Cassandra Koole

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation

Background: The ECL2 of family B GPCRs has been suggested to contribute to biological activity. Results: Mutation of most ECL2 residues to alanine results in changes in binding and/or efficacy of GLP-1 peptide agonists. Conclusion: The ECL2 of the GLP-1R is critical for GLP-1 peptide-mediated receptor activation and selective signaling. Significance: This work reveals broad significance for ECL2 in maintaining receptor conformations driving selective signaling. The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1–36)-NH2 or GLP-1(7–36)-NH2 binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca2+ (Ca2+i) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1–36)-NH2- and GLP-1(7–36)-NH2-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7–36)-NH2-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.

Polymorphism and Ligand Dependent Changes in Human Glucagon-Like Peptide-1 Receptor (GLP-1R) Function: Allosteric Rescue of Loss of Function Mutation

The glucagon-like peptide-1 receptor (GLP-1R) is a key physiological regulator of insulin secretion and a major therapeutic target for the treatment of type II diabetes. However, regulation of GLP-1R function is complex with multiple endogenous peptides that interact with the receptor, including full-length (1–37) and truncated (7–37) forms of GLP-1 that can exist in an amidated form (GLP-1(1–36)NH2 and GLP-1(7–36)NH2) and the related peptide oxyntomodulin. In addition, the GLP-1R possesses exogenous agonists, including exendin-4, and the allosteric modulator, compound 2 (6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline). The complexity of this ligand-receptor system is further increased by the presence of several single nucleotide polymorphisms (SNPs) that are distributed across the receptor. We have investigated 10 GLP-1R SNPs, which were characterized in three physiologically relevant signaling pathways (cAMP accumulation, extracellular signal-regulated kinase 1/2 phosphorylation, and intracellular Ca2+ mobilization); ligand binding and cell surface receptor expression were also determined. We demonstrate both ligand- and pathway-specific effects for multiple SNPs, with the most dramatic effect observed for the Met149 receptor variant. At the Met149 variant, there was selective loss of peptide-induced responses across all pathways examined, but preservation of response to the small molecule compound 2. In contrast, at the Cys333 variant, peptide responses were preserved but there was attenuated response to compound 2. Strikingly, the loss of peptide function at the Met149 receptor variant could be allosterically rescued by compound 2, providing proof-of-principle evidence that allosteric drugs could be used to treat patients with this loss of function variant.

Differential impact of amino acid substitutions on critical residues of the human glucagon-like peptide-1 receptor involved in peptide activity and small-molecule allosterys

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein–coupled receptor that has a critical role in the regulation of glucose homeostasis, principally through the regulation of insulin secretion. The receptor system is highly complex, able to be activated by both endogenous [GLP-1(1–36)NH2, GLP-1(1–37), GLP-1(7–36)NH2, GLP-1(7–37), oxyntomodulin], and exogenous (exendin-4) peptides in addition to small-molecule allosteric agonists (compound 2 [6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline], BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]). Furthermore, the GLP-1R is subject to single-nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants previously reported to impact peptide-mediated receptor activity (M149) and small-molecule allostery (C333). These residues were mutated to a series of alternate amino acids, and their functionality was monitored across physiologically significant signaling pathways, including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation, and intracellular Ca2+ mobilization, in addition to peptide binding and cell-surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small-molecule allosteric agonist compound 2. Conversely, mutation of residue 333 had little impact on peptide-mediated receptor activation, but this activity could not be modulated by compound 2 to the same extent as that observed at the wild-type receptor. These results provide insight into the importance of residues 149 and 333 in peptide function and highlight the complexities of allosteric modulation within this receptor system.

Allosteric Ligands of the Glucagon-Like Peptide 1 Receptor (GLP-1R) Differentially Modulate Endogenous and Exogenous Peptide Responses in a Pathway-Selective Manner: Implications for Drug Screening

