Constitutive activity and ligand selectivity of human, guinea pig, rat, and canine histamine H2 receptors

Previous studies revealed pharmacological differences between human and guinea pig histamine H(2) receptors (H(2)Rs) with respect to the interaction with guanidine-type agonists. Because H(2)R species variants are structurally very similar, comparative studies are suited to relate different properties of H(2)R species isoforms to few molecular determinants. Therefore, we systematically compared H(2)Rs of human (h), guinea pig (gp), rat (r), and canine (c). Fusion proteins of hH(2)R, gpH(2)R, rH(2)R, and cH(2)R, respectively, and the short splice variant of G(salpha), G(salphaS), were expressed in Sf9 insect cells. In the membrane steady-state GTPase activity assay, cH(2)R-G(salphaS) but neither gpH(2)R-G(salphaS) nor rH(2)R-G(salphaS) showed the hallmarks of increased constitutive activity compared with hH(2)R-G(salphaS), i.e., increased efficacies of partial agonists, increased potencies of agonists with the extent of potency increase being correlated with the corresponding efficacies at hH(2)R-G(salphaS), increased inverse agonist efficacies, and decreased potencies of antagonists. Furthermore, in membranes expressing nonfused H(2)Rs without or together with mammalian G(salphaS) or H(2)R-G(salpha) fusion proteins, the highest basal and GTP-dependent increases in adenylyl cyclase activity were observed for cH(2)R. An example of ligand selectivity is given by metiamide, acting as an inverse agonist at hH(2)R-G(salphaS), gpH(2)R-G(salphaS), and rH(2)R-G(salphaS) in the GTPase assay in contrast to being a weak partial agonist with decreased potency at cH(2)R-G(salphaS). In conclusion, the cH(2)R exhibits increased constitutive activity compared with hH(2)R, gpH(2)R, and rH(2)R, and there is evidence for ligand-specific conformations in H(2)R species isoforms.     

Probing ligand-specific histamine H1- and H2-receptor conformations with NG-acylated Imidazolylpropylguanidines

Impromidine (IMP) and arpromidine (ARP)-derived guanidines are more potent and efficacious guinea pig (gp) histamine H(2)-receptor (gpH(2)R) than human (h) H(2)R agonists and histamine H(1)-receptor (H(1)R) antagonists with preference for hH(1)R relative to gpH(1)R. We examined N(G)-acylated imidazolylpropylguanidines (AIPGs), which are less basic than guanidines, at hH(2)R, gpH(2)R, rat H(2)R (rH(2)R), hH(1)R, and gpH(1)R expressed in Sf9 cells as probes for ligand-specific receptor conformations. AIPGs were similarly potent H(2)R agonists as the corresponding guanidines IMP and ARP, respectively. Exchange of pyridyl in ARP against phenyl increased AIPG potency 10-fold, yielding the most potent agonists at the hH(2)R-G(salpha) fusion protein and gpH(2)R-G(salpha) identified so far. Some AIPGs were similarly potent and efficacious at hH(2)R-G(salpha) and gpH(2)R-G(salpha). AIPGs stabilized the ternary complex in hH(2)R-G(salpha) and gpH(2)R-G(salpha) differently than the corresponding guanidines. Guanidines, AIPGs, and small H(2)R agonists exhibited distinct agonist properties at hH(2)R, gpH(2)R, and rH(2)R measuring adenylyl cyclase activity. In contrast to ARP and IMP, AIPGs were partial H(1)R agonists exhibiting higher efficacies at hH(1)R than at gpH(1)R. This is remarkable because, so far, all bulky H(1)R agonists exhibited higher efficacies at gpH(1)R than at hH(1)R. Collectively, our data suggest that AIPGs stabilize different active conformations in hH(2)R, gpH(2)R, and rH(2)R than guanidines and that, in contrast to guanidines, AIPGs are capable of stabilizing a partially active state of hH(1)R.     

