Activities of hH2R-GsS, gpH2R-GsS, rH2R-GsS, and cH2R-GsS in the GTPase Assay. As was reported for a constitutively activated mutant of the 2AR (Samama et al., 1993), the following major hallmarks distinguish constitutively active GPCRs from not (quiescent) or less constitutively active GPCRs. First, the efficacies of partial agonists are increased at the more constitutively active receptor. To uncover differences in the constitutive activities among H2R-GsS species, efficacies of partial and full agonists 1 to 16 and inverse agonist efficacies of antagonists 17 to 21 were compared at hH2R-GsS with gpH2R-GsS, rH2R-GsS, and cH2R-GsS, respectively (Fig. 4, A, C, and E). Second, constitutively active receptors exhibit an increased affinity for agonists but not antagonists, with the extent of affinity increase being correlated with the efficacy of the ligand (Lefkowitz et al., 1993). Essentially, the potencies in the GTPase assay represent apparent affinities and can be therefore related, as logEC50 differences between hH2R-GsS and the other H2R species isoforms, to the corresponding efficacies at hH2R-GsS (Fig. 4, B, D, and F). Finally, at receptors with increased constitutive activity inverse agonists have an elevated inhibitory effect on GTP hydrolysis (Seifert et al., 1998b).
|
S and hH2R-GsS (Fig. 4A). As Fig. 4B illustrates, a poor but significant correlation (r2 = 0.27; p = 0.016) was observed between the log (potency ratio) of these species and the efficacies of compounds 1 to 21 at hH2R-GsS. However, this correlation was determined by ligand-specific interactions, namely, the high potencies of guanidines (8 to 10) and NG-acylguanidines (11 to 16) at gpH2R-GsS (Kelley et al., 2001; Xie et al., 2006a,b), and disappeared if only compounds 1 to 7 and 17 to 21 were considered (r2 = 0.04; p = 0.527). The efficacies of compounds 1 to 21 at rH2R-GsS and hH2R-GsS were almost identical (Fig. 4C). Moreover, no correlation between the log (potency ratio) and the efficacies at hH2R-GsS was evident (r2 = 0.16; p = 0.077) (Fig. 4D). Thus, in the steady-state GTPase assay, rH2R-GsS and hH2R-GsS exhibited similar levels of constitutive activities. By contrast, cH2R-GsS showed the hallmarks of a GPCR with increased constitutive activity compared with hH2R-GsS. Specifically, partial agonists 5 to 7 and 14 were considerably more efficacious at cH2R-GsS and the inverse agonist efficacies of antagonists 18 to 21 were increased compared with hH2R-GsS (Fig. 4E). A highly significant correlation between the log (potency ratio) and the efficacies of compounds 1 to 21 at hH2R-GsS was determined (r2 = 0.77; p < 0.0001; Fig. 4F). It is noteworthy that this correlation was independent of distinct interactions of guanidines and NG-acylguanidines with cH2R-GsS as omitting compounds 8 to 16 did not change the fit (r2 = 0.75; p = 0.0003).
Ambiguous Response of Metiamide (22) in the GTPase Assay. At hH2R-GsS, metiamide (22) decreased the basal GTPase signal by 8 ± 1% and thus acted as weak inverse agonist (Table 1; Fig. 5). At gpH2R-GsS and rH2R-GsS, metiamide inhibited the basal GTPase signals by 6 ± 1 and 4 ± 1%, respectively, and was 2-fold more potent than at hH2R-GsS. Intriguingly, at cH2R-GsS metiamide did not act as an inverse agonist but rather as a very weak partial agonist (efficacy of 6 ± 1%). This is in marked contrast to the results of antagonists 18 to 21 reducing the basal GTPase signal at cH2R-GsS (increased constitutive activity) more effectively than at the other less constitutively active species. Furthermore, the potency of 22 was lowered by approximately 15-fold compared with hH2R-GsS and not increased as would have been expected for a partial agonist (Samama et al., 1993). Attempts to detect changes in AC activity upon stimulation with metiamide in membranes coexpressing cH2R and GsS failed due to the much lower sensitivity of this system compared with the GTPase activity assay using fusion proteins (data not shown).
