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 [³⁵S]GTPγS assays). The human H₁R was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hH₁R and insect cell Gα_q. By contrast, functional expression of the hH₂R required the generation of an hH₂R-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hH₂R to the long (Gsα_L) or short (Gsα_S) splice variant of Gα_s resulted in comparable constitutive hH₂R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH₁R/hH₂R and their guinea pig orthologues gpH₁R/gpH₂R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH₃R with Gα_i1, Gα_i2, Gα_i3, and Gα_i/o 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 hH₃R state. Potential sodium binding sites in the hH₃R protein were analyzed by expressing specific hH₃R mutants in Sf9 cells. In contrast to the hH₃R, the hH₄R preferentially couples to co-expressed Gα_i2 in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hH₄R shows structural instability and adopts a G protein-independent high-affinity state. A detailed characterization of affinity and activity of a series of hH₄R antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hH₄R. 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 H_xR pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general.

Keywords: GPCRs; Histamine receptors; Radioligand binding; Sf9 insect cells; Steady-state GTPase assay; [35S]GTPγS binding.

Fig. 1

Preparation of baculoviruses for the expression of a GPCR (example for H₄R-Gα_i2 fusion protein). The gene of interest (in this case a FLAG-tagged hH₄R fused to Gα_i2 via a His₆ linker) is cloned into a pVl1392 transfer vector. The plasmid and the missing part of the baculovirus DNA (BaculoGold™ DNA) are co-transfected into Sf9 cells. The full baculovirus genome is completed in Sf9 cells by homologous recombination. The cells start to release virus particles into the surrounding medium

Fig. 2

Stimulation of Gα_i-proteins by the histamine H₄R and resulting G protein cycle. The numbers designate the different stages of the cycle and are explained in detail in Sect. 1.2.1

Fig. 3

Structure of a GPCR-Gα fusion protein. The GPCR is N-terminally tagged with a FLAG epitope, which allows detection by an anti-FLAG antibody, and connected to the N-terminus of a Gα-subunit via a His6 linker. Gα proteins are anchored in the plasma membrane via their acylation sites. The interaction between Gα_s proteins and the plasma membrane is only weak in co-expression systems, but can be significantly improved in GPCR-Gαs fusion proteins

Fig. 4

Two-state model of receptor activation and factors stabilizing the active (R*) and inactive (R) receptor conformation. Every GPCR population exists in an equilibrium of active and inactive receptor conformations. Full agonists produce a maximum shift towards the active side, while inverse agonists cause a maximum stabilization of the inactive GPCR conformation. Partial agonists and partial inverse agonists induce only an incomplete shift towards either side. Neutral antagonists bind to all receptor states with the same affinity and therefore do not change the equilibrium. G proteins stabilize the active conformation, while sodium ions usually uncouple GPCRs from their G proteins by shifting the equilibrium towards the inactive side. It should be noted that, despite its usefulness, the two-state model is very simplistic and does not account for the numerous distinct ligand- and G protein-specific receptor conformations occurring in reality. Adapted from Schneider and Seifert (2010a)

Fig. 5

Schematic depiction of the hH₄R-RGS fusion protein. The C-terminus of the hH₄R is fused to the N-terminus of the RGS protein by a hexahistidine linker. This brings the RGS protein into close proximity to the heterotrimeric G protein. Adapted from Schneider and Seifert (2010c)

Fig. 6

Scaffold structure of three classes of H₄R antagonists/inverse agonists. The numbers in brackets indicate the number of compounds tested