Multiple differences in agonist and antagonist pharmacology between human and guinea pig histamine H1-receptor

Species isoforms of histamine H2-, H3-, and H4-receptors differ in their pharmacological properties. The study aim was to dissect differences between the human H1R (hH1R) and guinea pig H1R (ghH1R). We coexpressed hH1R and gpH1R with regulators of G-protein signaling in Sf9 insect cells and analyzed the GTPase activity of Gq-proteins. Small H1R agonists showed similar effects at hH1R and gpH1R, whereas bulkier 2-phenylhistamines and histaprodifens were up to approximately 10-fold more potent at gpH1R than at hH1R. Most 2-phenylhistamines and histaprodifens were more efficacious at gpH1R than at hH1R. Several first-generation H1R antagonists were approximately 2-fold, and arpromidine-type H1R antagonists up to approximately 10-fold more potent at gpH1R than at hH1R. [3H]Mepyramine competition binding studies confirmed the potency differences of the GTPase studies. Phe-153-->Leu-153 or Ile-433-->Val-433 exchange in hH1R (hH1R-->gpH1R) resulted in poor receptor expression, low [3H]mepyramine affinity, and functional inactivity. The Phe-153-->Leu-153/Ile-433-->Val-433 double mutant expressed excellently but only partially changed the pharmacological properties of hH1R. Small H1R agonists and 2-phenylhistamines interacted differentially with human and guinea pig H2R in terms of potency and efficacy, respectively. Our data show the following: 1) there are differences in agonist- and antagonist-pharmacology of hH1R and gpH1R encompassing diverse classes of bulky ligands. These differences may be explained by higher conformational flexibility of gpH1R relative to hH1R; 2) Phe-153 and Ile-433 are critical for proper folding and expression of hH1R; and 3) H2R species isoforms distinguish between H1R agonists.     

Pharmacological profile of histaprodifens at four recombinant histamine H1 receptor species isoforms

There are differences in the pharmacological properties of phenylhistamines and histaprodifens between guinea pig histamine H(1) receptor (gpH(1)R) and human histamine H(1) receptor (hH(1)R). The aim of this study was to analyze species differences in more detail, focusing on histaprodifen derivatives and including the bovine histamine H(1) receptor (bH(1)R) and rat histamine H(1) receptor (rH(1)R). H(1)R species isoforms were coexpressed with the regulator of G protein signaling RGS4 in Sf9 insect cells. We performed [(3)H]mepyramine binding assays and steady-state GTPase assays. For a novel class of histaprodifens, the chiral histaprodifens, unique species differences between hH(1)R, bH(1)R, rH(1)R, and gpH(1)R were observed. The chiral histaprodifens 8R and 8S were both partial agonists at gpH(1)R, but only 8R was a partial agonist at the other H(1)R species isoforms. An additional phenyl group in chiral histaprodifens 10R and 10S, respectively, resulted in a switch from agonism at gpH(1)Rto antagonism at hH(1)R, bH(1)R, and rH(1)R. In general, histaprodifens showed the order of potency hH(1)R < bH(1)R < rH(1)R < gpH(1)R. An active-state model of gpH(1)R was generated with molecular dynamics simulations. Dimeric histaprodifen was docked into the binding pocket of gpH(1)R. Hydrogen bonds and electrostatic interactions were detected between dimeric histaprodifen and Asp-116, Ser-120, Lys-187, Glu-190, and Tyr-432. We conclude the following: 1) chiral histaprodifens interact differentially with H(1)R species isoforms; 2) gpH(1)R and rH(1)R, on one hand, and hH(1)R and bH(1)R, on the other hand, resemble each other structurally and pharmacologically; and 3) histaprodifens interact with H(1)R at multiple sites.     