|
-like G proteins), in membranes coexpressing H2R and mammalian GsS, and in membranes expressing H2R-GsS fusion proteins. Basal AC activities were similar in membranes expressing hH2R, gpH2R, and rH2R (Table 3) and 2-fold higher in the case of the cH2R. GTP (10 µM) by itself increased AC activities at all four H2R species by 2-fold above the basal level. HA (1) further increased, and IAPT (21) inhibited this GTP-dependent signal increase, indicative for constitutive activity of all four H2R species isoforms in Sf9 membranes (Fig. 6, A–D). These observations are in agreement with previous studies at the 2AR (Seifert et al., 1998a). The stimulatory effects of GTP, as determined by relating the effects of GTP (10 µM) to the effects of HA (100 µM) plus GTP (10 µM), were largest at cH2R and rH2R, compared with hH2R and gpH2R. Both the high basal AC activity and the strong stimulation with GTP indicate an elevated level of constitutive activity in membranes expressing the cH2R relative to membranes expressing hH2R, gpH2R, and rH2R. It is noteworthy that the constitutive activity of rH2R seemed to be slightly increased compared with hH2R and gpH2R.
|
|
The GPCR/G protein stoichiometry affects the magnitude of response (Kenakin, 2001). In H2R membranes coexpressing mammalian GsS, 5- to 18-fold increased basal levels of AC activity were measured relative to membranes expressing H2R alone (Table 3). Basal AC activities were 6-fold higher at cH2R plus GsS and 2-fold higher at rH2R plus GsS, respectively, compared with hH2R plus GsS. With gpH2R plus GsS, the basal AC activity was somewhat lower than with hH2R plus GsS. As was observed in membranes expressing H2R alone, the highest stimulatory effects of GTP in the coexpression system were observed with cH2R and rH2R compared with gpH2R and hH2R. The inverse agonist IAPT (10 µM) decreased the GTP-dependent increases of AC activity at all species isoforms (Fig. 6, E–H), but even strongly reduced basal AC activities at the lowest concentrations of added GTP. These effects were probably due to traces of GDP being converted to GTP by the action of nucleoside diphosphate kinase and were most prominent in membranes expressing cH2R plus GsS (69% reduction below basal) and rH2R plus GsS (59% reduction), compared with hH2R plus GsS (29% reduction) and gpH2R plus GsS (23% reduction). Taken together, among H2R species isoforms coexpressed with GsS, cH2R was the most constitutively active GPCR.
Due to the efficient coupling of the signaling partners in GPCR-Gs fusion proteins (Seifert et al., 1999), in membranes expressing H2R-GsS, strongly elevated basal AC activities were measured, compared with membranes expressing nonfused H2Rs coexpressing GsS (Table 3). In agreement with the results obtained for membranes expressing nonfused H2Rs, among the four species isoforms, cH2R-GsS and rH2R-GsS exhibited the highest basal AC activities. As shown in Fig. 6, K and L, GTP increased AC activity in those membranes so effectively that HA could not produce a further increase, reflecting exhaustion of the limiting pool of AC molecules (Seifert at al., 1998a). At hH2R-GsS and gpH2R-GsS, GTP induced only smaller increases, allowing HA to further enhance AC activity. By contrast, in the absence of added GTP, HA (100 µM) yielded a reduction of basal AC activities at all four species (Fig. 6, I–L). Very similar effects were observed previously for the 2AR-GsS fusion protein (Seifert et al., 1998b) and are due to dissociation of GDP from GsS following agonist binding to the receptor without subsequent binding of GTP. Because Gs-GDP is more effective in activating AC than nucleotide-free Gs, AC activity was reduced below basal. Due to much less efficient coupling in membranes coexpressing receptors and GsS (Seifert et al., 1998a; Houston et al., 2002; Gille and Seifert, 2003), in this case HA did not reduce basal AC activity (Fig. 6, E–H). Similar differences in the coupling efficiencies between fusion proteins and nonfused expression systems were observed in terms of ternary complex formation, guanosine 5'-O-(3-thio)triphosphate binding, GTP hydrolysis, and AC activation in the presence of GTP (Seifert et al., 1998a; Wenzel-Seifert et al., 2002; Gille and Seifert, 2003). Thus, in membranes expressing H2R-GsS fusion proteins the apparent constitutive activities were considerably higher than in membranes expressing nonfused H2Rs. In the case of cH2R-GsS and rH2R-GsS, saturation of AC molecules became manifest upon agonist (HA) stimulation.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
fusion protein in combination with the determination of GTPase activity in Sf9 cell membranes was previously shown to be an appropriate system to quantify constitutive activity (Seifert et al., 1998b; Seifert and Wenzel-Seifert, 2002). This system fixes GPCR/G protein coupling and stays at a proximal level, thus avoiding potential bias caused by more downstream effects, such as effector activation or changes in gene expression. Moreover, due to the defined 1:1 stoichiometry of receptor and Gs 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 (Seifert et al., 1999; Milligan, 2000).
