title: GPCR proteomics: mass spectrometric and functional analysis of histamine H1 receptor after baculovirus-driven and in vitro cell free expression.
abstract: The human histamine H1 Receptor (hH1R) belongs to the family of G-protein coupled receptors (GPCRs), an attractive and proven class of drug targets in a wide range of therapeutic areas. However, due to the low amount of available purified protein and the hydrophobic nature of GPCRs, limited structural information is available on ligand-receptor interaction especially for the transmembrane (TM) domain regions where the majority of ligand-receptor interactions occur. During the last decades, proteomic techniques have increasingly become an important tool to reveal detailed information on the individual GPCR class, including post-translational modifications and characterizations of GPCRs binding pocket. Herein, we report the successful functional production and mass spectrometric characterization of the hH1R, after baculovirus-driven and in vitro cell-free expression. Using only MALDI-ToF, sequence coverage of more than 80%, including five hydrophobic TM domains was achieved. Moreover, we have identified an asparagine residue in the hH1R protein that is subject to N-linked glycosylation. This information would be valuable for drug discovery efforts by allowing us to further study H1R-ligand interactions using histaminergic ligands that covalently bind the hH1R, and eventually revealing binding sites of hH1R and other GPCRs.
authors.affiliation: Leiden/Amsterdam Center for Drug Research (LACDR), Vrije Universiteit Amsterdam, Department of Medicinal Chemistry, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands.
authors.forename: Kamonchanok
authors.initials: K
authors.lastname: Sansuk
authors.forename: Crina I A
authors.initials: CI
authors.lastname: Balog
authors.forename: Anne M
authors.initials: AM
authors.lastname: van der Does
authors.forename: Raymond
authors.initials: R
authors.lastname: Booth
authors.forename: Willem J
authors.initials: WJ
authors.lastname: de Grip
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Remko A
authors.initials: RA
authors.lastname: Bakker
authors.forename: Rob
authors.initials: R
authors.lastname: Leurs
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hensbergen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000502:physiology*; D002474:Cell-Free System; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods; D011969:Receptors, Histamine H1 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera
chemical_list: D011969:Receptors, Histamine H1