Marcel Hoffmann

Constitutive Activity and Structural Instability of the Wild‐Type Human H2 Receptor

Abstract: Stable expression of the human H2 receptor in Chinese hamster ovary cells resulted in an increase in basal cyclic AMP (cAMP) production, which was inhibited by the inverse agonists cimetidine, famotidine, and ranitidine with potencies similar to those found for the rat H2 receptor. Burimamide, a neutral antagonist at the rat H2 receptor, behaved as a weak partial agonist at the human H2 receptor. Burimamide competitively antagonized both the histamine‐induced increase in cAMP and the cimetidine‐induced reduction of the basal cAMP level with apparent KB values that were similar to its H2 receptor affinity. Investigation of the modulation of receptor expression after long‐term drug treatment revealed that at low concentrations histamine induced a significant reduction in H2 receptor expression, whereas at high concentrations receptor expression was slightly increased. The partial agonist burimamide induced, like inverse agonists, an upregulation of the human H2 receptor after prolonged treatment. These findings suggest a structural instability of the constitutively active human H2 receptor in transfected Chinese hamster ovary cells. Occupation of the H2 receptor by any ligand reduces the instability, thus resulting in higher cellular expression levels.

Molecular properties and signalling pathways of the histamine H1 receptor

Using a guinea pig model of allergic asthma, we investigated the effects of the inhaled, highly selective nonpeptide tachykinin NK1 and NK2 receptor antagonists SR 140333 and SR 48968, respectively, on allergen‐induced early (EAR) and late (LAR) asthmatic reactions, airway hyperreactivity (AHR) after these reactions, and infiltration of inflammatory cells in the airways. Both SR 140333 (100 nM, 3 min) and SR 48968 (100 nM, 3 min) had no effect on the severity of the EAR, while the NK2 receptor antagonist SR 48968, but not the NK1 receptor antagonist SR 140333, caused significant inhibition of the LAR. SR 140333 significantly reduced the allergen‐induced AHR to histamine, both after the EAR and the LAR. By contrast, SR 48968 did not affect the AHR after the EAR, but significantly attenuated the AHR after the LAR. Bronchoalveolar lavage studies performed after the LAR indicated that SR 140333 caused significant inhibition of allergen‐induced infiltration of eosinophils, neutrophils and lymphocytes, while SR 48968 attenuated the infiltration of neutrophils and lymphocytes, but not of eosinophils. Both NK receptor antagonists tended to reduce the accumulation of ciliated epithelial cells in the airways. These results indicate that NK1 and NK2 receptors are importantly, but differentially, involved in the development of allergen‐induced airways obstruction, AHR and infiltration of inflammatory cells in the airways. Therefore, both NK1 and NK2 receptor antagonists, or dual NK1 and NK2 antagonists, could be useful in the treatment of allergic asthma.

High antagonist potency of GT 2227 and GT-2331, new histamine H3 receptor antagonists, in two functional models.

GT-2227 (4-(6-cyclohexylhex-cis-3-enyl)imidazole) and GT-2331 ((1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole) were developed as new potent histamine H3 receptor antagonists. The functional activity of these ligands on the histamine H3 receptor-mediated inhibition of neurogenic contraction of the guinea-pig jejunum and histamine H3 receptor-mediated inhibition of norepinephrine release from guinea-pig heart synaptosomes were investigated. GT-2227 and GT-2331 both antagonized the inhibitory effects of (R)-alpha-methylhistamine on the contraction induced by electrical field stimulation in the guinea-pig jejunum with pA2 values of 7.9+/-0.1 and 8.5+/-0.03, respectively. In addition, GT-2227 and GT-2331 antagonized the inhibition of norepinephrine release in cardiac synaptosomes by GT-2203 ((1R,2R)-trans-2-(1H-imidazol-4-yl)cyclopropylamine), a histamine H3 receptor agonist. The current results demonstrate the antagonist activity for both GT-2227 and GT-2331 in two functional assays for histamine H3 receptors.

The C terminal tail of the histamine H2 receptor contains positive and negative signals important for signal transduction and receptor down-regulation.

Abstract: To examine the role of the C terminal tail in H2 receptor regulation, three cDNAs, encoding truncated histamine H2 receptor mutants (H2T295, H2T307, and H2T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H2 receptor binding of the H2T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H2T307) did not affect the binding properties of H2 antagonists and histamine or histamine‐induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H2T341), which was able to form a ternary complex but was unable to fully activate the Gs protein on histamine exposure. Agonist‐induced but not the cyclic AMP‐dependent H2 receptor down‐regulation was more profound for the H2T307 receptor, indicating that different structural elements of the H2 receptor protein are involved in the cyclic AMP‐dependent and independent pathways of H2 receptor down‐regulation. Taken together, in this study we identified regions in the C terminal tail of the H2 receptor that act as positive and/or negative signals in H2 receptor signaling and down‐regulation.

Recombinant Semliki Forest virus for over-expression and pharmacological characterisation of the histamine H(2) receptor in mammalian cells

We describe the use of recombinant Semliki Forest virus (SFV) vectors for efficient expression of the rat histamine H(2) (rH(2)) receptor in COS-7 (African green monkey kidney cells) cells. Recombinant SFV-infected COS-7 cells express the histamine rH(2) receptor in a time-dependent fashion with a maximum expression level of 50 pmol mg(-1) after 40 h. SFV-mediated histamine rH(2) receptor expression shows similar pharmacological properties as the receptor expressed transiently or stably in mammalian cells. In addition, we demonstrate the pharmacological and functional characterisation of the D(115)N mutated histamine rH(2) receptor. It has been shown that the D(115)N mutation renders the receptor constitutively active and structurally unstable. The rapid onset of and high maximal expression levels obtained from SFV-infected COS-7 cells enabled us to characterise this mutant receptor. We prove that recombinant SFV vectors are powerful tools for heterologous expression of G-protein-coupled receptors and that one can achieve both the high-level gene expression described for baculovirus-infected insect cells and the use of mammalian cells as hosts.

