The influence of peptide context on signalling and trafficking of glucagon-like peptide-1 receptor biased agonists

Abstract

Signal bias and membrane trafficking have recently emerged as important considerations in the therapeutic targeting of the glucagon-like peptide-1 receptor (GLP-1R) in type 2 diabetes and obesity. In the present study, we have evaluated a peptide series with varying sequence homology between native GLP-1 and exendin-4, the archetypal ligands on which approved GLP-1R agonists are based. We find notable differences in agonist-mediated signalling, endocytosis and recycling, dependent both on the introduction of a His → Phe switch at position 1 and the specific mid-peptide helical regions and C-termini of the two agonists. These observations were linked to insulin secretion in a beta cell model and provide insights into how ligand factors influence GLP-1R function at the cellular level.

Signal bias at glucagon family receptors: rationale and downstream impacts

“…Overall, these results highlight how the N-termini of each ligand play important roles in receptor activation. However, it should be noted that the magnitude of signal bias with the GLP-1 analogues tested here is smaller than for exendin-4-derived biased GLP-1R agonists, a finding that is consistent with our recent exploration of GLP-1/exendin-4 chimeric peptides [13].…”

“…This study builds on our earlier work using biased GLP-1R agonists derived from exendin-4 and GLP-1 bearing amino acid substitutions close to the N-terminus [12,13]. In the former study [12], we found that a number of changes, including dHis1, dTyr1, Phe1 and dGln3 (as included in the present investigation) led to marked reductions in recruitment of β-arrestin-1 and -2, substantially reduced GLP-1R endocytosis, increased insulin secretion, and improved anti-hyperglycaemic effect in mice.…”

“…The assay was adapted from a previous description [13]. HEK293T cells were seeded in black, clear bottom, plates coated with 0.1% poly-D-lysine and assayed 24 hours after transfection with plasmid DNA.…”

“…In contrast, GIPR internalisation with GIP was less extensive, and no change in DERET signal could be detected with GCGR stimulated by GCG (Figure 2A). Endocytic profiles were confirmed using an alternative approach based on reversible SNAP-tag labelling, in which the fluorescent probe BG-S-S-649 is cleaved from residual surface receptors after agonist-induced internalisation using the cellimpermeant reducing agent Mesna [13,36]. A time course study showed rapid and extensive loss of surface SNAP-GLP-1R when cells were treated with GLP-1, whereas SNAP-GIPR and SNAP-GCGR internalisation with their cognate agonists were, respectively, more limited and virtually absent ( Figure 2B).…”

“…A number of examples of signal bias at the GLP-1R have been described for naturally occurring [8,9] and pharmacological [10,11] orthosteric agonists. We recently described how biased GLP-1R agonists derived from exendin-4, characterised by reduced recruitment of β-arrestins, lead to increases in sustained insulin secretion through avoidance of GLP-1R desensitisation, reduction of GLP-1R endocytosis and resultant attenuation of GLP-1R downregulation over pharmacologically relevant time periods [12,13].…”