We comprehensively characterized the human, guinea pig, rat, and canine H2R species isoforms in steady-state GTPase assays in Sf9 cell membranes expressing H2R-GsS fusion proteins. Structurally diverse H2R full and partial agonists and antagonists unmasked considerable differences in the constitutive activities of the receptors. Specifically, cH2R-GsS but neither rH2R-GsS nor gpH2R-GsS displayed the hallmarks of increased constitutive activity compared with hH2R-GsS (Lefkowitz et al., 1993; Samama et al., 1993): 1) increased efficacies of partial agonists, 2) increased potencies of agonists with the extent of potency increase being correlated with the efficacy, and 3) increased inverse agonist efficacies and decreased potencies of antagonists.
The determination of AC activity in Sf9 cell membranes is an alternative and sensitive method to investigate constitutive activity of GPCRs (Seifert et al., 1998a). With respect to AC, differences in the basal activity and in the magnitudes of signal increases upon stimulation with GTP are indicators for various levels of constitutive activity. In the AC activity assay with membranes expressing nonfused H2R species isoforms either without or together with mammalian GsS, both effects were most pronounced for canine relative to human, guinea pig, and rat, corroborating the outstanding role of cH2R in terms of constitutive activity.
However, our analysis of AC activity in membranes expressing H2R species isoforms also illustrates the limitations of this system. Most importantly, the low concentration levels of AC molecules constrain the maximal signal output, thereby yielding large stimulatory effects of GTP and large inhibitory effects of inverse agonists on AC activity. In contrast, the stimulatory effects of the agonist HA are small, if at all detectable. In addition, in the case of the rH2R, basal AC activities and the increases of AC activity upon stimulation with GTP were moderately higher compared with the hH2R, whereas in the GTPase activity assay rH2R-GsS and hH2R-GsS showed similar constitutive activity. The accumulation of rH2R in Sf9 cell membrane microdomains rich in AC molecules could be an explanation for the observed effects (Ostrom and Insel, 2004).
It is now widely accepted that GPCR activation involves disruption of an ionic lock between Asp(3.49) and Arg(3.50) of the highly conserved (E/D)RY motif in TM3 and Glu(6.30) in the cytoplasmatic extension of TM6 (Ballesteros et al., 2001; Visiers et al., 2002). The effects of mutations in the DRY motif on constitutive activity and structural instability of the rat H2R were shown previously (Alewijnse et al., 2000). Asp-115(3.49), Arg-116(3.50), and Glu-228/229(6.30) are conserved among all H2R species isoforms. However, preceding Glu-229(6.30) in hH2R and gpH2R and the corresponding Glu-228(6.30) in rH2R, human, guinea pig, and rat H2Rs exhibit an arginine (6.29), compared with a glycine (6.29), in cH2R. Strikingly, many class A GPCRs contain a basic amino acid at the corresponding position, and accordingly, a stabilizing role of this residue in the network of ionic interactions was proposed (Ballesteros et al., 2001). Hence, the lack of this additional constraint in cH2R could facilitate the transition from the inactive to the active state, resulting in the observed enhancement in constitutive activity.
