The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism

Abstract

SummaryLigand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach

“…The selected WT CLR was simulated as previously described (Wootten et al, 2016a;Wootten et al, 2016b) using all-atom molecular dynamics as implemented in ACEMD (Harvey et al, 2009). The receptor was embedded in a POPC bilayer and solvated with TIP3 water at anionic strength of 0.15 M. The whole system was energy minimised and followed by a 160 ps heating protocol from 0 K to 300 K in the NVT ensemble.…”

“…Similarly, the basic residue at the start of ECL2 R/K 4.64b is also important for ligand binding and signal transduction in a number of family B GPCRs (Gkountelias et al, 2009;Koole et al, 2012;. However, it is important to stress that the role of ECL2 is receptor-, ligand-and pathway-specific (Coin et al, 2013;Gkountelias et al, 2009;Koole et al, 2012;Siu et al, 2013;Wootten et al, 2016b). Thus the absolutely conserved tryptophan in the centre of ECL2 has distinct features in CLR compared to the glucagon receptor.…”

“…This has been shown through biochemical techniques and has been supported by the many crystal structures of GPCRs bound to both agonists and antagonists. The importance of ECL2 in family B GPCRs has been shown with ECL2 alanine scans of a number of receptors, including the calcitonin gene related peptide (CGRP) receptor, glucagon-like peptide-1 receptor (GLP-1R) and corticotropin-releasing factor receptor 1 (CRFR1) (Gkountelias et al, 2009;Koole et al, 2012;Wootten et al, 2016b). CGRP is a potent vasodilatory peptide that has been implicated in migraine, but it is also cardioprotective and is important in the establishment of hypertension .…”

“…The existence of distinct ECL2 structures in the two GCGR structures (4L6R and 5EE7) demonstrates the flexibility of ECL2 at its C-terminal end, perhaps meaning that it can adopt different conformations when binding distinct agonists. Interestingly, mutations in ECL2 at the GLP-1 receptor have been suggested to differentially alter ligand bias (Wootten et al, 2016b).…”

Characterization of signal bias at the GLP-1 receptor induced by backbone modification of GLP-1

“…Binding of GLP-1 to the extracellular surface of the GLP-1R promotes recruitment of several G proteins, including Gαs, Gαq, Gαi and Gαo, 7–9 as well as β-arrestin 1 and β-arrestin 2, to the cytoplasmic surface of the receptor. 9, 10 While Gs stimulation is principally linked to activation of adenylate cyclase and cAMP formation, the canonical driver of GLP-1-stimulated insulin secretion, 6 Gs, Gq and Gi/o proteins can each lead to mobilization of intracellular calcium and/or ERK1/2 phosphorylation, in a ligand- and cell-type-specific manner.…”