title: Large-scale purification and characterization of human parathyroid hormone-1 receptor stably expressed in HEK293S GnTI- cells.
abstract: Human parathyroid hormone-1 receptor (hPTHR1) belongs to class II of the G protein-coupled receptor (GPCR) family, whose members all contain a seven-transmembrane helix domain. The receptor regulates bone metabolism through interactions with its ligand, human parathyroid hormone (hPTH). For structural studies of the hPTHR1/hPTH complex, we constructed a mammalian cell line to stably express recombinant hPTHR1 in large-scale. The receptor was solubilized with dodecyl maltoside and purified with affinity chromatography. The purified receptor displayed restricted N-glycosylation as expected. Functionality was demonstrated: the hPTHR1 retained affinity for bPTH-(1-34) and specifically cross-linked to a radioiodinated bPTH-(1-34) analog. This work describes an approach for preparing milligram-scale quantities of receptor for elucidation of the structural biology of this seven-transmembrane GPCR.
authors.affiliation: Department of Physiology, Tufts University, School of Medicine, 136 Harrison Avenue, Boston, MA 02111-1800, USA.
authors.forename: Lu
authors.initials: L
authors.lastname: Gan
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Alexander
authors.forename: Angela
authors.initials: A
authors.lastname: Wittelsberger
authors.forename: Beena
authors.initials: B
authors.lastname: Thomas
authors.forename: Michael
authors.initials: M
authors.lastname: Rosenblatt
mesh_terms: D019149:Bioreactors; D002460:Cell Line; D049109:Cell Proliferation; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: C473449:PTH1R protein, human; D044168:Receptor, Parathyroid Hormone, Type 1; D011994:Recombinant Proteins