Other differences in amino acid sequences could contribute to the differences in constitutive activity as well. Specifically, in G649, an allelic variant of the hH2R, Asn-217 in i3, is replaced by Asp-217. This mutant displays low basal activity and is resistant to up-regulation upon antagonist exposure (Fukushima et al., 2001). Intriguingly, Asn-217 is conserved within hH2R, gpH2R, and rH2R, but it is replaced by a histidine in cH2R. Moreover, major variations in the sequences of H2R species isoforms occur in the C-terminal domain. Because the C terminus of H2R is important for Gs protein activation (Smit et al., 1996), the observed variations in the constitutive activities may alternatively or additionally be due to differences in this domain. In fact, an influence of the C terminus on the constitutive activities of various GPCRs was described previously (Prezeau et al., 1996; Wenzel-Seifert and Seifert, 2003).
Ligand-Specific Interactions at H2R Species Isoforms. In the GTPase activity assay, N-[3-(1H-imidazol-4-yl)propyl]guanidines and their NG-acylated analogs were more potent and more efficacious at gpH2R-GsS than at hH2R-GsS, which is in agreement with previous studies (Kelley et al., 2001; Xie et al., 2006a). Because both species isoforms exhibit similar constitutive activities, our present data further support the concept of distinct interactions as a rationale for this species selectivity. As was predicted by molecular modeling studies and subsequently verified by site-directed mutagenesis, the species selectivity of guanidine-type agonists is based on two distinct amino acids, Tyr-17(1.31) in TM1 and Asp-271(7.36) in TM7 in the gpH2R, presumably interacting via a charge assisted H-bond and thereby stabilizing an active agonist-bound conformation (Kelley et al., 2001). In the hH2R [Cys-17(1.31), Ala-271(7.36)] and the rH2R [Leu-17(1.31), Gly-270(7.36)], this interaction is impossible. Consistently, the guanidine-type agonists were similarly efficacious and potent at rH2R-GsS and hH2R-GsS. Both cH2R and hH2R contain Cys-17(1.31) and Ala-271(7.36) and the differences in potencies and efficacies of the compounds between cH2R-GsS and hH2R-GsS were not specific to the guanidines. Thus, these differences can be explained by the increased constitutive activity of cH2R-GsS rather than by distinct ligand/GPCR interactions.
Recently, certain NG-acylated guanidines have shown to be more efficacious than HA at gpH2R-GsS in the GTPase assay (Xie et al., 2006b), similar to the observations made with UR-PG222A (13) in the present study. These effects can be attributed to the concept of ligand-specific gpH2R conformations as well, i.e., these compounds stabilize active gpH2R conformations that lead to more efficient interactions with GsS than achieved with the endogenous ligand HA. By analogy, at the 2AR labeled with a fluorescent probe, the synthetic ligand isoproterenol induced a stronger change in fluorescence intensity than the endogenous ligand norepinephrine (Swaminath et al., 2004).
A further example of ligand-specific interactions at H2R species isoforms is given by metiamide, acting as a weak partial agonist with low potency at cH2R-GsS in the GTPase assay compared with being an inverse agonist with increased potency at human, guinea pig, and rat H2R-GsS. Moreover, in contrast to increased inverse agonist efficacies of antagonists 18 to 21 at cH2R-GsS relative to hH2R-GsS, gpH2R-GsS, and rH2R-GsS, the inverse agonist efficacies of cimetidine (17), a cyanoguanidine analog of metiamide, were similar at all four species whereas its potency was significantly decreased at cH2R-GsS. Presumably because of the common 2[(5-methylimidazol-4-yl)methylthio]ethyl moiety, both metiamide and cimetidine stabilize distinct conformations in cH2R relative to the other species isoforms, thus leading to an altered interaction with GsS, which, in the extreme case of metiamide, causes weak partial agonism rather than increased inverse agonism.
