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

The human histamine H-4-receptor (hH(4)R) is expressed in mast cells and eosinophils and mediates histamine (HA)-induced chemotaxis via G(i)-proteins. For a detailed investigation of hH(4)R/G(i)-protein interaction, we coexpressed the hH(4)R with G alpha(i2) and G beta(1)gamma(2) as well as an hH(4)R-G alpha(i2) fusion protein with G beta(1)gamma(2) in Sf9 insect cells. The agonist radioligand [H-3]HA showed a K-D value of similar to 10 nM at hH(4)R and hH(4)R-G alpha(i2). The high-affinity states of hH(4)R and hH(4)R-G alpha(i2) were insensitive to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). The affinity of [H-3]HA for hH(4)R was retained in the absence of mammalian G(i)-proteins. In steady-state GTPase- and [S-35]GTP gamma S-binding assays, hH(4)R exhibited high constitutive activity and uncommon insensitivity to Na+. Thioperamide (THTO) was only a partial inverse agonist. Addition of HA or THIO to baculovirus-infected (hH(4)R + G alpha(i2) + G beta(1)gamma(2)) Sf9 cells increased the B-max in [H-3]HA binding, but not in immunoblots, suggesting conformational instability and ligand-induced stabilization of membrane-integrated hH(4)R. No effect was observed on hH(4)R-G alpha(i2) expression, neither in [H-3]HA binding nor in immunoblot. However, the expression level of hH(4)R-G alpha(i2) was consistently higher compared to hH(4)R, suggesting chaperone-like or stabilizing effects of G alpha(i2) on hH(4)R. In 37 degrees C stability assays, HA stabilized hH(4)R, and THIO even restored misfolded [H-3]HA binding sites. Inhibition of hH(4)R glycosylation by tunicamycin reduced the [H-3]HA binding B-max value. In conclusion, (i) hH(4)R shows high constitutive activity and structural instability; (ii) hH(4)R shows a G-protein-independent high-affinity state; (iii) hH(4)R conformation is stabilized by agonists, inverse agonists and G-proteins; (iv) hH(4)R glycosylation is essential for cell-surface expression of intact hH(4)R.