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

Project description

Following the passing of the baton in the race to stimulate insulin secretion

G-protein-coupled receptors (GPCRs) are a large and ubiquitous family of transmembrane proteins that mediate the majority of cellular responses to external stimuli. The glucagon-like peptide-1 receptor (GLP-1R) is one of these, an important target in the treatment of type 2 diabetes mellitus, whose prevalence has risen dramatically irrespective of income levels. Binding to its agonist causes GLP-1R to be internalised in the cell, leading to stimulation of insulin secretion. However, the mechanisms remain largely unknown. INTERGLP1 is developing cell lines to study GPCR signalling and GLP-1R internalisation. Making the 'tools' available to the scientific community should significantly speed the development of treatments for diabetes and a host of other GPCR-related conditions.

Objective

Glucagon-like peptide-1 is a key regulator of insulin and blood glucose release. The cognate receptor, the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR is a major drug target for type 2 diabetes. Agonist-induced internalization is a major regulatory mechanism for GPCR signalling. Interestingly, GLP-1R internalizes via a mechanism that is distinct from the canonical beta-arrestin mediated pathway. However, the details of the exact mechanism is largely conflicting and poorly understood for GPCRs in general. The present project aims to develop genetic and pharmacological tool box to dissecting the intracellular pathways leading to GLP-1R internalisation. To do this, I will generate a series of HEK293 cells where key proteins involved in a range of GPCR internalization pathways have been systematically removed by CRISPR/Cas9 genome editing. These cell lines will complement the already existing G protein alpha subunit and beta-arrestin knockout HEK293 cell lines to generate a highly efficient toolbox to study pathways and mechanisms involved in GPCR signalling, internalization and regulation. In the present project, I will utilise theses cell lines to dissect pathways leading to GLP-1R internalization and endosomal signalling. To understand the role of bias in the complex signalling profile of GPCR, I will also develop conformationally selective nanobodies using the yeast surface display platform. Importantly, we will make the cell lines and pharmacological tool compounds available to the scientific community enabling similar studies on a much wider range of GPCRs.

Coordinator

KOBENHAVNS UNIVERSITET
Net EU contribution
€ 207 312,00
Address
NORREGADE 10
1165 Kobenhavn
Denmark

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Region
Danmark Hovedstaden Byen København
Activity type
Higher or Secondary Education Establishments
Links
Total cost
€ 207 312,00