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

ended

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.