Engineered mini-G proteins block the internalization of cognate GPCRs and disrupt downstream intracellular signaling.

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 Gα-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-G, a mini-G protein derived from Gα, blocked β-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 Gα and Gα 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 Gα:GPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling.