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 Galpha(i2) and Gbeta(1)gamma(2) as well as an hH(4)R-Galpha(i2) fusion protein with Gbeta(1)gamma(2) in Sf9 insect cells. The agonist radioligand [(3)H]HA showed a K(D) value of approximately 10 nM at hH(4)R and hH(4)R-Galpha(i2). The high-affinity states of hH(4)R and hH(4)R-Galpha(i2) were insensitive to guanosine 5'-[gamma-thio]triphosphate (GTPgammaS). The affinity of [(3)H]HA for hH(4)R was retained in the absence of mammalian G(i)-proteins. In steady-state GTPase- and [(35)S]GTPgammaS-binding assays, hH(4)R 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 (hH(4)R + Galpha(i2) + Gbeta(1)gamma(2)) Sf9 cells increased the B(max) in [(3)H]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-Galpha(i2) expression, neither in [(3)H]HA binding nor in immunoblot. However, the expression level of hH(4)R-Galpha(i2) was consistently higher compared to hH(4)R, suggesting chaperone-like or stabilizing effects of Galpha(i2) on hH(4)R. In 37 degrees C stability assays, HA stabilized hH(4)R, and THIO even restored misfolded [(3)H]HA binding sites. Inhibition of hH(4)R glycosylation by tunicamycin reduced the [(3)H]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.