Exploring the Structure, Function & Regulation of the Human Glucagon-Like Peptide-1 Receptor

Glucagon-like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion and promotes β-cell function via its receptor (GLP-1R), which therefore is a validated target for the treatment of type 2 diabetes. Due to difficulties with peptide therapeutics, it is important to find small-molecule GLP-1R agonists. This leads to a need to understand the structure, function and regulation of the receptor, particularly, differences between agonisms mediated by GLP-1 (orthosteric agonist) and small molecules. The GLP-1R contains a putative N-terminal signal peptide sequence, which is assessed here by recombinantly expressing several epitope-tagged GLP-1R constructs in HEK293 cells. The findings demonstrate that the GLP-1R is expressed predominately at the plasma membrane and also slightly cytosolic. Only fully glycosylated, mature form of the receptor is able to traffic to the cell surface and performs the function. The signal peptide sequence of the GLP-1R is essential for synthesis. After fulfilling the function, this sequence is cleaved and thus not part of the mature protein. The cleavage of signal peptide is critical for processing and trafficking of the GLP-1R. Based on one of these constructs generated here, a cell line (HEK293: GLP-1R-EGFP) with stable expression of the visible GLP-1R is established, which allows observations and determinations for ligand-mediated receptor internalisation in real time. Compound 2 (6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline) has been described as a GLP-1R allosteric modulator and agonist. Findings here that compound 2-mediated agonisms on both the wild-type (WT) GLP-1R and the mutant with removal of the N-terminal domain provide direct evidence for the allosteric agonism. Interestingly, compound 2-mediated cAMP response is enhanced by orthosteric antagonist exendin 9-39, but the latter inhibits receptor internalisation mediated by compound 2. Recently, it has been hypothesised that the binding of GLP-1 allows a sequence of NRTFD (Asn63-Asp67) in the N-terminus of the GLP-1R to interact with another part of the receptor and cause agonism. This was examined here by generating receptor mutants and synthetic peptides. Findings here that Asp67 plays a key role in stabilising the N-terminal structure of the GLP-1R and thus is critical for processing and trafficking of the receptor protein do not support such hypothesis although synthetic NRTFD mediates a weak and partial agonism on the WTGLP-1R.