|
Conclusions |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
). Thus, differences in constitutive activities of GPCRs regulating H+ production may reflect species-specific requirements for "basal" and peak H+ secretion in the stomach. For example, following stimulation with HA, gastric acid secretion rates in dogs exceed those of the human (Kararli, 1995). Moreover, by studying the H2R antagonist metiamide, further evidence for ligand-specific conformations of H2R species isoforms was obtained. Our present study validates the notion that quantitative comparison of species isoforms of GPCRs provides unique insights into the molecular mechanisms of GPCR activation and ligand/GPCR interactions. |
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: H2R, histamine H2 receptor; TM, transmembrane domain of a G protein-coupled receptor; H1R, histamine H1 receptor; gpH2R, guinea pig histamine H2 receptor; gpH2R-GsS, fusion protein of the guinea pig histamine H2 receptor and the short splice variant of Gs; hH2R, human histamine H2 receptor; hH2R-GsS, fusion protein of the human histamine H2 receptor and the short splice variant of Gs; Gs, -subunit of the Gs, protein that mediates adenylyl cyclase activation; GsS, short splice variant of the Gs protein Gs; HA, histamine; DIM, dimaprit; AMT, amthamine; BET, betahistine; IMP, impromidine; ARP, arpromidine; CIM, cimetidine; RAN, ranitidine; FAM, famotidine; APT, aminopotentidine; IAPT, iodoaminopotentidine; AC, adenylyl cyclase; GPCR, G protein-coupled receptor; rH2R, rat histamine H2 receptor; rH2R-GsS, fusion protein of the rat histamine H2 receptor and the short splice variant of Gs; S, signal peptide from influenza hemagglutinin; F, FLAG epitope; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; cH2R, canine histamine H2 receptor; cH2R-GsS, fusion protein of the canine histamine H2 receptor and the short splice variant of Gs; AR, adrenoceptor; ANOVA, analysis of variance.
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
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
Alewijnse AE, Smit MJ, Hoffmann M, Verzijl D, Timmerman H, and Leurs R (1998) Constitutive activity and structural instability of the wild-type human H2 receptor. J Neurochem 71: 799–807.[Medline]
Alewijnse AE, Timmerman H, Jacobs EH, Smit MJ, Roovers E, Cotecchia S, and Leurs R (2000) The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H2 receptor. Mol Pharmacol 57: 890–898.
Ballesteros JA, Jensen AD, Liapakis G, Rasmussen SG, Shi L, Gether U, and Javitch JA (2001) Activation of the 2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6. J Biol Chem 276: 29171–29177.
Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22: 3099–3108.[CrossRef][Medline]
Dove S, Elz S, Seifert R, and Buschauer A (2004) Structure-activity relationships of histamine H2 receptor ligands. Mini Rev Med Chem 4: 941–954.[Medline]
Elz S, Kramer K, Pertz HH, Detert H, ter Laak AM, Kühne R, and Schunack W (2000) Histaprodifens: synthesis, pharmacological in vitro evaluation, and molecular modeling of a new class of highly active and selective histamine H1-receptor agonists. J Med Chem 43: 1071–1084.[CrossRef][Medline]
Fukushima Y, Asano T, Saitoh T, Anai M, Funaki M, Ogihara T, Katagiri H, Matsuhashi N, Yazaki Y, and Sugano K (1997) Oligomer formation of histamine H2 receptors expressed in Sf9 and COS7 cells. FEBS Lett 409: 283–286.[CrossRef][Medline]
Fukushima Y, Oka Y, Saitoh T, Katagiri H, Asano T, Matsuhashi N, Takata K, van Breda E, Yazaki Y, and Sugano K (1995) Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action. Biochem J 310: 553–558.[Medline]
Fukushima Y, Saitoh T, Anai M, Tsukuda K, Onishi Y, Sakoda H, Inukai K, Ogihara T, Funaki M, Ono H, et al. (2001) G649, an allelic variant of the human H2 receptor with low basal activity, is resistant to upregulation upon antagonist exposure. Pharmacogenomics J 1: 78–83.[Medline]
Gantz I, DelValle J, Wang LD, Tashiro T, Munzert G, Guo YJ, Konda Y, and Yamada T (1992) Molecular basis for the interaction of histamine with the histamine H2 receptor. J Biol Chem 267: 20840–20843.
Gantz I, Munzert G, Tashiro T, Schäffer M, Wang L, DelValle J, and Yamada T (1991a) Molecular cloning of the human histamine H2 receptor. Biochem Biophys Res Commun 178: 1386–1392.[CrossRef][Medline]
Gantz I, Schäffer M, DelValle J, Logsdon C, Campbell V, Uhler M, and Yamada T (1991b) Molecular cloning of a gene encoding the histamine H2 receptor. Proc Natl Acad Sci USA 88: 429–433.
