Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.

Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking

“…We also used tetramethylrhodamine (TMR)-tagged conjugates of each ligand, with the fluorophore installed at position K12, previously shown to be well tolerated by exendin-4 (Clardy et al ., 2014; Jones et al ., 2018; Pickford et al ., 2020) and validated for GLP-1R binding by TR-FRET (Supplementary Figure 2B) and cAMP measurements (Supplementary Figure 2C), with the latter showing modest reductions in potency for both TMR-conjugates. Using the tetracycline-inducible T-REx system to modulate SNAP-GLP-1R expression (Fang, Chen, Manchanda, et al ., 2020), both fluorescent ligands were shown to bind specifically to the receptor and demonstrated extensive uptake into the endosomal compartment (Figure 2C).…”

The MafA-target gene PPP1R1A regulates GLP1R-mediated amplification of glucose-stimulated insulin secretion in β-cells

“…These mechanisms are critical for β-cell function and the target of several type 2 diabetes drugs that aim to improve β-cell function [13,14]. Unfortunately, up to 60% of patients are unresponsive to these treatments for not completely understood reasons resulting in an urgent need to better understand the intracellular signaling pathways used by this receptor [15][16][17][18].…”

The Interplay of Glucagon-Like Peptide-1 Receptor Trafficking and Signalling in Pancreatic Beta Cells

“…Post-endocytic GPCR trafficking occurs through a series of dynamically interconnected organelles crucial for the sorting of receptors to different intracellular compartments ( 86 ). The passage of GLP-1R through different beta cell endosomal compartments has been examined using electron microscopy ( 55 , 87 ). Sorting endosomes, also known as early endosomes (EE), are the first organelles receiving the endocytosed receptor-containing vesicles, and are typically marked by Rab5 ( 88 ).…”