title: Studying the regulation of endosomal cAMP production in GPCR signaling.
abstract: We describe methods based on live cell fluorescent microscopy and mass spectrometry to characterize the mechanism of endosomal cAMP production and its regulation using the parathyroid hormone (PTH) type 1 receptor as a prime example. These methods permit to measure rapid changes of cAMP levels in response to PTH, kinetics of endosomal ligand-receptor interaction, pH changes associated with receptor trafficking, and to identify the endosomal receptor interactome.
authors.affiliation: Molecular Mechanisms of Mycobacterial Infection, Center for Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Gidon
authors.affiliation: Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Timothy N
authors.initials: TN
authors.lastname: Feinstein
authors.affiliation: Laboratory for GPCR Biology, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Kunhong
authors.initials: K
authors.lastname: Xiao
authors.affiliation: Laboratory for GPCR Biology, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Vilardaga
mesh_terms: D000595:Amino Acid Sequence; D000242:Cyclic AMP / Q000096:biosynthesis*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D021381:Protein Transport; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry / Q000378:metabolism*; D015290:Second Messenger Systems
chemical_list: D044168:Receptor, Parathyroid Hormone, Type 1; D000242:Cyclic AMP
keywords: Arrestin; Endosomal GPCR proteomics; Endosomal GPCR signaling; FRET; GPCR trafficking; PTH receptor; Receptor dynamics; TIRF