title: High constitutive activity and a G-protein-independent high-affinity state of the human histamine H(4)-receptor.
abstract: 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.
authors.affiliation: Department of Pharmacology and Toxicology, University of Regensburg, Universitatsstrasse 31, D-93040 Regensburg, Germany. erich.schneider@chemie.uni-regensburg.de
authors.forename: Erich H
authors.initials: EH
authors.lastname: Schneider
authors.forename: David
authors.initials: D
authors.lastname: Schnell
authors.forename: Dan
authors.initials: D
authors.lastname: Papa
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D000818:Animals; D002460:Cell Line; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007313:Insecta; D007700:Kinetics; D010880:Piperidines / Q000494:pharmacology; D055550:Protein Stability / Q000187:drug effects; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011968:Receptors, Histamine / Q000378:metabolism*; D000074040:Receptors, Histamine H4; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: C418607:HRH4 protein, human; D010880:Piperidines; D043562:Receptors, G-Protein-Coupled; D011968:Receptors, Histamine; D000074040:Receptors, Histamine H4; D011993:Recombinant Fusion Proteins; D014415:Tunicamycin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012965:Sodium Chloride; D006632:Histamine; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C052075:thioperamide