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Cell, Volume 169, Issue 2, 6 April 2017, Pages 338-349.e11 | http://doi.org/10.1016/j.cell.2017.03.028
Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling
Jaeho Paek1, 5, Marian Kalocsay2, 5, Dean P. Staus3, 4, Laura Wingler3, 4, Roberta Pascolutti1, Joao A. Paulo2, Steven P. Gygi2, Andrew C. Kruse1, 6
1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
2 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
3 Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
4 Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA
5 Co-first author
6 Lead Contact
Corresponding author : Andrew C. Kruse
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.
논문정보
- Research article
- 2017년 04월 (BRIC 등록일 2017-04-13)
- 국외 연구진
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