Oligomerization of recombinant and endogenously expressed human histamine H(4) receptors.
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.
van Rijn
Richard M
RM
Leiden/Amsterdam Center for Drug Research, Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Chazot
Paul L
PL
Shenton
Fiona C
FC
Sansuk
Kamonchanok
K
Bakker
Remko A
RA
Leurs
Rob
R
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019281:Dimerization; D006031:Glycosylation; D017442:Histamine Agonists / Q000494:pharmacology; D006801:Humans; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000502:physiology; D011968:Receptors, Histamine / Q000737:chemistry* / Q000502:physiology; D011969:Receptors, Histamine H1 / Q000737:chemistry; D000074040:Receptors, Histamine H4; D011994:Recombinant Proteins / Q000737:chemistry
C418607:HRH4 protein, human; D017442:Histamine Agonists; D043562:Receptors, G-Protein-Coupled; D011968:Receptors, Histamine; D011969:Receptors, Histamine H1; D000074040:Receptors, Histamine H4; D011994:Recombinant Proteins