Ghorai P (2005) Arpromidine-Related Acylguanidines: Synthesis and Structure-Activity Relationships of a New Class of Guanidine-Type Histamine H2 Receptor Agonists with Reduced Basicity. Ph.D. thesis. University of Regensburg, Regensburg, Germany.
Gille A and Seifert R (2003) Co-expression of the 2-adrenoceptor and dopamine D1-receptor with Gs proteins in Sf9 insect cells: limitations in comparison with fusion proteins. Biochim Biophys Acta 1613: 101–114.[Medline]
Hill SJ, Ganellin CR, Timmerman H, Schwartz JC, Shankley NP, Young JM, Schunack W, Levi R, and Haas HL (1997) International Union of Pharmacology. XIII. Classification of histamine receptors. Pharmacol Rev 49: 253–278.
Houston C, Wenzel-Seifert K, Bürckstümmer T, and Seifert R (2002) The human histamine H2-receptor couples more efficiently to Sf9 insect cell Gs-proteins than to insect cell Gq-proteins: limitations of Sf9 cells for the analysis of receptor/Gq-protein coupling. J Neurochem 80: 678–696.[CrossRef][Medline]
Kararli TT (1995) Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm Drug Dispos 16: 351–380.[Medline]
Kelley MT, Bürckstümmer T, Wenzel-Seifert K, Dove S, Buschauer A, and Seifert R (2001) Distinct interaction of human and guinea pig histamine H2-receptor with guanidine-type agonists. Mol Pharmacol 60: 1210–1225.
Kenakin T (2001) Inverse, protean, and ligand-selective agonism: matters of receptor conformation. FASEB J 15: 598–611.
Kopin AS, McBride EW, Schaffer K, and Beinborn M (2000) CCK receptor polymorphisms: an illustration of emerging themes in pharmacogenomics. Trends Pharmacol Sci 21: 346–353.[CrossRef][Medline]
Lefkowitz RJ, Cotecchia S, Samama P, and Costa T (1993) Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci 14: 303–307.[CrossRef][Medline]
Milligan G (2000) Insights into ligand pharmacology using receptor-G-protein fusion proteins. Trends Pharmacol Sci 21: 24–28.[CrossRef][Medline]
Nederkoorn PH, van Gelder EM, Donne-Op den Kelder GM, and Timmerman H (1996) The agonistic binding site at the histamine H2 receptor. II. Theoretical investigations of histamine binding to receptor models of the seven -helical transmembrane domain. J Comput Aided Mol Des 10: 479–489.[CrossRef][Medline]
Ostrom RS and Insel PA (2004) The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology. Br J Pharmacol 143: 235–245.[CrossRef][Medline]
Preuss H, Ghorai P, Kraus A, Dove S, Buschauer A, and Seifert R (2007) Mutations of Cys-17 and Ala-271 in the human histamine H2 receptor determines the species selectivity of guanidine-type agonists and increase constitutive activity. J Pharmacol Exp Ther 321: 975–982.
Prezeau L, Gomeza J, Ahern S, Mary S, Galvez T, Bockaert J, and Pin JP (1996) Changes in the carboxyl-terminal domain of metabotropic glutamate receptor 1 by alternative splicing generate receptors with differing agonist-independent activity. Mol Pharmacol 49: 422–429.[Abstract]
Ruat M, Traiffort E, Arrang JM, Leurs R, and Schwartz JC (1991) Cloning and tissue expression of a rat histamine H2-receptor gene. Biochem Biophys Res Commun 179: 1470–1478.[CrossRef][Medline]
Samama P, Cotecchia S, Costa T, and Lefkowitz RJ (1993) A mutation-induced activated state of the 2-adrenergic receptor. Extending the ternary complex model. J Biol Chem 268: 4625–4636.
