title: Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling.
abstract: G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays.
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America; Department of Advanced Clinical Science and Therapeutics, The University of Tokyo, Tokyo, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Kumagai
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Ikeda
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Motozawa
authors.affiliation: Department of Gastroenterology, Graduate Scholl of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Fujishiro
authors.affiliation: Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Max Plank- The University of Tokyo Center for Integrative Inflammology, The University of Tokyo, Tokyo, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Okamura
authors.affiliation: Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Keishi
authors.initials: K
authors.lastname: Fujio
authors.affiliation: Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Okazaki
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Seitaro
authors.initials: S
authors.lastname: Nomura
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Norifumi
authors.initials: N
authors.lastname: Takeda
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Mutsuo
authors.initials: M
authors.lastname: Harada
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Haruhiro
authors.initials: H
authors.lastname: Toko
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Eiki
authors.initials: E
authors.lastname: Takimoto
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Akazawa
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Morita
authors.affiliation: Department of Advanced Clinical Science and Therapeutics, The University of Tokyo, Tokyo, Japan.
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Suzuki
authors.affiliation: Clinical Research Support Center, The University of Tokyo, Tokyo, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Issei
authors.initials: I
authors.lastname: Komuro
authors.affiliation: Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America; International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Yanagisawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay; D001710:Biotin / Q000737:chemistry; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003412:Cricetulus; D004705:Endocytosis / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D064446:Orexin Receptors / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering; D021381:Protein Transport; D018004:Receptors, Bombesin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013194:Staining and Labeling / Q000379:methods; D014161:Transduction, Genetic
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; C578626:HCRTR2 protein, human; D008024:Ligands; D064446:Orexin Receptors; D018004:Receptors, Bombesin; D011993:Recombinant Fusion Proteins; C107120:bombesin receptor subtype 3; D001710:Biotin; D006867:Hydrolases