title: Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling.
abstract: G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the β2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of β2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Jaeho
authors.initials: J
authors.lastname: Paek
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Marian
authors.initials: M
authors.lastname: Kalocsay
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA; Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Dean P
authors.initials: DP
authors.lastname: Staus
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA; Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Wingler
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Roberta
authors.initials: R
authors.lastname: Pascolutti
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Joao A
authors.initials: JA
authors.lastname: Paulo
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: andrew_kruse@hms.harvard.edu.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Kruse
mesh_terms: D060387:Ascorbate Peroxidases / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000032:analysis; D057809:HEK293 Cells; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D015202:Protein Engineering; D044140:Receptor, Angiotensin, Type 1 / Q000819:agonists / Q000032:analysis* / Q000378:metabolism*; D015398:Signal Transduction; D013194:Staining and Labeling / Q000379:methods*; D000071557:beta-Arrestins / Q000737:chemistry
chemical_list: D009842:Oligopeptides; D044140:Receptor, Angiotensin, Type 1; D000071557:beta-Arrestins; D001710:Biotin; D060387:Ascorbate Peroxidases; D019204:GTP-Binding Proteins; C555534:Sar-Arg-Val-Tyr-Ile-His-Pro-Ala-OH
keywords: APEX; G-protein-coupled receptor; GPCR; isobaric tagging; mass spectrometry; proximity labeling; signal transduction