Differential efficacies of somatostatin receptor agonists for G-protein activation and desensitization of somatostatin receptor subtype 4-mediated responses

Both the histamine H1-receptor (H1R) and H2-receptor (H2R) exhibit pronounced species selectivity in their pharmacological properties; i.e., bulky agonists possess higher potencies and efficacies at guinea pig (gp) than at the corresponding human (h) receptor isoforms. In this study, we examined the effects of NG-acylated imidazolylpropylguanidines substituted with a single phenyl or cyclohexyl substituent on H1R and H2R species isoforms expressed in Sf9 insect cells. N1-(3-Cyclohexylbutanoyl)-N2-[3-(1H-imidazol-4-yl)propyl]guanidine (UR-AK57) turned out to be the most potent hH2R agonist identified so far (EC50 of 23 nM in the GTPase assay at the hH2R-Gsalpha fusion protein expressed in Sf9 insect cells). UR-AK57 was almost a full-hH2R agonist and only slightly less potent and efficacious than at gpH2R-Gsalpha. Several NG-acylated imidazolylpropylguanidines showed similar potency at hH2R and gpH2R. Most unexpectedly, UR-AK57 exhibited moderately strong partial hH1R agonism with a potency similar to that of histamine, whereas at gpH1R, UR-AK57 was only a very weak partial agonist. Structure/activity relationship studies revealed that both the alkanoyl chain connecting the aromatic or alicyclic substituent with the guanidine moiety and the nature of the carbocycle (cyclohexyl versus phenyl ring) critically determine the pharmacological properties of this class of compounds. Collectively, our data show that gpH1R and gpH R do not necessarily exhibit preference for bulky agonists (2) compared with hH1R and hH2R, respectively, and that UR-AK57 is a promising starting point for the development of both potent and efficacious hH1R and hH2R agonists.     

Ligand-specific contribution of the N terminus and E2-loop to pharmacological properties of the histamine H1-receptor

There are species differences between human histamine H(1) receptor (hH(1)R) and guinea pig (gp) histamine H(1) receptor (gpH(1)R) for phenylhistamines and histaprodifens. Several studies showed participation of the second extracellular loop (E2-loop) in ligand binding for some G protein-coupled receptors (GPCRs). Because there are large species differences in the amino acid sequence between hH(1)R and gpH(1)R for the N terminus and E2-loop, we generated chimeric hH(1)Rs with gp E2-loop (h(gpE2)H(1)R) and gp N terminus and gp E2-loop (h(gpNgpE2)H(1)R). hH(1)R, gpH(1)R, and chimeras were expressed in Sf9 insect cells. [(3)H]Mepyramine binding assays and steady-state GTPase assays were performed. In the series hH(1)R > h(gpE2)H(1)R > h(gpNgpE2)H(1)R, we observed a significant decrease in potency of histamine 1 in the GTPase assay. For phenoprodifen 5 and the chiral phenoprodifens 6R and 6S, a significant decrease in affinity and potency was found in the series hH(1)R > h(gpE2)H(1)R > h(gpNgpE2)H(1)R. In addition, we constructed new active-state H(1)R models based on the crystal structure of the human beta(2)-adrenergic receptor (hbeta(2)AR). Compared with the H(1)R active-state models based on the crystal structure of bovine rhodopsin, the E2-loop differs in its contact to the ligand bound in the binding pocket. In the bovine rhodopsin-based model, the backbone carbonyl of Lys187 (gpH(1)R) interacts with large histaprodifens in the binding pocket, but in the hbeta(2)AR-based model, Lys187 (gpH(1)R) is located distantly from the binding pocket. In conclusion, the differences in N terminus and E2-loop between hH(1)R and gpH(1)R exert an influence on affinity and/or potency for histamine and phenoprodifens 5, 6R, and 6S.     

Similar apparent constitutive activity of human histamine H(2)-receptor fused to long and short splice variants of G(salpha).

Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The beta(2)-adrenoceptor (beta(2)AR) fused to the long splice variant of G(salpha) (G(salphaL)) exhibits a higher apparent constitutive activity than the beta(2)-adrenoceptor fused to the short splice variant of G(salpha) (G(salphaS)). Experimentally, this results in higher efficacy and potency of partial agonists and in higher efficacy of inverse agonists at the beta(2)AR fused to G(salphaL) relative to the beta(2)AR fused to G(salphaS), indicating that the agonist-free beta(2)AR and the beta(2)AR occupied by partial agonists promote GDP dissociation from G(salphaL) more efficiently than from G(salphaS). In fact, the GDP affinity of G(salphaS) fused to the beta(2)AR is higher than the GDP affinity of G(salphaL) fused to the beta(2)AR. We asked the question whether the histamine H(2)-receptor (H(2)R) exhibits similar coupling to G(salpha) splice variants as the beta(2)AR. To address this question, we studied H(2)R-G(salpha) fusion proteins expressed in Sf9 cells. In contrast to beta(2)AR-G(salpha) fusion proteins, the potencies and efficacies of partial agonists and the efficacies of inverse agonists were similar at the H(2)R fused to G(salphaL) and G(salphaS) as assessed by guanosine-5'-O-(3-thio)triphosphate binding and/or steady-state GTPase activity. However, the time course analysis of guanosine-5'-O-(3-thio)triphosphate binding indicated that G(salphaS) fused to the H(2)R possesses a higher GDP-affinity than G(salphaL) fused to the H(2)R. Our data show that the H(2)R fused to G(salphaL) and G(salphaS) possesses similar constitutive activity and is insensitive to differences in GDP affinity of G(salpha) splice variants. Thus, GDP affinity of G proteins does not generally determine constitutive activity of receptors.     

Inverse agonism at heptahelical receptors: concept, experimental approach and therapeutic potential

Inverse agonists (negative antagonists) are ligands that stabilize the inactive conformation (R) of receptors according to the two-state receptor model. The active conformation (R*) of heptahelical receptors, i.e. G protein-coupled receptors, has high affinity for G proteins. According to ternary complex models of receptor activation, the R*G complex is in equilibrium with R + G, with spontaneous activity in the absence of agonist. Inverse agonists, having a higher affinity for R, shift R*G towards R + G, decreasing the spontaneous activity of receptors. Agonists have the opposite effect, with a higher affinity for R*. Neutral antagonists have the same affinity for R and R* and compete for both agonists and inverse agonists. Inverse agonists have been recently proposed for a variety of heptahelical receptors. Methods to detect inverse agonists among antagonists are based on the determination of ligand affinity at R and R* with binding experiments, and on the modulation of G protein activity (GTP binding and hydrolysis) or of effector activity. Receptor inverse agonists, but also G protein antagonists and GTPase inhibitors, decrease spontaneous G protein activity corresponding to R*G. Receptor agonists, G protein agonists and GTPase inhibitors increase effector basal activity, but receptor inverse agonists decrease it. The therapeutic potential of inverse agonists is proposed in human diseases ascribed to constitutively active mutant receptors and may be extended to diseases related to wild-type receptor over-expression leading to the increase of R*. Some of the therapeutic effects of presently used receptor antagonists may be related to their inverse agonist properties. Inverse agonists lead to receptor upregulation, offering new approaches to tolerance and dependence to drugs.     

Evaluation of histamine H1-, H2-, and H3-receptor ligands at the human histamine H4 receptor: identification of 4-methylhistamine as the first potent and selective H4 receptor agonist

The histamine H(4) receptor (H(4)R) is involved in the chemotaxis of leukocytes and mast cells to sites of inflammation and is suggested to be a potential drug target for asthma and allergy. So far, selective H(4)R agonists have not been identified. In the present study, we therefore evaluated the human H(4)R (hH(4)R) for its interaction with various known histaminergic ligands. Almost all of the tested H(1)R and H(2)R antagonists, including several important therapeutics, displaced less than 30% of specific [(3)H]histamine binding to the hH(4)R at concentrations up to 10 microM. Most of the tested H(2)R agonists and imidazole-based H(3)R ligands show micromolar-to-nanomolar range hH(4)R affinity, and these ligands exert different intrinsic hH(4)R activities, ranging from full agonists to inverse agonists. Interestingly, we identified 4-methylhistamine as a high-affinity H(4)R ligand (K(i) = 50 nM) that has a >100-fold selectivity for the hH(4)R over the other histamine receptor subtypes. Moreover, 4-methylhistamine potently activated the hH(4)R (pEC(50) = 7.4 +/- 0.1; alpha = 1), and this response was competitively antagonized by the selective H(4)R antagonist JNJ 7777120 [1-[(5-chloro-1H-indol-2-yl)-carbonyl]-4-methylpiperazine] (pA(2) = 7.8). The identification of 4-methylhistamine as a potent H(4)R agonist is of major importance for future studies to unravel the physiological roles of the H(4)R.     

An 80-amino acid deletion in the third intracellular loop of a naturally occurring human histamine H3 isoform confers pharmacological differences and constitutive activity

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

Endogenous regulator of g protein signaling proteins reduce {mu}-opioid receptor desensitization and down-regulation and adenylyl cyclase tolerance in C6 cells

Chronic exposure of cells to mu-opioid agonists leads to tolerance which can be measured by a reduced ability to activate signaling pathways in the cell. Cell signaling through inhibitory G proteins is negatively regulated by RGS (regulator of G protein signaling) proteins. Here we examine the hypothesis that the GTPase accelerating activity of RGS proteins, by altering the lifetime of Galpha and Gbetagamma, plays a role in the development of cellular tolerance to mu-opioids. C6 glioma cells were stably transfected with mu-opioid receptor and pertussis toxin (PTX)-insensitive Galpha(o) that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous Galpha proteins followed by exposure to the mu-opioid agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) or morphine. Receptor desensitization as measured by agonist-stimulated [(35)S]GTPgammaS binding and receptor down-regulation as measured by [(3)H]diprenorphine binding were increased in cells expressing RGS-insensitive Galpha(o). Exposure to high concentrations of morphine or the peptidic mu-opioid agonist DAMGO led to a tolerance to inhibit adenylyl cyclase activity in both cell types with a rapid (30 min) and a slower component. Using a submaximal concentration of DAMGO to induce a reduced level of tolerance, a shift in the concentration-effect curve for DAMGO to inhibit adenylyl cyclase activity was seen in the cells expressing RGS-insensitive Galpha(o), but not in the cells expressing RGS-sensitive Galpha(o), which can be partly explained by an increased supersensitization of the adenylyl cyclase response. The results show that RGS proteins endogenously expressed in C6 cells reduce agonist-induced mu-opioid receptor desensitization, down-regulation, and sensitivity to tolerance to inhibit adenylyl cyclase activity.     

Molecular characterization of quinolone-resistant Neisseria gonorrhoeae in Hong Kong

In Hong Kong, ParC changes among high-level quinolone-resistant Neisseria gonorrhoeae (QRNG) isolates at Ser-87-->Arg were associated with a higher level of resistance than a Ser-87-->Ile alteration. Two previously undescribed mutations in clinical isolates occurring in gyrA, conferring Ala-92-->Pro and Asp-95-->Tyr changes, were detected. Nine different outer membrane lipoprotein (Lip) repeat classes-11 to 19 repeats-were identified, with repeat lengths of 16 and 17 the most common, indicating considerable strain diversity.     

Constitutive activation of the gastrin-releasing peptide receptor expressed by the nonmalignant human colon epithelial cell line NCM460.

