INTERGLP1 · Dissecting GLP-1 receptor internalization pathways using genetic and pharmacological tools

Dissecting GLP-1 receptor internalization pathways using genetic and pharmacological tools

The importance of the incretin hormone glucagon-like peptide-1 (GLP-1) in regulating post-prandial blood glucose levels has been known for decades and has led to the development of stable GLP-1 analogs for the treatment of type 2 diabetes. On the pancreatic ß-cell, GLP-1 activates GLP-1 receptors (GLP-1R) to mediate insulin release after food intake. One major pathway controlling insulin release is the internalisation of GLP1R after activation. In collaboration with Novo Nordisk scientists, my host laboratory has used a state-of-the-art real-time internalization assay to show that the GLP-1R receptor is rapidly internalized and subsequently recycled to the cell surface. Yet, the mechanism of GLP-1R internalization remains unclear. For most G protein-coupled receptors, the process is commonly thought to be mediated via the canonical ß-arrestin dependent pathway, but it has also been shown that the GLP-1R can interact directly with AP2 (thus surpassing the ß-arrestins) and internalize via the distinct caveolae pathway, demonstrating the need for further studies. Internalization pathways have traditionally been studied using siRNA knockdown, co-expression of dominant negative mutants and/or use of pharmacological inhibitors. However, while they are important and widely used tools they have also suffered from caveats such as partial shutdown of pathways and/or limited selectivity. Highly efficient and selective genetic of the components of the different internalization pathways is thus highly warranted to dissect the pathway(s) employed by each receptor. Given the important therapeutic and physiological role of GLP-1R, the overall aim of the present application is to expand the pharmacological and genetic toolbox for GLP-1R receptor to dissect the mechanism of signalling and internalisation pathways. Specific objectives include: 1. Use CRISPR/Cas9 gene editing to create a HEK293 cell line tool box enabling dissection of GPCR internalization pathways 2. Employ the HEK293 cell lines to delineate pathway(s) mediating GLP-1R internalization and study cell membrane versus endosome mediated signaling 3. Develop GLP-1R bias nanobodies using the yeast platform to dissect G protein signaling and internalization. In conclusion, using a combination of genetic and pharmacological approaches, I was able to decipher the internalisation mechanism of GLP1R. The receptor internalises using an atypical mechanism, highly distinct from the canonical arrestin-dependent internalisation pathway. This highlights that not all GPCRs internalises in the same method and suggests that the internalisation method of other GPCRs should be thoroughly investigated to aid in drug discovery efforts.

Data: CORDIS, © European Union