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 H-4 receptor (hH(4)R), co-expressed with G alpha(i2) and G beta(1 gamma 2) in Sf9 cells, is highly constitutively active. In the steady-state GTPase assay, the full agonist histamine (HA) induces only a relatively small signal (similar to 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 RCS19 (GAIP) were investigated. RGS4 and GAIP were fused to the C-terminus of hH(4)R or co-expressed with non-fused hH(4)R, always combined with G alpha(i2) and G beta(1 gamma 2). The non-fused RGS proteins did not significantly increase the relative effect of HA. With the hH(4)R-RGS4 fusion protein the absolute GTPase activities, but not the relative HA-induced signal were increased. Fusion of hH(4)R with GAIP caused a selective increase of the HA signal, resulting in an enhanced signal-to-noise ratio. A detailed characterization of the hH(4)R-GAIP fusion protein (co-expressed with G alpha(i2) and G(beta 1 gamma 2)) and a comparison with the data obtained for the non-fused hH(4)R (co-expressed with G alpha(i2) and G(beta 1 gamma 2)) led to the following results: (i) the relative agonist- and inverse agonist-induced signals at hH(4)R-GAIP are markedly increased. (ii) Compared to the wild-type hH(4)R, standard ligands show unaltered potencies and efficacies at hH(4)R-GAIP. (iii) Like hH(4)R, hH(4)R-GAIP shows high and NaCl-resistant constitutive activity. (iv) hH(4)R-GAIP shows the same G-protein selectivity profile as the non-fused hH(4)R. Collectively, hH(4)R-GAIP provides a sensitive test system for the characterization of hH(4)R ligands and can replace the non-fused hH(4)R in steady-state GTPase assays. (C) 2009 Elsevier Inc. All rights reserved.