Details

Autor(en) / Beteiligte

• Paek, Jaeho
• Kalocsay, Marian
• Staus, Dean P.
• Wingler, Laura
• Pascolutti, Roberta
• Paulo, Joao A.
• Gygi, Steven P.
• Kruse, Andrew C.

Titel

Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling

Ist Teil von

• Cell, 2017-04, Vol.169 (2), p.338-349.e11

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2017

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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. [Display omitted] •GPCR agonist response tracked by time-resolved labeling of receptor-proximal proteins•GPCRs co-localize with a variety of G proteins even before activation•G proteins are largely separated from receptors upon receptor endocytosis•In-cell time-resolved labeling technique is generalizable to multiple receptor types Using APEX proximity labeling to monitor GPCR signaling provides both spatial and temporal insight into receptor signaling and internalization in response to both balanced and biased GPCR ligands.