Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

Abstract

Mini-G proteins are engineered thermostable variants of Gα subunits designed to specifically stabilise G protein-coupled receptors (GPCRs) in their active conformation for structural analyses. Due to their smaller size and ease of use, they have become popular tools in recent years to assess specific GPCR behaviours in cells, both as reporters of receptor coupling to each G protein subtype and for in-cell assays designed to quantify compartmentalised receptor signalling from a range of subcellular locations. Here, we describe a previously unappreciated consequence of the co-expression of mini-G proteins with their cognate GPCRs, namely a profound disruption in GPCR trafficking and intracellular signalling caused by the co-expression of the specific mini-G subtype coupled to the affected receptor. We studied the Gαs-coupled pancreatic beta cell class B GPCR glucagon-like peptide-1 receptor (GLP-1R) as a model to describe in detail the molecular consequences derived from this effect, including a complete halt in β-arrestin-2 recruitment and receptor internalisation, despite near-normal levels of receptor GRK2 recruitment and lipid nanodomain segregation, as well as the disruption of endosomal GLP-1R signalling by mini-Gs co-expression. We also extend our analysis to a range of other prototypical GPCRs covering the spectrum of Gα subtype coupling preferences, to unveil a widely conserved phenomenon of GPCR internalisation blockage by specific mini-G proteins coupled to a particular receptor. Our results have important implications for the design of methods to assess intracellular GPCR signalling. We also present an alternative adapted bystander intracellular signalling assay for the GLP-1R in which we substitute the mini-Gs by a nanobody, Nb37, with specificity for active Gαs:GPCR complexes and no deleterious effect on the capacity for GLP-1R internalisation.

In vitro assays for the functional characterization of (psychedelic) substances at the serotonin receptor 5‐HT2AR

“…However, it needs to be taken into account that the miniGα q protein is engineered and may not always represent the physiological situation, despite extensive validation. Indeed, recent research has uncovered that GPCR trafficking and signaling properties may be altered by the expression of miniG proteins (Carpenter & Tate, 2016; Manchanda, 2021; Nehme et al, 2017; Wan et al, 2018). Furthermore, the addition of tags, such as the (fluorescent/luminescent) protein(s) (fragments), can theoretically influence the properties of a protein.…”

An examination of the divergent spatiotemporal signaling of GLP-1R versus GIPR in pancreatic beta cells

“…We also employed a different approach to quantify plasma membrane and endosomal activity from both incretin receptors based on a NanoBRET assay to measure the recruitment of NanoLuc-fused mini-Gs proteins to either KRAS- or Rab5-Venus constructs (Supplementary Figure 7C). We have recently shown that co-expression of mini-Gs with a cognate GPCR results in the inhibition of the normal level of receptor internalization in response to agonist stimulation [28]. For this reason, we were only able to perform this assay by using supra-physiological levels (100 nM) of GLP-1 and GIP and we could not obtain concentration response curves.…”

Investigating G-protein coupled receptor signalling with light-emitting biosensors

“…In addition to a BRET pair, the mini-G assay can be adapted to use the NanoLuc complementation assay NanoBiT ( Section 3.3 ) by fusing LgBiT to the mini-G protein and SmBiT to the receptor of interest ( Benkel et al, 2022 ). Although confocal microscopy and BRET spectroscopy have demonstrated mini-G recruitment to GPCRs at internal membranes such as the Golgi and endosomes ( Wan et al, 2018 ), mini-G protein binding to a GPCR have been reported to abolish β-arr recruitment and subsequent receptor internalisation ( Manchanda et al, 2021 ), which, if corroborated, would make them unsuitable to study internalised receptor signalling or biased agonism.…”