The glucagon-like peptide-1 (GLP-1) receptor is a key regulator of insulin secretion and a major therapeutic target for treatment of diabetes. However, GLP-1 receptor function is complex, with multiple endogenous peptides that can interact with the receptor, including full-length (1–37) and truncated (7–37) forms of GLP-1 that can each exist in an amidated form and the related peptide oxyntomodulin. We have investigated two GLP-1 receptor allosteric modulators, Novo Nordisk compound 2 (6,7-dichloro2-methylsulfonyl-3-tert-butylaminoquinoxaline) and quercetin, and their ability to modify binding and signaling (cAMP formation, intracellular Ca2+ mobilization, and extracellular signal-regulated kinase 1/2 phosphorylation) of each of the naturally occurring endogenous peptide agonists, as well as the clinically used peptide mimetic exendin-4. We identified and quantified stimulus bias across multiple endogenous peptides, with response profiles for truncated GLP-1 peptides distinct from those of either the full-length GLP-1 peptides or oxyntomodulin, the first demonstration of such behavior at the GLP-1 receptor. Compound 2 selectively augmented cAMP signaling but did so in a peptide-agonist dependent manner having greatest effect on oxyntomodulin, weaker effect on truncated GLP-1 peptides, and negligible effect on other peptide responses; these effects were principally driven by parallel changes in peptide agonist affinity. In contrast, quercetin selectively modulated calcium signaling but with effects only on truncated GLP-1 peptides or exendin and not oxyntomodulin or full-length peptides. These data have significant implications for how GLP-1 receptor targeted drugs are screened and developed, whereas the allosterically driven, agonist-selective, stimulus bias highlights the potential for distinct clinical efficacy depending on the properties of individual drugs.

Modulation of the Glucagon-Like Peptide-1 Receptor Signaling by Naturally Occurring and Synthetic Flavonoids

The glucagon-like peptide 1 receptor (GLP-1R) is a promising target for the treatment of type II diabetes mellitus because of its role in metabolic homeostasis. In recent years, difficulties with peptide therapies have driven the search for small-molecule compounds to modulate the activity of this receptor. We recently identified quercetin, a naturally occurring flavonoid, as a probe-dependent, pathway-selective allosteric modulator of GLP-1R-mediated signaling. Using Chinese hamster ovary cells expressing the human GLP-1R, we have now extended this work to identify the structural requirements of flavonoids to modify GLP-1R binding and signaling (cAMP formation and intracellular Ca2+ mobilization) of each of the GLP-1R endogenous agonists, as well as the clinically used exogenous peptide mimetic exendin-4. This study identified a chemical series of hydroxyl flavonols with the ability to selectively augment calcium (Ca2+) signaling in a peptide agonist-specific manner, with effects only on truncated GLP-1 peptides [GLP-1(7–36)NH2 and GLP-1(7–37)] and exendin-4, but not on oxyntomodulin or full-length GLP-1 peptides [GLP-1(1–36)NH2 and GLP-1(1–37)]. In addition, the 3-hydroxyl group on the flavone backbone (i.e., a flavonol) was essential for this activity, however insufficient on its own, to produce the allosteric effects. In contrast to hydroxyl flavonols, catechin had no effect on peptide-mediated Ca2+ signaling but negatively modulated peptide-mediated cAMP formation in a probe-dependent manner. These data represent a detailed examination of the action of different flavonoids on peptide agonists at the GLP-1R and may aid in the development of future small molecule compounds targeted at this receptor.

Second Extracellular Loop of Human Glucagon-like Peptide-1 Receptor (GLP-1R) Differentially Regulates Orthosteric but Not Allosteric Agonist Binding and Function

Background: The ECL2 of the GLP-1R is critical for GLP-1 peptide-mediated selective signaling. Results: Mutation of most ECL2 residues to alanine results in changes in binding and/or efficacy of oxyntomodulin and exendin-4 but not allosteric agonists. Conclusion: ECL2 of the GLP-1R has ligand-specific as well as general effects on peptide agonist-mediated receptor activation. Significance: This work provides insight into control of family B GPCR activation transition. The glucagon-like peptide-1 receptor (GLP-1R) is a prototypical family B G protein-coupled receptor that exhibits physiologically important pleiotropic coupling and ligand-dependent signal bias. In our accompanying article (Koole, C., Wootten, D., Simms, J., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2012) J. Biol. Chem. 287, 3642–3658), we demonstrate, through alanine-scanning mutagenesis, a key role for extracellular loop (ECL) 2 of the receptor in propagating activation transition mediated by GLP-1 peptides that occurs in a peptide- and pathway-dependent manner for cAMP formation, intracellular (Ca2+i) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). In this study, we examine the effect of ECL2 mutations on the binding and signaling of the peptide mimetics, exendin-4 and oxyntomodulin, as well as small molecule allosteric agonist 6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline (compound 2). Lys-288, Cys-296, Trp-297, and Asn-300 were globally important for peptide signaling and also had critical roles in governing signal bias of the receptor. Peptide-specific effects on relative efficacy and signal bias were most commonly observed for residues 301–305, although R299A mutation also caused significantly different effects for individual peptides. Met-303 was more important for exendin-4 and oxyntomodulin action than those of GLP-1 peptides. Globally, ECL2 mutation was more detrimental to exendin-4-mediated Ca2+i release than GLP-1(7–36)-NH2, providing additional evidence for subtle differences in receptor activation by these two peptides. Unlike peptide activation of the GLP-1R, ECL2 mutations had only limited impact on compound 2 mediated cAMP and pERK responses, consistent with this ligand having a distinct mechanism for receptor activation. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition of the receptor by peptide agonists.

