Histamine H receptor-RGS fusion proteins expressed in Sf9 insect cells: A sensitive and reliable approach for the functional characterization of histamine H receptor ligands
Résumé
The human histamine H receptor (hHR), co-expressed with Gα and Gβγ 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 regulator of G-protein signaling (RGS) proteins RGS4 and RGS19 (GAIP) were investigated. RGS4 and GAIP were fused to the C-terminus of hHR or co-expressed with non-fused hHR, always combined with Gα and Gβγ. The non-fused RGS proteins did not significantly increase the relative effect of HA. With the hHR-RGS4 fusion protein the absolute GTPase activities, but not the relative HA-induced signal were increased. Fusion of hHR with GAIP caused a selective increase of the HA signal, resulting in an enhanced signal-to-noise ratio. A detailed characterization of the hHR-GAIP fusion protein (co-expressed with Gα and Gβγ) and a comparison with the data obtained for the non-fused hHR (co-expressed with Gα and Gβγ) led to the following results: (i) The relative agonist- and inverse agonist-induced signals at hHR-GAIP are markedly increased. (ii) Compared to the wild-type hHR, standard ligands show unaltered potencies and efficacies at hHR-GAIP. (iii) Like hHR, hHR-GAIP shows high and NaCl-resistant constitutive activity. (iv) hHR-GAIP shows the same G-protein selectivity profile as the non-fused hHR. Collectively, hHR-GAIP provides a sensitive test system for the characterization of hHR ligands and can replace the non-fused hHR in steady-state GTPase assays.
Origine : Fichiers produits par l'(les) auteur(s)