Constitutive Activation and Trafficking of the Glucagon-like Peptide-1 Receptor Using Tethered Pharmacology

Presentation Number: SUN 610
Date of Presentation: April 2nd, 2017

Tom Podewin1, Johannes Broichhagen1, Nicholas H.F. Fine2, Daniela Nasteska2, Philipp Leippe1, Teresa Buenaventura3, Nisha Kanda3, Sarah Janik1, Alejandra Tomas3, Dirk Trauner1, Anja Hoffmann-Röder1 and David Jonathan Hodson*2
1LMU Munich, Munich, Germany, 2University of Birmingham, Birmingham, United Kingdom, 3Imperial College London, London, United Kingdom


In response to ligand binding, the glucagon-like peptide-1 receptor (GLP-1R) induces 3'-5'-cyclic adenosine monophosphate (cAMP) synthesis, intracellular Ca2+ fluxes and insulin secretion (1; 2). These processes are thought to be regulated by post-endocytotic receptor trafficking (3). However, GLP-1R signaling may continue following receptor internalization. How this influences GLP-1R function is poorly understood. Therefore, we sought to develop a tethered agonist for the SNAP-tag-directed activation and trafficking of the GLP-1R (4; 5). To do this, Exenatide (Byetta®) was derivatized to produce ExONatide in which the C-terminal Ser39 is substituted with Cys39, before attachment of a PEG4-linked benzyl-guanine (BG) bioconjugation handle via a disulfide bridge. ExONatide was able to covalently bind the SNAP-tag moiety via its BG, as shown by a competitive reduction in TMR-Star labeling in AD293-SNAP-GLP-1R cells. While the EC50 for cAMP generation was right-shifted compared to Exendin4(1-39) (19 pm vs. 16.2 nm), maximal responses were almost 100%, implying full agonism. ExONatide induced long-lasting cAMP and Ca2+ rises in MIN6B1-SNAP-GLP-1R, which persisted even following washout (P<0.01 before and after washout versus control). Similar results were seen for incretin-stimulated insulin secretion (P<0.01 before and after washout versus control). ExONatide internalized the GLP-1R (P<0.01 at 3 hrs), but cell surface expression was not restored using the specific antagonist Exendin4(9-39). In all cases, the effects of ExONatide could be reversed following application of beta-mercaptoethanol, a disulfide-cleaving cell-permeable reducing agent (P<0.01 versus control). Thus, ExONatide provides a template for the design and production of agonists for the constitutive yet reversible activation of receptor proteins bearing an N-terminal fused SNAP-tag. This or similar compounds may be useful for understanding GLP-1R signaling both in vitro and in vivo, with broad applicability to other GPCRs.


Nothing to Disclose: TP, JB, NHFF, DN, PL, TB, NK, SJ, AT, DT, AH, DJH