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.     

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.     

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.     

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.     

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.     

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.     

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.     

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

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 (gpH(2)R-G(salphaS)) than human (hH(2)R-G(salphaS)) histamine H(2) receptor, coupled to the short splice variant of G(salpha), G(salphaS). Whereas Ala-271 (hH(2)R) and Asp-271 (gpH(2)R) 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 gpH(2)R suggested that an H-bond between Tyr-17 and Asp-271 stabilizes an active receptor conformation of the gpH(2)R. In the present study, we generated a mutant hH(2)R-G(salphaS) with Cys-17--> Tyr-17/Ala-271--> Asp-271 exchanges (hH(2)R-->gpH(2)R) that exhibited an enhanced level of constitutive GTPase activity and adenylyl cyclase activity compared with wild-type hH(2)R-G(salphaS) and gpH(2)R-G(salphaS). Potencies and efficacies of guanidines and N(G)-acylguanidines were increased at this mutant receptor compared with hH(2)R-G(salphaS), but they were still lower than at gpH(2)R-G(salphaS), suggesting that aside from Tyr-17 and Asp-271 additional amino acids contribute to the distinct pharmacological profiles of both species isoforms. Another hH(2)R-G(salphaS) mutant with a Cys-17--> Tyr-17 exchange showed inefficient coupling to G(salphaS) 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 H(2)R-G(salphaS) fusion proteins.     

Cloning of rat histamine H(3) receptor reveals distinct species pharmacological profiles

The recent cloning and characterization of the human histamine H(3) receptor cDNA marked a significant step toward a greater understanding of the role of this receptor in the central nervous system. We now report the cloning of the rat histamine H(3) receptor cDNA and demonstrate distinct pharmacological species differences. The rat cDNA clone encodes a putative 445-amino acid protein with 93% identity to the human H(3) receptor. Northern blot analysis revealed a major single entity of 2.7-kb in length expressed only in brain. Transfection of the rat receptor cDNA into SK-N-MC cells conferred an ability to inhibit forskolin-stimulated cAMP formation in response to histamine and other H(3) agonists. N-[(3)H]methylhistamine saturably bound to transfected cells with high affinity (K(d) = 0.8 nM). All agonists tested had low or subnanomolar K(i) values similar to that for the human recombinant receptor. The antagonists thioperamide and clobenpropit also bound with high affinity (K(i) = 4 and 0.4 nM, respectively). This is in contrast to the antagonist profile obtained for the human recombinant receptor that showed K(i) values of 58 and 0.6 nM for thioperamide and clobenpropit, respectively. These data suggest that the low affinity of thioperamide for the human H(3) receptor represents a species difference in pharmacology and not a unique pharmacological subtype. It also was found that chloroproxyfan behaved as a full agonist at the rat recombinant receptor. These findings highlight the significance of validating the pharmacology of experimental compounds at both the rat and human H(3) receptors.     

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.     

Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain

Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites, the molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have synthesized and assayed three conformationally restricted GHB analogs for binding against the GHB-specific ligand [3H]NCS-382 [(E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene-)acetic acid] in rat brain homogenate. The cyclohexene and cyclopentene analogs, 3-hydroxycyclohex-1-enecarboxylic acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported to date. Neither of the cycloalkenes showed any affinity (IC50 > 1 mM) for GABA(A) or GABA(B) receptors. These compounds show excellent potential as lead structures and novel tools for studying specific GHB receptor-mediated pharmacology.     

Molecular determinants of melanocortin 4 receptor ligand binding and MC4/MC3 receptor selectivity

The molecular basis of ligand recognition by the melanocortin 4 receptor (MC4R) has not been fully defined. In this study, we investigated the molecular determinants of MC4R ligand binding, employing a large array of ligands, using three approaches. First, molecular modeling of the receptor was used to identify Phe284, in transmembrane (TM) 7, as a potential site of ligand interaction. Mutation of Phe284 to alanine reduced binding affinity and potency of peptides containing L-Phe by up to 71-fold but did not appreciably affect binding of linear peptides containing D-Phe, consistent with a hydrophobic interaction between the Phe7 of alpha-melanocyte-stimulating hormone and Phe284. Second, we examined the effect of a naturally occurring mutation in TM3 (I137T) that is linked to obesity. This mutation decreased affinity and potency of cyclic, rigid peptides but not more flexible peptides, consistent with an indirect effect of the mutation on the tertiary structure of the receptor. Third, we examined the residues that support ligand selectivity for the MC4R over the MC3R. Mutation of Ile125 (TM3) of the MC4R to the equivalent residue of the MC3R (phenylalanine) selectively decreased affinity and potency of MC4R-selective ligands. This effect was mirrored by the reciprocal MC3R mutation F157I. The magnitude of this effect indicates that this locus is not of major importance. However, it is considered that an isoleucine/phenylalanine mutation may affect the orientation of Asp122, which has been identified as a major determinant of ligand binding affinity. Thus, this study provides further characterization of the MC4R binding pocket.     