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

Summary Ligand-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.

Recent advances in understanding GLP-1R (glucagon-like peptide-1 receptor) function.

Type 2 diabetes is a major global health problem and there is ongoing research for new treatments to manage the disease. The GLP-1R (glucagon-like peptide-1 receptor) controls the physiological response to the incretin peptide, GLP-1, and is currently a major target for the development of therapeutics owing to the broad range of potential beneficial effects in Type 2 diabetes. These include promotion of glucose-dependent insulin secretion, increased insulin biosynthesis, preservation of β-cell mass, improved peripheral insulin sensitivity and promotion of weight loss. Despite this, our understanding of GLP-1R function is still limited, with the desired spectrum of GLP-1R-mediated signalling yet to be determined. We review the current understanding of GLP-1R function, in particular, highlighting recent contributions in the field on allosteric modulation, probe-dependence and ligand-directed signal bias and how these behaviours may influence future drug development.

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

The glucagon-like peptide-1 receptor (GLP-1R) is a family B G protein-coupled receptor and an important drug target for the treatment of type II diabetes, with activation of pancreatic GLP-1Rs eliciting glucose-dependent insulin secretion. Currently, approved therapeutics acting at this receptor are peptide based, and there is substantial interest in small molecule modulators for the GLP-1R. Using a variety of resonance energy transfer techniques, we demonstrate that the GLP-1R forms homodimers and that transmembrane helix 4 (TM4) provides the primary dimerization interface. We show that disruption of dimerization using a TM4 peptide, a minigene construct encoding TM4, or by mutation of TM4, eliminates G protein-dependent high-affinity binding to GLP-1(7-36)NH2 but has selective effects on receptor signaling. There was <10-fold decrease in potency in cAMP accumulation or ERK1/2 phosphorylation assays but marked loss of intracellular calcium mobilization by peptide agonists. In contrast, there was near-complete abrogation of the cAMP response to an allosteric agonist, compound 2, but preservation of ERK phosphorylation. Collectively, this indicates that GLP-1R dimerization is important for control of signal bias. Furthermore, we reveal that two small molecule ligands are unaltered in their ability to allosterically modulate signaling from peptide ligands, demonstrating that these modulators act in cis within a single receptor protomer, and this has important implications for small molecule drug design.

Molecular mechanisms underlying physiological and receptor pleiotropic effects mediated by GLP‐1R activation

The incidence of type 2 diabetes in developed countries is increasing yearly with a significant negative impact on patient quality of life and an enormous burden on the healthcare system. Current biguanide and thiazolidinedione treatments for type 2 diabetes have a number of clinical limitations, the most serious long‐term limitation being the eventual need for insulin replacement therapy (Table 1). Since 2007, drugs targeting the glucagon‐like peptide‐1 (GLP‐1) receptor have been marketed for the treatment of type 2 diabetes. These drugs have enjoyed a great deal of success even though our underlying understanding of the mechanisms for their pleiotropic effects remain poorly characterized even while major pharmaceutical companies actively pursue small molecule alternatives. Coupling of the GLP‐1 receptor to more than one signalling pathway (pleiotropic signalling) can result in ligand‐dependent signalling bias and for a peptide receptor such as the GLP‐1 receptor this can be exaggerated with the use of small molecule agonists. Better consideration of receptor signalling pleiotropy will be necessary for future drug development. This is particularly important given the recent failure of taspoglutide, the report of increased risk of pancreatitis associated with GLP‐1 mimetics and the observed clinical differences between liraglutide, exenatide and the newly developed long‐acting exenatide long acting release, albiglutide and dulaglutide.