High Constitutive Activity and a G-Protein-Independent High-Affinity State of the Human Histamine H4-Receptor

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• 作者：Erich H. Schneider ; David Schnell ; Dan Papa ; Roland Seifert
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：February 17, 2009
• 年：2009
• 卷：48
• 期：6
• 页码：1424-1438
• 全文大小：336K
• 年卷期：v.48,no.6(February 17, 2009)
• ISSN：1520-4995

文摘

The human histamine H₄-receptor (hH₄R) is expressed in mast cells and eosinophils and mediates histamine (HA)-induced chemotaxis via G_i-proteins. For a detailed investigation of hH₄R/G_i-protein interaction, we coexpressed the hH₄R with Gα_i2 and Gβ₁γ₂ as well as an hH₄R-Gα_i2 fusion protein with Gβ₁γ₂ in Sf9 insect cells. The agonist radioligand [³H]HA showed a K_D value of 10 nM at hH₄R and hH₄R-Gα_i2. The high-affinity states of hH₄R and hH₄R-Gα_i2 were insensitive to guanosine 5′-[γ-thio]triphosphate (GTPγS). The affinity of [³H]HA for hH₄R was retained in the absence of mammalian G_i-proteins. In steady-state GTPase- and [³⁵S]GTPγS-binding assays, hH₄R exhibited high constitutive activity and uncommon insensitivity to Na⁺. Thioperamide (THIO) was only a partial inverse agonist. Addition of HA or THIO to baculovirus-infected (hH₄R + Gα_i2 + Gβ₁γ₂) Sf9 cells increased the B_max in [³H]HA binding, but not in immunoblots, suggesting conformational instability and ligand-induced stabilization of membrane-integrated hH₄R. No effect was observed on hH₄R-Gα_i2 expression, neither in [³H]HA binding nor in immunoblot. However, the expression level of hH₄R-Gα_i2 was consistently higher compared to hH₄R, suggesting chaperone-like or stabilizing effects of Gα_i2 on hH₄R. In 37 °C stability assays, HA stabilized hH₄R, and THIO even restored misfolded [³H]HA binding sites. Inhibition of hH₄R glycosylation by tunicamycin reduced the [³H]HA binding B_max value. In conclusion, (i) hH₄R shows high constitutive activity and structural instability; (ii) hH₄R shows a G-protein-independent high-affinity state; (iii) hH₄R conformation is stabilized by agonists, inverse agonists and G-proteins; (iv) hH₄R glycosylation is essential for cell-surface expression of intact hH₄R.