Constitutive activity and inverse agonism at the M2 muscarinic acetylcholine receptor

Introduction of a single-point mutation (Asn to Tyr) at position 410 at the junction between transmembrane domain 6 and the third extracellular loop of the human M(2) muscarinic acetylcholine (mACh) receptor generated a mutant receptor (N410Y) that possesses many of the hallmark features of a constitutively active mutant receptor. These included enhanced agonist binding affinity and potency, in addition to agonist-independent accumulation of [(3)H]inositol phosphates in cells coexpressing the chimeric Galpha(qi5) protein and the N410Y mutant M(2) mACh receptor. Constitutive activity was sensitive to inhibition by a range of muscarinic ligands, including those used clinically in the management of overactive bladder (oxybutynin, tolterodine, and darifenacin), indicating that these ligands behave as inverse agonists at the M(2) mACh receptor. Long-term (24-h) treatment of Chinese hamster ovary cells expressing the N410Y mutant M(2) mACh receptor with certain mACh receptor inverse agonists (atropine, darifenacin, and pirenzepine) elicited a concentration-dependent up-regulation of cell surface receptor expression. However, not all ligands possessing negative efficacy in the [(3)H]inositol phosphate accumulation assays were capable of significantly up-regulating receptor expression, perhaps indicating a spectrum of negative efficacies among ligands traditionally defined as mACh receptor antagonists. Finally, structurally distinct agonists exhibited differences in their relative potencies for the activation of Galpha(i/o) versus Galpha(s), consistent with agonist-directed trafficking of signaling at the N410Y mutant, but not at the wild-type M(2) mACh receptor. This indicates that the N410Y mutation of the M(2) mACh receptor alters receptor-G-protein coupling in an agonist-dependent manner, in addition to generating a constitutively active receptor phenotype.     

Differences in the properties of muscarinic cholinergic receptors in the developing chick myocardium

The number of muscarinic cholinergic receptors detected with [3H]quinuclidinyl benzilate (QNB) was constant in embryonic chick heart membranes but increased 2.5-fold by 3 days posthatching; the KD for [3H]QNB did not change. The affinities of the muscarinic receptors for agonists, as determined in in vitro [3H]QNB competition experiments, differed during development. The IC50 values were lowest for 10- to 14-day embryonic heart receptors, intermediate for 18- to 20-day embryonic receptors and highest for 3- to 7-day newborn heart receptors. These apparent differences in agonist affinity were not overcome by guanine nucleotides or monovalent cations alone, but were greatly diminished in the presence of a combination of guanylylimidodiphosphate plus NH4+. Modeling of [3H]QNB/oxotremorine competition curves indicated the presence of three agonist affinity states in membranes from embryonic heart and two in newborn hearts. The KD values for oxotremorine increased during development. The proportion of receptors displaying the highest affinity for oxotremorine was constant at all ages tested whereas the proportion displaying the lowest affinity decreased from 16% in membranes from 12-day embryonic hearts to zero in newborn hearts. The physiological significance of the differences in the properties of the receptors was investigated by assessing their ability to attenuate adenylate cyclase. The efficacy of agonists to attenuate basal adenylate cyclase increased 1.5-fold after birth, whereas the potency of agonists to produce this effect was similar at all ages tested.     

Constitutive activity of histamine h(3) receptors stably expressed in SK-N-MC cells: display of agonism and inverse agonism by H(3) antagonists.

Agonist-independent activity of G-protein-coupled receptor, also referred to as constitutive activity, is a well-documented phenomenon and has been reported recently for both the histamine H(1) and H(2) receptors. Using SK-N-MC cell lines stably expressing the human and rat H(3) receptors at physiological receptor densities (500-600 fmol/mg of protein), we show that both the rat and human H(3) receptors show a high degree of constitutive activity. The forskolin-mediated cAMP production in SK-N-MC cells is inhibited strongly upon expression of the G(i)-coupled H(3) receptor. The cAMP production can be further inhibited upon agonist stimulation of the H(3) receptor and can be enhanced by a variety of H(3) antagonists acting as inverse agonists at the H(3) receptor. Thioperamide, clobenpropit, and iodophenpropit raise the cAMP levels in SK-N-MC cells with potencies that match their receptor binding affinities. Surprisingly, impentamine and burimamide act as effective H(3) agonists. Modification of the amine group of impentamine dramatically affected the pharmacological activity of the ligand. Receptor affinity was reduced slightly for most impentamine analogs, but the functional activity of the ligands varied from agonist to neutral antagonist and inverse agonist, indicating that subtle changes in the chemical structures of impentamine analogs have major impact on the (de)activation steps of the H(3) receptor. In conclusion, upon stable expression of the rat and human H(3) receptor in SK-N-MC cells constitutive receptor activity is detected. In this experimental system, H(3) receptors ligands, previously identified as H(3) antagonists, cover the whole spectrum of pharmacological activities, ranging from full inverse agonists to agonists.     

