Received for publication November 22, 2005.
Revised April 13, 2006.
Accepted for publication April 27, 2006.

Oligomerization of recombinant and endogenously expressed human histamine H₄ receptors

Abstract

In this study we report the homo- and hetero-oligomerization of the human histamine H₄R (h H₄R) by both biochemical (Western blot and immobilized metal affinity chromatography) and biophysical (BRET and tr-FRET) techniques. The H₄R receptor is the most recent 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 HEK 293 and COS-7 cells heterologously overexpressing H4Rs, as well as putative native H₄R oligomers in human PHA blasts endogenously expressing H₄Rs. Moreover, we show that H₄R homo-oligomers are formed constitutively, are formed at low receptor densities (300 fmol/mg protein) and are present at the cell surface, as detected by tr-FRET. The formation of these oligomers is independent of N-glycosylation and not modulated by H₄R ligands, covering the full spectrum of agonists, neutral antagonists and inverse agonists. Whereas 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.

Key words: Histamine, Gi family, Fluorescence techniques, Receptor binding studies

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