Dataset Information

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 ?2 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 ?2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.

SUBMITTER: Paek J

PROVIDER: S-EPMC5514552 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling.

Paek Jaeho J Kalocsay Marian M Staus Dean P DP Wingler Laura L Pascolutti Roberta R Paulo Joao A JA Gygi Steven P SP Kruse Andrew C AC

Cell 20170401 2

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 ...[more]

PMID: 28388415

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Project description:Proximity labeling can be defined as an enzymatic "in-cell" chemical reaction that catalyzes the proximity-dependent modification of biomolecules in live cells. Since the modified proteins can be isolated and identified via mass spectrometry, this method has been successfully utilized for the characterization of local proteomes such as the sub-mitochondrial proteome and the proteome at membrane contact sites, or spatiotemporal interactome information in live cells, which are not "accessible" via conventional methods. Currently, proximity labeling techniques can be applied not only for local proteome mapping but also for profiling local RNA and DNA, in addition to showing great potential for elucidating spatial cell-cell interaction networks in live animal models. We believe that proximity labeling has emerged as an essential tool in "spatiomics," that is, for the extraction of spatially distributed biological information in a cell or organism.Proximity labeling is a multidisciplinary chemical technique. For a decade, we and other groups have engineered it for multiple applications based on the modulation of enzyme chemistry, chemical probe design, and mass analysis techniques that enable superior mapping results. The technique has been adopted in biology and chemistry. This "in-cell" reaction has been widely adopted by biologists who modified it into an in vivo reaction in animal models. In our laboratory, we conducted in vivo proximity labeling reactions in mouse models and could successfully obtain the liver-specific secretome and muscle-specific mitochondrial matrix proteome. We expect that proximity reaction can further contribute to revealing tissue-specific localized molecular information in live animal models.Simultaneously, chemists have also adopted the concept and employed chemical "photocatalysts" as artificial enzymes to develop new proximity labeling reactions. Under light activation, photocatalysts can convert the precursor molecules to the reactive species via electron transfer or energy transfer and the reactive molecules can react with proximal biomolecules within a definite lifetime in an aqueous solution. To identify the modified biomolecules by proximity labeling, the modified biomolecules should be enriched after lysis and sequenced using sequencing tools. In this analysis step, the direct detection of modified residue(s) on the modified proteins or nucleic acids can be the proof of their labeling event by proximal enzymes or catalysts in the cell. In this Account, we introduce the basic concept of proximity labeling and the multidirectional advances in the development of this method. We believe that this Account may facilitate further utilization and modification of the method in both biological and chemical research communities, thereby revealing unknown spatially distributed molecular or cellular information or spatiome.

Dataset Information

Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling.

Publications

Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling.

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