Schneider, Erich H. and Seifert, Roland (2009) 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. BIOCHEMICAL PHARMACOLOGY, 78 (6). pp. 607-616. ISSN 0006-2952,
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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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | G-ACYLATED IMIDAZOLYLPROPYLGUANIDINES; HIGH CONSTITUTIVE ACTIVITY; PHARMACOLOGICAL CHARACTERIZATION; MOLECULAR ANALYSIS; HUMAN EOSINOPHILS; CLONING; ACTIVATION; REGULATORS; STATE; G(Q)-PROTEINS; Histamine H-4 receptor; Fusion protein; RGS protein; Steady-state GTPase assay; Constitutive activity; G(i)-proteins |
| Subjects: | 600 Technology > 615 Pharmacy |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy > Pharmacology and Toxicology (Prof. Schlossmann, formerly Prof. Seifert) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 07 Sep 2020 06:39 |
| Last Modified: | 07 Sep 2020 06:39 |
| URI: | https://pred.uni-regensburg.de/id/eprint/28395 |
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