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

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitisation and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to promote albumin binding in order to prolong their pharmacokinetics, but the potential for these ligand changes to influence GLP-1R signalling has rarely been investigated beyond potency assessments for cyclic adenosine monophosphate (cAMP). In this work we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analogue, exendin-4-C16, for their relative propensities to recruit and activate G proteins and β-arrestins, endocytic and post-endocytic trafficking profiles, and interactions with model and cellular membranes. Both ligands had similar cAMP potency but the exendin-4-C16 showed ~2.5-fold bias towards G protein recruitment and a ~60% reduction in β-arrestin-2 recruitment efficacy compared to exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting towards recycling pathways. These effects were associated with a reduced ability to promote the movement of the GLP-1R extracellular domain, as determined using a conformational biosensor approach, and a ~70% increase in insulin secretion. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Overall, our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology.

Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

“…With this in mind, we performed recruitment experiments with mini-Gs-NLuc co-expressed with either the plasma membrane KRAS-Venus or the endosomal Rab5-Venus reporters in HEK293 cells stably expressing SNAP-GLP-1R (Figure 1A and B). Similarly to our previous observations (Lucey et al, 2021), whilst we could clearly detect mini-Gs plasma membrane recruitment following GLP-1 stimulation, the pattern of recruitment was sustained and did not subside over time, as would be expected from the known kinetics as well as our own examination of GLP-1R internalisation times (Figure 1C). This effect correlated with an almost negligible detection of mini-Gs recruitment to the early endosomal compartment (Figure 1A and B).…”

“…This project stems from an initial observation of strikingly sustained mini-Gs retention at the plasma membrane after GLP-1R stimulation, which appears incompatible with the previously known fast internalisation kinetics of this receptor (Shaaban et al, 2016). Specifically, in GLP-1R-expressing HEK293 cells, we have previously observed that NanoLuc-tagged mini-Gs remains associated with the plasma membrane after an initial agonist-mediated recruitment phase, contrasting with the rapid disappearance of NanoLuc-tagged GLP-1R from the cell surface measured in parallel in the same cell system, but without mini-Gs co-expression (Lucey et al, 2021).…”

Receptor Activity-Modifying Protein 2 (RAMP2) alters glucagon receptor trafficking in hepatocytes with functional effects on receptor signalling

“…Here, we co-express a plasma membrane (CAAX) or endosomal (Endofin) marker fused to the large BiT (LgBiT) subunit of the Nanoluc luciferase together with nanobody-37 (Nb37), a single domain antibody which binds specifically to nucleotide-free Gα s in complex with active receptors [ 35 ], fused to a complementary small subunit (SmBiT) of Nanoluc. Under this configuration, when the two nanoluciferase subunits are closely apposed, a quantitative luminescent signal is generated [ 36 ], indicating the presence of active Gα s in endosomal or plasma membrane compartments (see Figure 3 G for a schematic of the assay). In HEK293T cells in the absence of GCG stimulation, we observed a trend towards diminished basal levels of activation at the plasma membrane in the presence of RAMP2 (p = 0.08) but not at the endosomal compartment ( Supplementary Figure 3C,D ), in keeping with our previous finding of reduced surface GCGR levels in basal conditions.…”

Hepatocyte cholesterol content modulates glucagon receptor signalling

“…INS-1 cells (Buenaventura et al, 2019) 10 mM MβCD: ↓ exendin-4-induced cAMP efficacy and endocytosis HEK293 cells (Lucey et al, 2021) 10 mM MβCD: ↓ exendin-4-C16-induced cAMP potency and endocytosis Glucagon-like peptide 2 receptor (GLP-2R) DLD-1 and BHK cells (Estall et There are various mechanisms by which cholesterol and other lipids may alter the stability, ligand binding properties, and thus function of GPCRs (Reiter et al, 2017): direct competition with agonist binding at the orthosteric site; directly binding at an allosteric site to modulate receptor conformation and dynamics (Abiko et al, 2021); indirectly via a change in local membrane composition and properties; or a combination of the above (Guixà-González et al, 2017). Recent work has demonstrated that GCGR function can be affected by endogenous allosteric modulators (Han et al, 2015;McGlone et al, 2021).…”