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

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.