Specific cell surface labeling of GPCRs using split GFP.
Sci Rep
; 6: 20568, 2016 Feb 09.
Article
em En
| MEDLINE
| ID: mdl-26857153
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 ß-stands (ß-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 ß-strands (ß-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.
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Base de dados:
MEDLINE
Assunto principal:
Coloração e Rotulagem
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Proteínas Recombinantes de Fusão
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Engenharia de Proteínas
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Receptores Acoplados a Proteínas G
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Proteínas de Fluorescência Verde
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article