Gaucher disease, the most prevalent lysosomal storage disease, occurs in three subtypes, all resulting from mutations in the acid beta-glucosidase gene. Molecular studies in five severely affected type 1 and two type 2 Gaucher disease patients of non-Jewish descent identified six new mutations: K74X, W179X, G195E, S271N, V352L, and a two-base deletion in exon 10 (1450del2). Two additional mutations identified in these patients (R48W and G202R) have been reported previously, but were not expressed or characterized. Heterologous expression in Sf 9 cells using the baculovirus system revealed that the missense mutations, R48W and V352L, had 14 and 7%, respectively, of the specific activity based on cross-reacting immunologic material expressed by the normal allele. In contrast, the G195E, G202R, and S271N mutant alleles were more severely compromised with only 1-2% of the normal expressed specific activity based on cross-reacting immunologic material. Structural distortion at the active site was probed by comparing the interaction of the mutant enzymes with active site-directed inhibitors (castanospermine, conduritol B epoxide and deoxynojirimycin). R48W, G202R, and S271N were normally inhibited, whereas the V352L and G195E mutant enzymes had significantly decreased binding affinity. These mutations further expand the genetic heterogeneity in the lesions causing Gaucher disease types 1 and 2, and further delineate genotype/phenotype correlations and functional domains within the acid beta-glucosidase gene.     

Inverse agonist activity of selected ligands of the cysteinyl-leukotriene receptor 1

Cysteinyl leukotrienes (CysLTs) are associated with several inflammatory processes, including asthma. Due to this association, considerable effort has been invested in the development of antagonists to the CysLT receptors (CysLT(1)R). Many of these molecules have been shown to specifically interact with CysLT(1)R, but little is known about their impact on the conformation of the receptor and its activity. We were especially interested in possible inverse agonist activity of the antagonists. Using a constitutively active mutant (N106A) of the human CysLT(1)R and the wild-type (WT) receptor coexpressed with the G(alphaq) subunit of the trimeric G protein, we were able to address this issue with ligands commonly used in therapy. We demonstrated that some of these molecules are inverse agonists, whereas others act as partial agonists. In cells expressing the CysLT(1)R mutant N106A exposed to Montelukast, Zafirlukast, or 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), the basal inositol phosphate production was reduced by 53 +/- 6, 44 +/- 3, and 54 +/- 4%, respectively. On the other hand, 6(R)-(4-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (BayU9773) and 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazole-5-YL)-butoxy]-phenyl ethanone] (LY171883) acted as partial agonists and alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV 5901) as a neutral antagonist. However, in cells expressing CysLT(1)R and G(alphaq), all antagonists used had inverse agonist activity. The decrease in basal inositol phosphate production by ligands with inverse agonist activity could be inhibited by a more neutral antagonist, confirming the specificity of the reaction. We demonstrate here that Montelukast, MK571, and Zafirlukast can act as inverse agonists on the human CysLT(1) receptor.     

Functional differences between full and partial agonists: evidence for ligand-specific receptor conformations

The interaction of an agonist-bound G-protein-coupled receptor (GPCR) with its cognate G-protein initiates a sequence of experimentally quantifiable changes in both the GPCR and G-protein. These include the release of GDP from G(alpha), the formation of a ternary complex between the nucleotide-free G-protein and the GPCR, which has a high affinity for agonist, followed by the binding of GTP to G(alpha), the dissociation of the GPCR/G-protein complex, and the hydrolysis of GTP. The efficacy of an agonist is a measure of its ability to activate this cascade. It has been proposed that efficacy reflects the ability of the agonist to stabilize the active state of the GPCR. We examined a series of beta(2)-adrenoceptor (beta(2)AR) agonists (weak partial agonists to full agonists) for their efficacy at promoting two different steps of the G-protein activation/deactivation cycle: stabilizing the ternary complex (high-affinity, GTP-sensitive agonist binding), and steady-state GTPase activity. We obtained results for the wild-type beta(2)AR and a constitutively active mutant of the beta(2)AR (beta(2)AR(CAM)) using fusion proteins between the GPCRs and G(salpha) to facilitate GPCR/G-protein interactions. There was no correlation between efficacy of ligands in activating GTPase and their ability to stabilize the ternary complex at beta(2)AR(CAM). Our results suggest that the GPCR state that optimally promotes the GDP release and GTP binding is different from the GPCR state that stabilizes the ternary complex. By strongly stabilizing the ternary complex, certain partial agonists may reduce the rate of G-protein turnover relative to a full agonist.     

