Oligomerization of Recombinant and Endogenously Expressed Human Histamine H₄Receptors

Abstract

In this study, we report the homo-and hetero-oligomerization of the human histamine H 4 R 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 H 4 R 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 H 4 R, we demonstrate the presence of H 4 R oligomers in human embryonic kidney 293 and COS-7 cells heterologously overexpressing H 4 Rs and putative native H 4 R oligomers in human phytohaemagglutinin blasts endogenously expressing H 4 Rs. Moreover, we show that H 4 R 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 H 4 R ligands, covering the full spectrum of agonists, neutral antagonists, and inverse agonists. Although we show H 4 R homo-oligomer formation at physiological expression levels, the detection of H 1 R-H 4 R hetero-oligomers was achieved only at higher H 1 R expression levels and are most likely not physiologically relevant.

Histamine receptor expression in human renal tubules: a comparative pharmacological evaluation

“…The resultant conjugate was used to generate polyclonal antibodies in rabbits. The selectivity of the anti-hH 4 R antibody was confirmed by blockade with the C-terminal peptide of the H 4 R and a lack of cross-reactivity with the human H 3 R, the most closely related G-coupled receptor [21] …”

“…The final concentration of the organic solvent was less than 0.1%, which had no effect on cell viability. Antibodies H 3 R and H 4 R were detected by using the anti-hH 3 R (349-358) and the anti-hH 4 R (374-390) antibodies produced and validated for detecting both human and rodent H 3 R and H 4 R in the School of Biological and Biomedical Sciences, Durham University [21][22][23][24][25][26][27]. Briefly, anti-hH 3 R antibody was generated coupling the peptide corresponding to the amino acids RLSRDRKVAK Cys of the human and rat H 3 R to thyroglobulin by m-maleimidobenzoic acid N-hydroxysuccinimide ester coupling method as described previously [6] and antibodies were raised in rabbits to the resultant conjugate.…”

Overexpression of histamine H4 receptors in the kidney of diabetic rat

“…The selectivity of the anti-h H 4 receptor antibody was confirmed by blockade with the C-terminal peptide of the H 4 receptor and a lack of cross reactivity with the human H 3 receptor, the most related G-coupled receptor [26]. anti-rabbit polyclonal antibodies were from Molecular Probes (Leiden, the Netherlands).…”

The Four Major N- and C-Terminal Splice Variants of the Excitatory Amino Acid Transporter GLT-1 Form Cell Surface Homomeric and Heteromeric Assemblies

“…Pair-wise combinations of HAand FLAG-tagged GLT-1 isoforms were transfected into HEK-293 cells. As a positive control, cells cotransfected with HA-and FLAG-tagged histamine H 4 receptor subunits were used because the H 4 receptor has previously been shown to form multimers (van Rijn et al, 2006). Whole cells were suspended and subjected to tr-FRET using Eu 3ϩ -labeled anti-FLAG as the donor fluorophore and XL-665-labeled anti-HA as the acceptor fluorophore.…”