Spatiotemporal GLP-1 and GIP receptor signaling and trafficking/recycling dynamics induced by selected receptor mono- and dual-agonists

Acylation of the Incretin Peptide Exendin-4 Directly Impacts Glucagon-Like Peptide-1 Receptor Signaling and Trafficking

“…Using a nanoluciferase tag at the GLP-1R C-terminus, we were also able to obtain BRET measurements of GLP-1R redistribution to early, late and recycling endosomes that corroborate the TR-FRET responses. This approach has been used recently to study the trafficking profiles of GLP-1R mono-agonists and dual GLP-1R/GIPR co-agonists (Fletcher et al, 2018;Novikoff et al, 2021), albeit using Renilla luciferase rather than the nanoluciferase we used in our study. The rank order of ligandinduced changes was consistent for "matched" TR-FRET and BRET approaches, with exendin-4-C16 showing reduced GLP-1R internalisation and lysosomal accumulation, but faster recycling.…”

Evaluation of efficacy- versus affinity-driven agonism with biased GLP-1R ligands P5 and exendin-F1

“…Recent preclinical studies have shown that GLP-1R agonists that favour G protein signalling and generation of cyclic adenosine monophosphate (cAMP) over β-arrestin recruitment are particularly effective at reducing blood glucose levels [8] , [9] , [10] , [11] , [12] . Moreover, it is suggested that the markedly reduced β-arrestin recruitment seen at the GLP-1R with the dual GLP-1R/glucose-dependent insulinotropic peptide receptor (GIPR) agonist Tirzepatide [13] , [14] , [15] may contribute to its superior anti-diabetic efficacy in clinical trials [16] . An appealing explanation for the effects of these “biased” GLP-1R agonists is that reductions in β-arrestin-mediated desensitisation, as well as a trafficking phenotype that favours preservation of GLP-1R at the plasma membrane, lead to prolonged intracellular signalling and cumulatively greater insulin release over time.…”

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

“…As mini-G proteins are being used to detect the presence of activated GPCRs at different subcellular compartments, with the interrogation of endocytic signalling being a common application (Crilly et al, 2021;Jimenez-Vargas et al, 2020;Wan et al, 2018), there is clear potential for any mini-G-mediated alteration of receptor trafficking responses to confound these measurements. In view of the present observations, we recommend exhaustive checks on specific receptor internalisation rates if a mini-G is to be utilised in any intracellular signalling assay, and to consider changing to other non-mini-G based bystander methods including the use of nanobodies against G protein:active GPCR pairs, amongst other possibilities, such as, for example, the use of fulllength G protein fusion constructs (Novikoff et al, 2021), or strategies to inhibit GPCR internalisation (Sposini et al, 2017).…”