Abstract

In this study, we report the homo- and hetero-oligomerization of the human histamine H₄R by both biochemical (Western blot and immobilized metal affinity chromatography) and biophysical [bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer (tr-FRET)] techniques. The H₄R receptor is the most recently discovered member of the histamine family of G-protein-coupled receptors. Using specific polyclonal antibodies raised against the C-terminal tail of the H₄R, we demonstrate the presence of H₄R oligomers in human embryonic kidney 293 and COS-7 cells heterologously overexpressing H₄Rs and putative native H₄R oligomers in human phytohaemagglutinin blasts endogenously expressing H₄Rs. Moreover, we show that H₄R homo-oligomers are formed constitutively, are formed at low receptor densities (300 fmol/mg of protein), and are present at the cell surface, as detected by tr-FRET. The formation of these oligomers is independent of N-glycosylation and is not modulated by H₄R ligands, covering the full spectrum of agonists, neutral antagonists, and inverse agonists. Although we show H₄R homo-oligomer formation at physiological expression levels, the detection of H₁R-H₄R hetero-oligomers was achieved only at higher H₁R expression levels and are most likely not physiologically relevant.