The role of transmembrane helix 5 in agonist binding to the human H3 receptor

We have used alanine scanning mutagenesis to identify residues in transmembrane domain 5 of the histamine H3 receptor that are important for agonist binding. All of the mutants generated were functionally expressed as demonstrated by their ability to bind [(125)I]iodoproxyfan with comparable affinity to the wild-type receptor and their ability to inhibit forskolin-stimulated cAMP formation when activated by histamine. Many mutations produced small changes in the potency of histamine, but the most pronounced reduction in potency and affinity of the agonists, histamine, R-alpha-methylhistamine, imetit, and impentamine, was seen with mutation of glutamate 206. Our modeling suggests that this residue plays a key role in ligand binding by interacting with the imidazole ring of histamine. Interestingly, L199A greatly reduced agonist potency in functional assays but had only minor effects on agonist affinity, implicating a role for this residue in the mechanism of receptor activation. We also studied the functional effects of the mutations by linking the receptor to calcium signaling using a chimeric G protein. A comparison of the two functional assays demonstrated contrasting effects on agonist activity. Histamine, imetit, and impentamine were full agonists in the cAMP assay, but imetit exhibited only partial agonist activity through the chimeric G protein. Furthermore, impentamine, another potent agonist in the cAMP assay, was only able to activate the E206A mutant in the calcium assay despite being inactive at the wild-type receptor. These observations suggest that the agonist receptor complexes formed by these three different H3 agonists are not conformationally equivalent.     

Characterization of specific opioid binding sites in neural membranes from the myenteric plexus of porcine small intestine

Delta- and kappa-opioid receptors (OPRs), but not micro-OPRs, are expressed in the myenteric plexus of the porcine distal small intestine. In a subpopulation of myenteric neurons, delta- and kappa-OPRs seem to be colocalized and may functionally interact. In this study, radioligand binding was used to characterize myenteric OPR populations in detail. The nonselective OPR antagonist [3H]diprenorphine bound to a single, high-affinity site in myenteric neural membrane homogenates. Naloxone displaced 65 and 59% of [3H]diprenorphine binding from this site in Na(+)-free Tris and Krebs-HEPES buffers, respectively. Naltrexone-derived delta- and kappa-OPR antagonists, including naltriben, 7-benzylidenenaltrexone, nor-binaltorphimine, and 5'-guanidinonaltrindole, displaced [3H]diprenorphine from two distinct binding sites to levels similar to that of naloxone. The selective delta-OPR ligands Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (TIPP), [D-Pen2,D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin II, and (+)-4-[(alphaR)-alpha((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl-3-methoxybenzyl)-N,N-diethylbenzamide (SNC-80) and the kappa-OPR agonist (D-(5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxoaspiro-(4,5)dec-8-yl) benzeneacetamide (U-69,593) displaced [3H]diprenorphine from three independent binding sites; these included high-affinity delta- and kappa-OPR sites, and a residual binding site. Residual [3H]diprenorphine binding was displaced by the selective kappa-OPR antagonist nor-binaltorphimine after saturation of delta and kappa sites, respectively, with DPDPE and U-69,593. The residual binding site displayed low affinity for delta- and kappa-OPR agonists and TIPP, as well as moderate affinity for naltrexone-derived ligands, properties reminiscent of delta-/kappa-OPR heterodimers.     

3H]LY341495 binding to group II metabotropic glutamate receptors in rat brain.