Seifert R, Lee TW, Lam VT, and Kobilka BK (1998a) Reconstitution of 2-adrenoceptor-GTP-binding-protein interaction in Sf9 cells: high coupling efficiency in a 2-adrenoceptor-Gs fusion protein. Eur J Biochem 255: 369–382.[Medline]
Seifert R and Wenzel-Seifert K (2002) Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors. Naunyn-Schmiedeberg's Arch Pharmacol 366: 381–416.[CrossRef][Medline]
Seifert R, Wenzel-Seifert K, Bürckstümmer T, Pertz HH, Schunack W, Dove S, Buschauer A, and Elz S (2003) Multiple differences in agonist and antagonist pharmacology between human and guinea pig histamine H1-receptor. J Pharmacol Exp Ther 305: 1104–1115.
Seifert R, Wenzel-Seifert K, and Kobilka BK (1999) GPCR-G fusion proteins: molecular analysis of receptor-G-protein coupling. Trends Pharmacol Sci 20: 383–389.[CrossRef][Medline]
Seifert R, Wenzel-Seifert K, Lee TW, Gether U, Sanders-Bush E, and Kobilka BK (1998b) Different effects of Gs splice variants on 2-adrenoreceptor-mediated signaling. The 2-adrenoreceptor coupled to the long splice variant of Gs has properties of a constitutively active receptor. J Biol Chem 273: 5109–5116.
Smit MJ, Timmerman H, Blauw J, Beukers MW, Roovers E, Jacobs EH, Hoffmann M, and Leurs R (1996) The C terminal tail of the histamine H2 receptor contains positive and negative signals important for signal transduction and receptor down-regulation. J Neurochem 67: 1791–1800.[Medline]
Swaminath G, Xiang Y, Lee TW, Steenhuis J, Parnot C, and Kobilka BK (2004) Sequential binding of agonists to the 2 adrenoceptor. Kinetic evidence for intermediate conformational states. J Biol Chem 279: 686–691.
Traiffort E, Vizuete ML, Tardivel-Lacombe J, Souil E, Schwartz JC, and Ruat M (1995) The guinea pig histamine H2 receptor: gene cloning, tissue expression and chromosomal localization of its human counterpart. Biochem Biophys Res Commun 211: 570–577.[CrossRef][Medline]
Visiers I, Ebersole B, Dracheva S, Ballesteros JA, Sealfon S, and Weinstein H (2002) Structural motifs as functional microdomains in G-protein-coupled receptors: Energetic considerations in the mechanism of activation of the serotonin 5-HT2A receptor by disruption of the ionic lock of the arginine cage. Int J Quantum Chem 88: 65–75.[CrossRef]
Wenzel-Seifert K, Liu HY, and Seifert R (2002) Similarities and differences in the coupling of human 1- and 2-adrenoceptors to Gs splice variants. Biochem Pharmacol 64: 9–20.[CrossRef][Medline]
Wenzel-Seifert K and Seifert R (2003) Functional differences between human formyl peptide receptor isoforms 26, 98, and G6. Naunyn-Schmiedeberg's Arch Pharmacol 367: 509–515.[CrossRef][Medline]
Xie SX, Ghorai P, Ye QZ, Buschauer A, and Seifert R (2006a) Probing ligand-specific histamine H1- and H2-receptor conformations with NG-acylated imidazolylpropylguanidines. J Pharmacol Exp Ther 317: 139–146.
Xie SX, Kraus A, Ghorai P, Ye QZ, Elz S, Buschauer A, and Seifert R (2006b) N1-(3-Cyclohexylbutanoyl)-N2-[3-(1H-imidazol-4-yl)propyl]guanidine (UR-AK57), a potent partial agonist for the human histamine H1- and H2-receptors. J Pharmacol Exp Ther 317: 1262–1268.
This article has been cited by other articles:
|
|
 |
|
  N. Weitl and R. Seifert Distinct Interactions of Human {beta}1- and {beta}2-Adrenoceptors with Isoproterenol, Epinephrine, Norepinephrine, and Dopamine J. Pharmacol. Exp. Ther., December 1, 2008; 327(3): 760 - 769. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Preuss, P. Ghorai, A. Kraus, S. Dove, A. Buschauer, and R. Seifert 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 J. Pharmacol. Exp. Ther., June 1, 2007; 321(3): 975 - 982. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
), HA (100 µM) (
), or IAPT (10 µM) (
). Data shown are the means ± S.E.M. of one representative experiment performed in duplicates. The statistical analysis of AC activities is provided in