Constitutive Activity and Ligand Selectivity of Human, Guinea Pig, Rat, and Canine Histamine H₂Receptors

Abstract

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 s␣ , G s␣S , were expressed in Sf9 insect cells. In the membrane steady-state GTPase activity assay, cH 2 R-G s␣S but neither gpH 2 R-G s␣S nor rH 2 R-G s␣S showed the hallmarks of increased constitutive activity compared with hH 2 R-G s␣S , 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 s␣S , increased inverse agonist efficacies, and decreased potencies of antagonists. Furthermore, in membranes expressing nonfused H 2 Rs without or together with mammalian G s␣S or H 2 R-G s␣ 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 s␣S , gpH 2 R-G s␣S , and rH 2 R-G s␣S in the GTPase assay in contrast to being a weak partial agonist with decreased potency at cH 2 R-G s␣S . 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.The histamine H 2 receptor (H 2 R) species isoforms of canine (Gantz et al., 1991b), human (Gantz et al., 1991a), rat (Ruat et al., 1991), and guinea pig (Traiffort et al., 1995) were cloned. The four H 2 R 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).Despite this high degree of structural similarity, N-[3-(1H-imidazol-4-yl)propyl]guanidines such as compounds 8 to 10 (Fig. 2) differentially activate guinea pig (gpH 2 R) and human (hH 2 R) H 2 receptors. In a membrane steady-state GTPase activity assay using fusion proteins of H 2 R and the short splice variant of G s␣ , G s␣S , such H 2 R-selective agonists are considerably more potent and efficacious at gpH 2 R-G s␣S than at hH 2 R-G s␣S (Kelley et al., 2001). By contrast, the small H 2 R agonists histamine (1, HA), dimaprit (2, DIM), amthamine (3, AMT), and betahistine (4, BET) are unselective between these species. Recently, a novel class of N G -acylated imida- Article, publication date, and citation information can be fo...

Cell Adhesion Receptor GPR133 Couples to Gs Protein

“…Adenylyl Cyclase Pathway-According to the current model of GPCR function (25,26), receptor overexpression can result in a constitutive activation of signaling pathways, which are normally activated after agonist stimulation (27)(28)(29)(30). Thus, the coupling abilities of several receptors, including "orphan" receptors, have been characterized by overexpression in the absence of an agonist (31)(32)(33).…”

Histamine H4 receptor–RGS fusion proteins expressed in Sf9 insect cells: A sensitive and reliable approach for the functional characterization of histamine H4 receptor ligands

“…Steady-state GTPase assays were essentially performed as previously described [29], Due to the displacement of [-32 P]GTP from the G subunit, the signal-to-noise ratio of the GTPase signal is reduced by unlabeled GTP. Therefore, unlabeled GTP was not used at concentrations higher than 1.5 µM.…”

“…All fusion protein sequences were cloned into the baculovirus expression vector, pVL1392. Infection of the cells with baculoviruses was performed as previously desribed [29]. The virus stocks were combined as described in the Results section.…”

Comparison of the pharmacological properties of human and rat histamine H3-receptors

“…The H 4 -receptor possesses very high constitutive activity as well. Similar constitutive activity renders the system well suited for the analysis of species-specific ligand effects, since differences in constitutive activity between GPCRs can alter their pharmacological profiles and lead to a further complication of data interpretation [40,41].…”

“…At the H 2 R, bulky agonists like the long-chained impromidine-and arpromidine-derived guanidines or N G -acylated imidazolylpropylguanidines (AIPGs), are more potent and efficacious at the gpH 2 R than at the hH 2 R [37,45]. Metiamide was identified to be an inverse agonist at the hH 2 R, gpH 2 R and rH 2 R, but a weak partial agonist at the cH 2 R [40]. At the H 4 R, which has the lowest sequence similarity between species, studies focusing on ligand-receptor interactions of agonists are beginning to emerge [27,46].…”