Metadata about: 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 [(35)S]GTPγS assays). The human H(1)R was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hH(1)R and insect cell Gα(q). By contrast, functional expression of the hH(2)R required the generation of an hH(2)R-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hH(2)R to the long (Gsα(L)) or short (Gsα(S)) splice variant of Gα(s) resulted in comparable constitutive hH(2)R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH(1)R/hH(2)R and their guinea pig orthologues gpH(1)R/gpH(2)R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH(3)R 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 hH(3)R state. Potential sodium binding sites in the hH(3)R protein were analyzed by expressing specific hH(3)R mutants in Sf9 cells. In contrast to the hH(3)R, the hH(4)R preferentially couples to co-expressed Gα(i2) in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hH(4)R shows structural instability and adopts a G protein-independent high-affinity state. A detailed characterization of affinity and activity of a series of hH(4)R antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hH(4)R. 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(x)R pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general.

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