A highly conserved aspartic acid (Asp-155) anchors the terminal amine moiety of tryptamines and is involved in membrane targeting of the 5-HT(2A) serotonin receptor but does not participate in activation via a "salt-bridge disruption" mechanism

Discovering the molecular and atomic mechanism(s) by which G-protein-coupled receptors (GPCRs) are activated by agonists remains an elusive goal. Recently, studies examining two representative GPCRs (rhodopsin and alpha(1b)-adrenergic receptors) have suggested that the disruption of a putative "salt-bridge" between highly conserved residues in transmembrane (TM) helix III, involving aspartate or glutamate, and helix VII, involving a basic residue, results in receptor activation. We have tested whether this is a general mechanism for GPCR activation by constructing a model of the 5-hydroxytryptamine (5-HT)(2A) receptor and characterizing several mutations at the homologous residues (Asp-155 and Asn-363) of the 5-HT(2A) serotonin receptor. All of the mutants (D155A, D155N, D155E, D155Q, and S363A) resulted in receptors with reduced basal activity; in no case was evidence for constitutive activity revealed. Structure-function studies with tryptamine analogs and various Asp-155 mutants demonstrated that Asp-155 interacts with the terminal, and not indole, amine moiety of 5-HT(2A) agonists. Interestingly, the D155E mutation interfered with the membrane targeting of the 5-HT(2A) receptor, and an inverse relationship was discovered when comparing receptor activation and targeting for a series of Asp-155 mutants. This represents the first known instance in which a charged residue located in a putative TM helix alters the membrane targeting of a GPCR. Thus, for 5-HT(2A) receptors, the TMIII aspartic acid (Asp-155) is involved in anchoring the terminal amine moiety of indole agonists and in membrane targeting and not in receptor activation by salt-bridge disruption.     

Cloning and nucleotide sequence of the DNA gyrase gyrA gene from the fish pathogen Aeromonas salmonicida.

The DNA gyrase gyrA gene from the fish pathogen Aeromonas salmonicida 2148/89 was cloned, and the nucleotide sequence was determined. An open reading frame of 2,766 nucleotides was identified and was found to encode a protein of 922 amino acids with a calculated molecular mass of 101.1 kDa. The derived amino acid sequence shared a high degree of identity with other DNA gyrase A proteins, in particular, with other gram-negative GyrA sequences. When the amino acid sequence of A. salmonicida GyrA was compared with that of Escherichia coli GyrA, a number of conserved residues were present at identical coordinates, including the catalytic Tyr residue at position 122 (Tyr-122) and residues whose substitution confers quinolone resistance, notably, Ser-83, Ala-67, Gly-81, Asp-87, Ala-84, and Gln-106. An intragenic region corresponding to 48 amino acids, which is not present in E. coli or other bacteria, was identified in the C-terminal part of A. salmonicida GyrA. This intragenic region shared sequence identity with various DNA-binding proteins of both prokaryotic and eukaryotic origins.     

In vitro activity of sitafloxacin against clinical strains of Streptococcus pneumoniae with defined amino acid substitutions in QRDRs of gyrase A and topoisomerase IV

OBJECTIVES: Fluoroquinolone-resistant Streptococcus pneumoniae are increasing worldwide rapidly. In vitro activities of sitafloxacin were evaluated against clinical isolates of S. pneumoniae resistant to levofloxacin (MIC of levofloxacin > or = 4 mg/L), which were characterized genetically. METHODS: The quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE of these strains were analysed by PCR-based sequencing. MICs of sitafloxacin and other quinolones were determined by a microdilution broth method. RESULTS: All 18 strains had at least one amino acid substitution in the QRDRs of GyrA and ParC, which included Ser-81-->Tyr/Phe and Glu-85-->Lys in GyrA and Ser-79-->Phe/Ile/Tyr, Asp-83-->Tyr, Asn-91-->Asp, Ser-107-->Phe, Lys-137-->Asn and Ala-142-->Ser in ParC. Most isolates had Asp-435-->Asn/Ile-460-->Val/Ala-596-->Thr substitutions in ParE, while no amino acid substitution in GyrB was noted in all isolates. Ten isolates for which levofloxacin MICs were 16 or 32 mg/L had multiple mutations in both GyrA and ParC. The MIC80 value of sitafloxacin for levofloxacin-resistant isolates was 0.25 mg/L. The range of MICs of sitafloxacin for isolates resistant to levofloxacin (MIC 4-32 mg/L) was 0.016-0.5 mg/L. CONCLUSIONS: These findings warrant further studies to evaluate the usefulness of sitafloxacin in the treatment of levofloxacin-resistant S. pneumoniae infection.     

