Histamine H(4) receptor-RGS fusion proteins expressed in Sf9 insect cells: a sensitive and reliable approach for the functional characterization of histamine H(4) receptor ligands.
The human histamine H(4) receptor (hH(4)R), co-expressed with Galpha(i2) and Gbeta(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 (approximately 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 hH(4)R or co-expressed with non-fused hH(4)R, always combined with Galpha(i2) and Gbeta(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 Galpha(i2) and Gbeta(1)gamma(2)) and a comparison with the data obtained for the non-fused hH(4)R (co-expressed with Galpha(i2) and Gbeta(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.
Schneider
Erich H
EH
University of Regensburg, Department of Pharmacology and Toxicology, Regensburg, Germany. erich.schneider@chemie.uni-regensburg.de
Seifert
Roland
R
D000818:Animals; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D007313:Insecta / Q000166:cytology; D008024:Ligands; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000502:physiology*; D011968:Receptors, Histamine / Q000502:physiology*; D000074040:Receptors, Histamine H4; D011993:Recombinant Fusion Proteins; D018411:Spodoptera / Q000166:cytology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014162:Transfection
C418607:HRH4 protein, human; D008024:Ligands; D020710:RGS Proteins; D043562:Receptors, G-Protein-Coupled; D011968:Receptors, Histamine; D000074040:Receptors, Histamine H4; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D006632:Histamine; D019204:GTP-Binding Proteins