The human δ opioid receptor activates Gi1α more efficiently than Go1α: Opioid receptor/G protein interactions

To assess the relative capacity of the human δ opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the α‐subunits of either Gi1 or Go1, containing point mutations to render them insensitive to the actions of pertussis toxin, were linked in‐frame with the C‐terminus of the receptor. Following transient and stable expression in HEK 293 cells, both constructs bound the antagonist [3H]naltrindole with high affinity. d‐ala2,d‐leu5 Enkephalin effectively inhibited forskolin‐stimulated adenylyl cyclase activity in intact cells in a concentration‐dependent, but pertussis toxin‐insensitive, manner. The high‐affinity GTPase activity of both constructs was also stimulated by d‐ala2,d‐leu5 enkephalin with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to d‐ala2,d‐leu5 enkephalin was more than three times greater for Gi1α than for Go1α. As the effect of agonist in both cases was to increase Vmax without increasing the observed Km for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of Gi1α compared with Go1α. An equivalent fusion protein between the human µ opioid receptor‐1 and Gi1α produced a similar d‐ala2,d‐leu5 enkephalin‐induced GTP turnover number as the δ opioid receptor−Gi1α fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of Gi1α.

Molecular properties and signaling pathways of the histamine H1 receptor.

Using a guinea pig model of allergic asthma, we investigated the effects of the inhaled, highly selective nonpeptide tachykinin NK1 and NK2 receptor antagonists SR 140333 and SR 48968, respectively, on allergen‐induced early (EAR) and late (LAR) asthmatic reactions, airway hyperreactivity (AHR) after these reactions, and infiltration of inflammatory cells in the airways. Both SR 140333 (100 nM, 3 min) and SR 48968 (100 nM, 3 min) had no effect on the severity of the EAR, while the NK2 receptor antagonist SR 48968, but not the NK1 receptor antagonist SR 140333, caused significant inhibition of the LAR. SR 140333 significantly reduced the allergen‐induced AHR to histamine, both after the EAR and the LAR. By contrast, SR 48968 did not affect the AHR after the EAR, but significantly attenuated the AHR after the LAR. Bronchoalveolar lavage studies performed after the LAR indicated that SR 140333 caused significant inhibition of allergen‐induced infiltration of eosinophils, neutrophils and lymphocytes, while SR 48968 attenuated the infiltration of neutrophils and lymphocytes, but not of eosinophils. Both NK receptor antagonists tended to reduce the accumulation of ciliated epithelial cells in the airways. These results indicate that NK1 and NK2 receptors are importantly, but differentially, involved in the development of allergen‐induced airways obstruction, AHR and infiltration of inflammatory cells in the airways. Therefore, both NK1 and NK2 receptor antagonists, or dual NK1 and NK2 antagonists, could be useful in the treatment of allergic asthma.

Methods to determine the constitutive activity of histamine H2 receptors

Publisher Summary The histamine H 2 receptor belongs to the large family of G protein-coupled receptors (GPCRs) and is positively coupled to adenylate cyclase. This chapter discusses a variety of experimental approaches that can be used to determine the constitutive activity of the H 2 receptor or other G s -coupled receptors. With the aid of different heterologous expression techniques, constitutive activity of the H2 receptor is detected easily in a wide variety of cell lines. Especially at high levels of receptor expression, the increase in basal CAMP production can be observed easily (either direct or via a reporter gene assay). Although modulation of GPCR expression is an important factor in the detection of constitutive signaling, the level of GPCR expression is also an important determinant for the extent of the constitutive responsiveness. The expression levels of the various partners in the GPCR signaling pathway also determine the final agonist-independent output. For the G q -coupled muscarinic m3 receptor, overexpression of G αq increases the basal activation of signal transduction pathways.

Molecular properties and signalling pathways of the histamine H1 receptor: Molecular properties and signalling pathways

With cloning of the gene encoding the histamine H1 receptor, a new area of histamine research has become reality. Finally, it seems feasible to study the target of the thera‐peutically important clans of antihistamine. Expression of the genes in mammalian cells allows detailed investigations of the various signal transduction routes of the histamine H1 receptor. Moreover, using molecular biological techniques, it is now possible to investigate ligand receptor interaction at the molecular level. Studies with mutant H1 receptors have shown that H1 antagonists bind to a specific amino acid residues in TM3 and 5. It is expected that these new developments will provide much fundamental knowledge on the ligand interaction with the H1 receptor.

Biochemical properties of the histamine H3 receptor

Publisher Summary This chapter discusses biochemical properties of the histamine H 3 receptor and potential strategies that may end in the cloning of the H 3 receptor cDNA. From radioligand binding studies in which guanyl-nucleotids have been shown to inhibit the binding of H 3 agonists to the H 3 receptor, the interaction with a G-protein is suggested in the chapter. The data from various binding studies are a strong indication for the involvement of G-proteins interacting with the Ha receptor. More evidence for the involvement of G-proteins is given in the chapter. It also discusses the cloning of histamine receptors genes. The cloned receptor cDNA encodes for a protein of 491 amino acids with all the characteristics of a typical G-proteins coupled receptor (GPCR), as there are 7 putative transmembrane domains, N-terminal glycosylation sites, and phosphorylation sites for protein kinase A and C. The cloning of gene encoding the H 3 receptor provides detailed insights on the molecular architecture, signal transduction, and putative receptor subtypes.