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

The human histamine H4 receptor (hH4R), co-expressed with Gαi2 and Gβ1γ2 in Sf9 cells, is highly constitutively active. In the steady-state GTPase assay, the full agonist histamine (HA) induces only a relatively small signal (∼20–30%), resulting in a low signal-to background ratio. In order to improve this system for ligand screening purposes, the effects of the regulators of G-protein signaling (RGS) RGS4 and RGS19 (GAIP) were investigated. RGS4 and GAIP were fused to the C-terminus of hH4R or co-expressed with non-fused hH4R, always combined with Gαi2 and Gβ1γ2. The non-fused RGS proteins did not significantly increase the relative effect of HA. With the hH4R–RGS4 fusion protein the absolute GTPase activities, but not the relative HA-induced signal were increased. Fusion of hH4R with GAIP caused a selective increase of the HA signal, resulting in an enhanced signal-to-noise ratio. A detailed characterization of the hH4R–GAIP fusion protein (co-expressed with Gαi2 and Gβ1γ2) and a comparison with the data obtained for the non-fused hH4R (co-expressed with Gαi2 and Gβ1γ2) led to the following results: (i) the relative agonist- and inverse agonist-induced signals at hH4R–GAIP are markedly increased. (ii) Compared to the wild-type hH4R, standard ligands show unaltered potencies and efficacies at hH4R–GAIP. (iii) Like hH4R, hH4R–GAIP shows high and NaCl-resistant constitutive activity. (iv) hH4R–GAIP shows the same G-protein selectivity profile as the non-fused hH4R. Collectively, hH4R–GAIP provides a sensitive test system for the characterization of hH4R ligands and can replace the non-fused hH4R in steady-state GTPase assays.

When co-expressed with Gi-proteins in Sf9 cells, the histamine H4 receptor shows only a low signal-to-background ratio. This ratio can be markedly enhanced by fusing the RGS protein GAIP to the receptor.Figure optionsDownload as PowerPoint slide