[3H]LY341495 is a highly potent and selective antagonist for group II metabotropic glutamate (mGlu) receptors (mGlu2 and mGlu3), which has been used to label these receptors in cells expressing recombinant receptor subtypes. In this study, we characterized the kinetics, pharmacology, and distribution of [3H]LY341495 binding to mGlu receptors in rat brain tissue. Equilibrium experiments in the rat forebrain demonstrated binding to a single site that was saturable, reversible, and of high affinity (Bmax, 3.9 +/- 0.65 pmol/mg of protein, Kd, 0.84 +/- 0.11 nM). The relative order of potencies for displacement of [3H]LY341495 by mGlu receptor ligands was LY341495 > L-glutamic acid > LY354740 > (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine > 4-(2R,4R)-aminopyrrolidine-2,4-dicarboxylate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (R,S)-alpha-methyl-4-phosphonophenylglycine > (R,S)3,5-dihydroxyphenylglycine > L-(+)-2-amino-4-phosphonobutyric acid. [3H]LY341495 was not displaced by the selective ionotropic glutamate receptor agonists N-methyl-D-aspartic acid, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, or kainate at concentrations up to 1 mM. Comparison of [3H]LY341495 binding in rat brain with recombinant mGlu receptor subtypes demonstrated a very high correlation with mGlu3 receptor binding (r2 = 0.957), a significant, but lower, correlation with mGlu2 receptor binding (r2 = 0.869), but no significant correlation to mGlu8 receptor binding (r2 = 0.284). Regional studies using autoradiography showed a similar distribution of [3H]LY341495 binding to that for group II mGlu receptors previously reported by others using immunocytochemical techniques. These studies indicate that [3H]LY341495 selectively labels group II (mGlu2/3) receptors, but under the conditions used, [3H]LY341495 may bind predominately to mGlu3 receptor populations in the rat forebrain.     

G-protein sensitivity of ligand binding to human dopamine D(2) and D(3) receptors expressed in Escherichia coli: clues for a constrained D(3) receptor structure

Human dopamine D(2) and D(3) receptors were expressed in Chinese hamster ovary (CHO) and Escherichia coli cells to compare their ligand binding properties in the presence or absence of G-proteins and to analyze their ability to interact with G(i/o)-proteins. Binding affinities of agonists (dopamine, 7-OH-DPAT, PD128907, lisuride) and antagonists/inverse agonists (haloperidol, risperidone, domperidone, spiperone, raclopride, nemonapride), measured using [(125)I]iodosulpride and [(3)H]7-OH-DPAT, were similar for hD(3) receptors in E. coli and CHO cell membranes. Both agonists and antagonists showed 2- to 25-fold lower binding affinities at hD(2) receptors in E. coli versus CHO cell membranes (measured with [(3)H]spiperone), but the rank order of potencies remained similar. Purported inverse agonists did not display higher affinities for G-protein-free receptors. In CHO membranes, GppNHp decreased high affinity agonist ([(3)H]7-OH-DPAT) binding at hD(2) receptors but not at hD(3) receptors. Also, [(3)H]7-OH-DPAT (nanomolar concentration range) binding was undetectable at hD(2) but clearly measurable at hD(3) receptors in E. coli membranes. Addition of a G(i/o)-protein mix to E. coli membranes increased high affinity [(3)H]7-OH-DPAT binding in a concentration-dependent manner at hD(2) and hD(3) receptors; this effect was reversed by addition of GppNHp. The potency of the G(i/o)-protein mix to reconstitute high affinity binding was similar for hD(2) and hD(3) receptors. Thus, agonist binding to D(3) receptors is only slightly affected by G-protein uncoupling, pointing to a rigid receptor structure. Furthermore, we propose that the generally reported lower signaling capacity of D(3) receptors (versus D(2) receptors) is not due to its lower affinity for G-proteins but attributed to its lower capacity to activate these G-proteins.     

G protein coupling and ligand selectivity of the D2L and D3 dopamine receptors

The human dopamine D(2L) receptor couples promiscuously to multiple members of the Galpha(i/o) subfamily. Despite the high homology of the D(2L) and D(3) receptors, the G protein coupling specificity of the human D(3) receptor is less clearly characterized. The primary aim of this study, then, was the parallel characterization of the G protein coupling specificity of the D(2L) and D(3) receptors. By using both receptor-G protein fusion proteins and stable cell lines in which pertussis toxin-resistant mutants of individual Galpha(i)-family G proteins were expressed in an inducible fashion, we demonstrated highly selective coupling of the D(3) receptor to Galpha(o1). Furthermore, by using the fusion proteins to ensure identical stoichiometry of receptor to G protein for each pairing, a range of ligands displayed higher potency and, for partial agonists, higher efficacy at the D(3) receptor when coupled to Galpha(o1) compared with the D(2L) receptor. The second aim of this study was to investigate the molecular basis of the above differential G protein coupling specificity. The importance of a 12-amino acid sequence from the C-terminal end of the third intracellular loop of the D(2L) receptor in providing promiscuity in G protein coupling was demonstrated using a chimeric D(3)/D(2) receptor in which the equivalent region of the D(3) receptor was exchanged for this sequence. This chimera displayed D(3)-like affinity for [(3)H]spiperone and potency for agonists but gained D(2)-like ability to couple to each of Galpha(i1-3) as well as Galpha(o1).