Cloning and characterization of dominant negative splice variants of the human histamine H sub(4) receptor
The H sub(4)R (histamine H sub(4) receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H sub(4)R is primarily expressed in eosinophils and mast cells and ha...
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Veröffentlicht in: | Biochemical journal 2008-01, Vol.414 (1), p.121-131 |
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Sprache: | eng |
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Zusammenfassung: | The H sub(4)R (histamine H sub(4) receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H sub(4)R is primarily expressed in eosinophils and mast cells and has the highest homology with the H sub(3)R. The occurrence of at least twenty different hH sub(3)R (human H sub(3)R) isoforms led us to investigate the possible existence of H sub(4)R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H sub(4)R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H sub(4)R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H sub(4)R-ligand induced signalling or constitutive activity for these H sub(4)R splice variants. However, when co-expressed with full-length H sub(4)R [H sub(4)R sub((390)) (H sub(4)R isoform of 390 amino acids)], the H sub(4)R splice variants have a dominant negative effect on the surface expression of H sub(4)R sub((390)). We detected H sub(4)R sub((390))-H sub(4)R splice varianthetero- oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H sub(4)R splice variants were detected in various cell types and expressed at similar levels to the full- length H sub(4)R sub((390)) mRNA in, for example, pre-monocytes. We conclude that the H sub(4)R splice variants described here have a dominant negative effect on H sub(4)R sub((390)) functionality, as they are able to retain H sub(4)R sub((390)) intracellularly and inactivate a population of H sub(4)R sub((390)), presumably via hetero-oligomerization. |
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ISSN: | 0264-6021 1470-8728 |