TY - JOUR
T1 - Quantitative measurement of GPCR endocytosis via pulse-chase covalent labeling
AU - Kumagai, Hidetoshi
AU - Ikeda, Yuichi
AU - Motozawa, Yoshihiro
AU - Fujishiro, Mitsuhiro
AU - Okamura, Tomohisa
AU - Fujio, Keishi
AU - Okazaki, Hiroaki
AU - Nomura, Seitaro
AU - Takeda, Norifumi
AU - Harada, Mutsuo
AU - Toko, Haruhiro
AU - Takimoto, Eiki
AU - Akazawa, Hiroshi
AU - Morita, Hiroyuki
AU - Suzuki, Jun Ichi
AU - Yamazaki, Tsutomu
AU - Yamamoto, Kazuhiko
AU - Komuro, Issei
AU - Yanagisawa, Masashi
PY - 2015/5/28
Y1 - 2015/5/28
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pone.0129394
DO - 10.1371/journal.pone.0129394
M3 - Article
C2 - 26020647
AN - SCOPUS:84934917094
SN - 1932-6203
VL - 10
JO - PLoS One
JF - PLoS One
IS - 5
M1 - e0129394
ER -