Journal
CELL
Volume 169, Issue 2, Pages 338-349Publisher
CELL PRESS
DOI: 10.1016/j.cell.2017.03.028
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Funding
- Smith Family Foundation
- Vallee Foundation
- Esther A. and Joseph Klingenstein Fund
- Biogen Idec
- NIH [R01HL016037, 1DP5OD021345, K01DK098285]
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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, timeresolved 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 beta 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 beta 2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.
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