Engineered mini-G proteins block the internalization of cognate GPCRs and disrupt downstream intracellular signaling.
Sci Signal
; 17(843): eabq7038, 2024 07 02.
Article
em En
| MEDLINE
| ID: mdl-38954638
ABSTRACT
Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein-coupled receptors (GPCRs) in their active conformations. Because of their small size and ease of use, they are popular tools for assessing GPCR behaviors in cells, both as reporters of receptor coupling to Gα subtypes and for cellular assays to quantify compartmentalized signaling at various subcellular locations. Here, we report that overexpression of mini-G proteins with their cognate GPCRs disrupted GPCR endocytic trafficking and associated intracellular signaling. In cells expressing the Gαs-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-Gs, a mini-G protein derived from Gαs, blocked ß-arrestin 2 recruitment and receptor internalization and disrupted endosomal GLP-1R signaling. These effects did not involve changes in receptor phosphorylation or lipid nanodomain segregation. Moreover, we found that mini-G proteins derived from Gαi and Gαq also inhibited the internalization of GPCRs that couple to them. Finally, we developed an alternative intracellular signaling assay for GLP-1R using a nanobody specific for active GαsGPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Engenharia de Proteínas
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Transdução de Sinais
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Receptores Acoplados a Proteínas G
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Receptor do Peptídeo Semelhante ao Glucagon 1
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article