title: Specific cell surface labeling of GPCRs using split GFP.
abstract: Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two β-stands (β-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine β-strands (β-stands 1-9) that carries the mature fluorophore, and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications.
authors: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071, China.
authors: Wen-Xue
authors: WX
authors: Jiang
authors: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071, China.
authors: Xu
authors: X
authors: Dong
authors: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071, China.
authors: Jing
authors: J
authors: Jiang
authors: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071, China.
authors: Yu-Hong
authors: YH
authors: Yang
authors: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071, China.
authors: Ju
authors: J
authors: Yang
authors: Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China.
authors: Yun-Bi
authors: YB
authors: Lu
authors: Core Facilities of Zhejiang University Institute of Neuroscience, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China.
authors: San-Hua
authors: SH
authors: Fang
authors: Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China.
authors: Er-Qing
authors: EQ
authors: Wei
authors: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071, China.
authors: Chun
authors: C
authors: Tang
authors: Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China.
authors: Wei-Ping
authors: WP
authors: Zhang
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
chemical_list: C121670:GPR17 protein, human; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins