title: An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells.
abstract: Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.
authors.affiliation: Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Braden T
authors.initials: BT
authors.lastname: Lobingier
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA.
authors.forename: Ruth
authors.initials: R
authors.lastname: Hüttenhain
authors.affiliation: Program in Biochemistry and Molecular Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Kelsie
authors.initials: K
authors.lastname: Eichel
authors.affiliation: Thermo Fisher Scientific, San Jose, CA 95134, USA.
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Miller
authors.affiliation: Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Ting
authors.affiliation: Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: mark.vonzastrow@ucsf.edu.
authors.forename: Mark
authors.initials: M
authors.lastname: von Zastrow
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA. Electronic address: nevan.krogan@ucsf.edu.
authors.forename: Nevan J
authors.initials: NJ
authors.lastname: Krogan
mesh_terms: D000818:Animals; D060387:Ascorbate Peroxidases / Q000737:chemistry*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D060066:Protein Interaction Maps; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011957:Receptors, Opioid / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D043562:Receptors, G-Protein-Coupled; D011957:Receptors, Opioid; D025801:Ubiquitin; D060387:Ascorbate Peroxidases
keywords: APEX; GPCR; adrenergic receptor; mass spectrometry; opioid receptor; proximity labeling