Ligand-dependent desensitisation and re-sensitisation of the GLP-1R

J Lu1, GB Willars2. 1University of Leicester, Leicester, UK, 2University of Leicester, Leicester, UK

Following nutrient ingestion, glucagon-like peptide-1 (GLP-1) is secreted from intestinal L-cells to mediate anti-diabetic effects, most notably enhancing glucose-dependent insulin release from pancreatic β-cells but also inhibiting glucagon release, promoting satiety and weight reduction and potentially enhancing or preserving β-cell mass. These effects are mediated through the GLP-1 receptor (GLP-1R), a Family B GPCR which is a target for the treatment of type 2 diabetes. Although GLP-1 analogues with extended plasma half-lives (eg. exendin-4) and orally active small molecule ligands have been investigated particularly in relation to their signalling properties, little is known regarding any potential differences in, or consequences of, altered receptor trafficking. We have shown previously that following desensitisation of the GLP-1R recombinantly expressed in HEK293 cells (HEK-GLP-1R) by GLP-1 7-36 amide, full re-sensitisation requires receptor recycling and the activity of endothelin-converting enzyme-1 (ECE-1), most likely within endosomes (Lu et al, 2013). Here we compared desensitisation and re-sensitisation of the GLP-1R following treatment with either GLP-1 7-36 amide, exendin-4 or the small-molecule ago-allosteric ligand, compound 2, and examined the role of ECE-1 in the re-sensitisation process.

Using a plate-reader to determine changes in fluorescence of fluo-4-loaded HEK-GLP-1R cells as an index of changes in the intracellular [Ca2+] we showed that both GLP-1 7-36 amide and exendin-4 increased intracellular [Ca2+] in a concentration-dependent manner (pEC50 9.63±0.24 and 10.41±0.03 (all data are mean±S.E.M., n>3) for GLP-1 7-36 amide and exendin-4 respectively). Pre-treatment with either 10 nM (Emax) GLP-1 7-36 amide or 1 nM (Emax) exendin-4 for 10 min induced desensitisation to subsequent stimulation with Emax concentrations of the same agonist. Following GLP-1 7-36 amide, Ca2+ responses fully re-sensitised within 3 h and this was significantly (p<0.05, Bonferroni’s test) inhibited by the ECE-1 inhibitor, SM19712 (10 µM). In contrast, there was little or no re-sensitisation following 3 h recovery in cells pre-challenged with exendin-4. Following 6 h recovery there was some re-sensitisation (50% of the response in untreated cells) but this was unaffected by inhibition of ECE-1. No re-sensitisation was apparent at 3 h following pre-treatment with higher concentrations of compound 2 (>10 μM). Following pre-treatment with a lower concentration of compound 2 (1 μM), responses recovered by 3 h but this was insensitive to inhibition of ECE-1.

Consistent with our earlier study (Lu et al, 2013), re-sensitisation of GLP-1R-mediated Ca2+ responses shows dependence on ECE-1 activity, which may degrade the ligand in the endosome and allow receptor recycling. Here we show that this is not true of other agonists, both peptide and small molecule ligands suggesting that GLP-1R trafficking might be ligand-dependent. Different ligands for GLP-1R may not only show bias for receptor activation (Wootten et al., 2013) but also trigger different pathways of receptor trafficking and this could be important for both re-sensitisation processes and signalling, particularly that mediated by scaffolding around β-arrestin.

(1) Lu J et al, pA2 Online 10:089P, 2012

(2) Lu J et al, Acta Pharmacologica Sinica 34:Suppl. S8.29, 2013

(3) Wootten D et al, Mol Pharmacol 83:822, 2013