Quantitative measurement of GPCR endocytosis via pulse-chase covalent labeling

Hidetoshi Kumagai, Yuichi Ikeda, Yoshihiro Motozawa, Mitsuhiro Fujishiro, Tomohisa Okamura, Keishi Fujio, Hiroaki Okazaki, Seitaro Nomura, Norifumi Takeda, Mutsuo Harada, Haruhiro Toko, Eiki Takimoto, Hiroshi Akazawa, Hiroyuki Morita, Jun Ichi Suzuki, Tsutomu Yamazaki, Kazuhiko Yamamoto, Issei Komuro, Masashi Yanagisawa

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


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.

Original languageEnglish (US)
Article numbere0129394
JournalPloS one
Issue number5
StatePublished - May 28 2015

ASJC Scopus subject areas

  • General


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