Reduced Clinical Efficacy of Pazufloxacin against Gonorrhea Due to High Prevalence of Quinolone-Resistant Isolates with the GyrA Mutation

Forty-two men with gonococcal urethritis were treated with an oral dosage of 200 mg of pazufloxacin, a new fluoroquinolone, three times daily for 3 days. Only 28 of the 42 men (66.7%) had negative culture results for Neisseria gonorrhoeae during follow-up. Of the 42 isolates, 41 could be recultured for antibiotic susceptibility testing and DNA sequencing. In 26 of the 41 isolates (63.4%), GyrA mutations with or without ParC mutations were identified. Among the 26 isolates, 23 contained a single GyrA mutation, 1 contained two GyrA mutations, and 2 contained three mutations including double GyrA and single ParC mutations. A single Ser-91-to-Phe mutation, which was detected in 14 of the 26 isolates, was the most common GyrA mutation, followed by an Ala-75 to Ser mutation and an Asp-95 to Asn or Gly mutation in GyrA. All three isolates with two or three mutations contained the Ser-91-to-Phe GyrA mutation. Eleven of the 14 isolates with the single Ser-91-to-Phe mutation within GyrA and all 3 isolates with two or three mutations persisted after pazufloxacin treatment. On the other hand, all 15 wild-type and 9 mutant isolates with a substitution at codon Ala-75 or Asp-95 were eradicated. The mean MIC of pazufloxacin for mutants with the single Ser-91-to-Phe mutation in GyrA was 66-fold higher than that for the wild type. The results obtained in this study suggest that a high prevalence of fluoroquinolone-resistant gonococcal isolates with the Ser-91-to-Phe mutation in GyrA reduced the efficacy of pazufloxacin as treatment for gonococcal urethritis.     

Identification of amino acid determinants of dopamine 2 receptor synthetic agonist function

The human dopamine 2 receptor (hD2R) modulates locomotor activity, hormone secretion, and neuropsychiatric function. Current knowledge of the hD2R structure is in large part derived from mutagenesis studies and molecular pharmacologic analysis together with homology modeling using bovine rhodopsin as a template. In this study, we utilized comparison of the Drosophila D2-like receptor (DD2R) with the hD2R as a novel approach for identifying candidate amino acids that are determinants of ligand potency and/or efficacy. We focused our studies on four dopaminergic ligands that are used in the treatment of Parkinson's disease: bromocriptine, pergolide, piribedil, and ropinirole. All four ligands are potent agonists at the wild-type hD2R, whereas only bromocriptine shows comparable function at the DD2R. We performed site-directed mutagenesis to replace hD2R amino acids (modeled to project into the ligand binding pocket) with corresponding fly residues, and vice versa. Substitution of three amino acids in the hD2R with the homologous DD2R residues (V91A, C118S, and L170I) led to a pronounced loss of pergolide potency and efficacy. A converse triple amino acid substitution of human residues into the fly receptor (DD2R-A133V/S160C/I211L) markedly enhanced pergolide efficacy and potency at the mutant DD2R. The same substitutions also converted piribedil and ropinirole, which lacked appreciable activity on the DD2R, to partial agonists. These findings show the important role of these three residues in drug-receptor interactions. Our study illustrates that comparison of a mammalian receptor with an invertebrate homolog complements previously described strategies for defining G protein-coupled receptor structure-function relationships.