Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutants in the Sf9 Cell Expression System

Abstract

A large problem of histamine receptor research is data heterogeneity. Various experimental approaches, the complex signaling pathways of mammalian cells, and the use of different species orthologues render it difficult to compare and interpret the published results. Thus, the four human histamine receptor subtypes were analyzed side-by-side in the Sf9 insect cell expression system, using radioligand binding assays as well as functional readouts proximal to the receptor activation event (steady-state GTPase assays and [S]GTPγS assays). The human HR was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hHR and insect cell Gα. By contrast, functional expression of the hHR required the generation of an hHR-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hHR to the long (Gsα) or short (Gsα) splice variant of Gα resulted in comparable constitutive hHR activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hHR/hHR and their guinea pig orthologues gpHR/gpHR. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hHR with Gα, Gα, Gα, and Gα in Sf9 cells revealed high constitutive activity and comparable interaction efficiency with all G protein isoforms. A comparison of various cations (Li, Na, K) and anions (Cl, Br, I) revealed that anions with large radii most efficiently stabilize the inactive hHR state. Potential sodium binding sites in the hHR protein were analyzed by expressing specific hHR mutants in Sf9 cells. In contrast to the hHR, the hHR preferentially couples to co-expressed Gα in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hHR shows structural instability and adopts a G protein-independent high-affinity state. A detailed characterization of affinity and activity of a series of hHR antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hHR. In summary, the Sf9 cell system permitted a successful side-by-side comparison of all four human histamine receptor subtypes. This chapter summarizes the results of pharmacological as well as medicinal chemistry/molecular modeling approaches and demonstrates that these data are not only important for a deeper understanding of HR pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general.

TRPV1 SUMOylation suppresses itch by inhibiting TRPV1 interaction with H1 receptors

“…Further supporting the involvement of TRPV1 deSUMOylation in histamine-induced increase in H1R-TRPV1 association, histamine did not alter the association of H1R with the SUMOylationdeficient FLAG-rTRPV1 K822R (Figures 5D and S5D). In line with H1R signaling through Ga q/11 (Yamashita et al, 1991;Schneider and Seifert, 2017), subcutaneous injection of a specific inhibitor of Ga q/11 , YM-254890 (0.2 mg/kg) (Takasaki et al, 2004;Taniguchi et al, 2004), abolished the scratching responses of WT mice to histamine (Figure 5E). YM-254890 also significantly, but incompletely, reduced the scratching responses of TRPV1 K823R KI mice to histamine (Figure 5E), indicating that either there is a Ga q/11 -independent histamine-induced itch that occurs through the deSUMOylated TRPV1 or some residual Ga q/11 activity still remained under our experimental conditions and was able to exert an effect when the H1R-TRPV1 interaction was strong.…”

The Function of the Histamine H4 Receptor in Inflammatory and Inflammation-Associated Diseases of the Gut

“…As a consequence, calcium ([Ca 2+ ] i ) is mobilized from intracellular stores and the concentration of cytosolic cAMP is diminished. Independent of G protein-signaling, β-arrestin 2 is recruited to agonist-bound H 4 R, initiating mitogen-activated protein kinase (MAPK) cascade activation as well as receptor desensitization and internalization [ 84 , 85 , 86 , 87 ]. Activation of the H 4 R signaling pathways lead to induction of pro-inflammatory AP-1 in Th2 cells and monocyte-derived dendritic cells (MoDC) [ 27 ].…”

Mouse Colonic Epithelial Cells Functionally Express the Histamine H4 Receptor

“…Finally, based upon these data, the molecular mechanisms at the bases of the inflammatory effects of the H 4 R still have to be elucidated in an appropriate epithelial model system as has been done before e.g. for analysis of G-protein coupling in Sf9 cells (Schneider and Seifert, 2017).…”