Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

The H4R (histamine H-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 H4R is primarily expressed in eosinophils and mast cells and has the highest homology with the H4R. The Occurrence of at least twenty different hH(3)R (human H3R) isoforms led us to investigate the possible existence of H4R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H4R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H4R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H4R-ligand induced signalling or constitutive activity for these H4R splice variants. However, when co-expressed with full-length H4R [H4R(390) (H4R isoform of 390 amino acids)], the H4R splice variants have a dominant negative effect on the Surface expression of H4R(390). We detected H4R(390)-H4R splice variant-hetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical I time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H,R splice variants were detected ill various cell types and expressed at similar levels to the full-length H4R(390) mRNA in, for example, pre-monocytes. We conclude that the H,R splice variants described here have a dominant negative effect on H4R(390) functionality, as they are able to retain H4R(390) intracellularly and inactivate a population of H4R(390), presumably via hetero-oligomerization.