Signalling, trafficking and glucoregulatory properties of glucagon‐like peptide‐1 receptor agonists exendin‐4 and lixisenatide

Abstract

Background and Purpose Amino acid substitutions at the N‐termini of glucagon‐like peptide‐1 (GLP‐1) receptor agonist peptides result in distinct patterns of intracellular signalling, sub‐cellular trafficking and efficacy in vivo. Here, we to determine whether sequence differences at the ligand C‐termini of clinically approved GLP‐1 receptor agonists exendin‐4 and lixisenatide lead to similar phenomena. Experimental Approach Exendin‐4, lixisenatide and N‐terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time‐resolved FRET approaches were developed to study agonist behaviours at the cellular and sub‐cellular level. Anti‐hyperglycaemic and anorectic effects of each parent ligand and their biased derivatives were assessed in mice. Key Results Lixisenatide and exendin‐4 showed equal binding affinity, but lixisenatide was fivefold less potent for cAMP signalling. Both peptides induced extensive GLP‐1 receptor clustering in the plasma membrane and were rapidly endocytosed, but the GLP‐1 receptor recycled more slowly to the cell surface after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti‐hyperglycaemic and anorectic effects in mice with lixisenatide. N‐terminal substitution of His1 by Phe1 to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose. Conclusion and Implications Changes to the C‐terminus of exendin‐4 affect signalling potency and GLP‐1 receptor trafficking via mechanisms unrelated to GLP‐1 receptor occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.

Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signaling at glucagon family receptors

“…This is in keeping with the consensus that glucagon family receptors are primarily coupled to cAMP signalling via Gαs, with systemdependent engagement with other Gα sub-types under some circumstances (22,23). Moreover, LgBiT-β-arrestin-2 recruitment responses could be detected in all cases but were more transient than for mini-G proteins, matching the pattern seen with pharmacological GLP-1R agonists (24). Notable differences between receptor types included 1) substantially greater amplitude for mini-Gs recruitment for GLP-1R than for GCGR and GIPR, with the latter also showing slower kinetics (t1/2 = 7.1 ± 0.4 min versus 1.5 ± 0.2 min for GLP-1R, p<0.05 by unpaired t-test); 2) mini-Gi and mini-Gq responses were virtually undetectable for GIPR; and 3) markedly reduced recruitment of β-arrestin-2 to GIPR compared to GLP-1R and GCGR, in keeping with another report (25).…”

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

“…After a 30 min preincubation at room temperature, cells were moved to 4 • C before agonist addition. Each experiment included a range of concentrations of the fluorescent antagonist probe exendin (9-39)-FITC [12], along with a range of concentrations of unlabelled test agonists in competition with 10 nM exendin (9-39)-FITC. After a 24 h incubation at 4 • C to reach equilibrium binding, the plate was read by TR-FRET in a Flexstation 3 instrument using the following settings: λ ex = 340 nm, λ em = 490 nm (cut-off 475 nm) and 520 nm (cut-off 515 nm), delay 50 µs, integration 300 µs.…”

“…Whilst in vitro optimisation of GLP-1R ligands has traditionally focussed on high binding affinity or signalling potency, recent work has highlighted that the balance between recruitment of signal-initiating versus signal-terminating effectors can allow low-affinity "biased" GLP-1R agonists to achieve high therapeutic efficacy [5][6][7][8]. Connected to this, agonist-specific trafficking patterns of the GLP-1R between different endomembrane compartments allow fine-tuning of the duration and spatial origin of intracellular signalling responses [9][10][11][12].…”

Deuterated rhodamines for protein labelling in nanoscopy

“…We next turned to cellular labelling and imaging and aimed to determine the behavior of our dyes on targets that are mainly expressed on the membrane, as well as targets that are found intracellularly, both regions that have been addressed for SNAP-tag labelling. [3] First, we employed CHO-K1 cells stably expressing SNAP-tagged glucagon-like peptide 1 receptor (SNAP-GLP1R:CHO-K1) [12] , a cell line intensely used to study the physiology of this blockbuster class B G protein-coupled receptor (GPCR) involved in glucose homeostasis and targeted in diabetes treatment [13,14] , as a benchmark for d12 performances. As such, cells were labelled with 1 μM BG-TMR/SiR(-d12) for 30 min, before washing and live imaging by confocal microscopy, which revealed staining of SNAP-GLP1R with all deuterated and parental dyes tested ( Fig.…”