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PLoS One. 2015 May 28;10(5):e0129394. doi: 10.1371/journal.pone.0129394. eCollection 2015.

Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling.

Author information

1
Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America; Department of Advanced Clinical Science and Therapeutics, The University of Tokyo, Tokyo, Japan.
2
Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
3
Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
4
Department of Gastroenterology, Graduate Scholl of Medicine, The University of Tokyo, Tokyo, Japan.
5
Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Max Plank- The University of Tokyo Center for Integrative Inflammology, The University of Tokyo, Tokyo, Japan.
6
Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
7
Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
8
Department of Advanced Clinical Science and Therapeutics, The University of Tokyo, Tokyo, Japan.
9
Clinical Research Support Center, The University of Tokyo, Tokyo, Japan.
10
Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America; International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan.

Abstract

G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays.

PMID:
26020647
PMCID:
PMC4447269
DOI:
10.1371/journal.pone.0129394
[Indexed for MEDLINE]
Free PMC Article

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