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

Informations projet

INTERGLP1

N° de convention de subvention: 846827

DOI

10.3030/846827

Projet clôturé

Date de signature de la CE 10 Avril 2019

Date de début 17 Février 2020

Date de fin 16 Février 2022

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 207 312,00

Contribution de l’UE

€ 207 312,00

Coordonné par

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 1 - INTERGLP1 (Dissecting GLP-1 receptor internalization pathways using genetic and pharmacological tools)

Période du rapport: 2020-02-17 au 2022-02-16

Résumé du contexte et des objectifs généraux du projet

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.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

WP1: In collaboration with other lab members, we generated HEK293 knockout cell lines using CRISPR/Cas9 gene editing. This method has been used successfully to make GRK2, GRK3, and GRK2/3 knockout cells, as well as novel PKC and conventional PKC knockout HEK293 cells. These cell lines have been validated to be specifically missing the protein(s) of interest, and are functionally not affected otherwise. The cell lines will be readily available to study other receptors in future projects. The results on the PKC knockout cell lines have been collected into a manuscript that is ready for submission.

WP2: Utilising a novel TR-FRET based assay recently developed by Cisbio and first published by the host laboratory in collaboration with Novo Nordisk, I managed to dissect the internalization pathways of GLP-1R and determined the contribution of individual components to this process. With the genome-edited HEK cell lines generated in WP1 and cell lines kindly gifted by Dr Asuka Inoue from Tohoku University, Japan, I was able to study the involvement of G proteins, arrestins, GRK and PKC. For other key internalisation components, I used dominant negative genes (caveolin, epsin and dynamin), knockdown with siRNA (clathrin and adaptors) and pharmacological inhibitors (clathrin, PKA, cholesterol). I was able to show that GLP1R agonist-induced internalisation is dependent on GRK and clathrin. Additionally, the clathrin adaptor may be able to bind directly to the receptor without the requirement of arrestins. In this WP, I also investigated the role of arrestins in GLP1R signalling and function. The work here is being prepared into an article for submission to a peer-reviewed journal.

WP3: In this WP, I intended to develop a complementary toolbox of bias nanobodies with the ability to stabilize specific GLP-1R conformation and potentially bias towards specific signaling pathways. We now have all the protocol, materials and equipment in place to generate nanobodies of the GLP1R using a yeast surface display platform. The resulting nanobodies will then be tested in pharmacological assays to investigate their signalling profile at the receptor.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

In this project, we managed to create cell lines that are devoid of GRK and PKC. The kinases, in addition to being involved in receptor regulation and endocytosis, are also involved in other key signalling processes making the cell lines valuable tools to study the role of these kinases. The cell lines are available for future projects and the wider scientific community. I also thoroughly investigated the internalisation mechanism of GLP1R and managed to uncover a unique internalisation mechanism that has never been reported before. This now allows for the individual components involved to be targeted specifically for the purpose of observing the pharmacology response when biased towards one pathway and aid in drug design. We now have a platform set up that can be used to study internalisation of most GPCRs and even other cell surface proteins. This is particularly relevant for receptors that have ambiguous internalisation mechanism, or have not been shown to follow the typical arrestin-dependent pathway. Finally, the discovery of conformationally selective nanobodies that are biased towards specific pathways will allow us to fully understand ligand-induced signaling bias and better understand the role of the receptor in health and disease.

Arrestin dependent and independent internalisation pathways