Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state

The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism

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

Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that plays a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and the type 1 transmembrane domain protein, receptor activity modifying protein (RAMP) 1. Herein, we report the 3.3 Å structure of the human CGRP receptor in complex with CGRP and the Gs40 protein heterotrimer determined by Volta phase plate cryo-electron microscopy. The RAMP transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilises CLR extracellular loop 2. RAMP1 makes only limited direct interaction with CGRP, consistent with allosteric modulation of CLR as its key function. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly the location of the CLR extracellular domain. The work provides novel insight into the control of G-protein-coupled receptor function

Pharmacological and structure-function analysis of the human glucagon-like peptide-1 receptor

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of insulin biosynthesis and secretion, and is one of the key therapeutic targets in the management of type II diabetes mellitus (DM). Like most GPCRs, the GLP-1R is pleiotropically coupled, with physiologically relevant signaling pathways including cAMP formation, intracellular calcium (iCa2+) mobilization and phosphorylation of extracellular signal regulated kinases 1 and 2 (pERK1/2). GLP-1R activity is further complicated by the ability to be activated by multiple endogenous peptide agonists, and the presence of single nucleotide polymorphisms (SNPs) within the receptor protein. Additionally, in the absence of complete crystal structures and limited knowledge of functionally relevant domains other than the N-terminus, this receptor system is in many respects unexplored. Consequently, the studies comprising this thesis are focused on illustrating distinct pharmacological concepts at the GLP-1R, while also addressing the role of a receptor core domain associated with biological activity. Specifically, the differential actions of allosteric modulators on endogenous peptide ligands (‘probe dependence’), the differential responses of receptor variants with respect to both orthosteric and allosteric ligands, and the capacity for both ligand- and pathway-specific effects (‘biased signaling’) are highlighted. While demonstrating the potential benefits of allosteric modulation, these studies significantly impact on the approaches and precautions that must be considered in the design, identification and development of allosteric modulators as therapeutics. In addition, these studies illustrate the importance of extracellular loop (ECL) 2 in the peptide-mediated activation transition of the GLP-1R. Collectively, this leads to enhanced understanding of the highly complex GLP-1R system, while also exemplifying the importance of extended pharmacological analysis of other GPCRs in order to gain a more comprehensive understanding of receptor systems

Pharmacological and structure-function analysis of the human glucagon-like peptide-1 receptor

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of insulin biosynthesis and secretion, and is one of the key therapeutic targets in the management of type II diabetes mellitus (DM). Like most GPCRs, the GLP-1R is pleiotropically coupled, with physiologically relevant signaling pathways including cAMP formation, intracellular calcium (iCa2+) mobilization and phosphorylation of extracellular signal regulated kinases 1 and 2 (pERK1/2). GLP-1R activity is further complicated by the ability to be activated by multiple endogenous peptide agonists, and the presence of single nucleotide polymorphisms (SNPs) within the receptor protein. Additionally, in the absence of complete crystal structures and limited knowledge of functionally relevant domains other than the N-terminus, this receptor system is in many respects unexplored. Consequently, the studies comprising this thesis are focused on illustrating distinct pharmacological concepts at the GLP-1R, while also addressing the role of a receptor core domain associated with biological activity. Specifically, the differential actions of allosteric modulators on endogenous peptide ligands (‘probe dependence’), the differential responses of receptor variants with respect to both orthosteric and allosteric ligands, and the capacity for both ligand- and pathway-specific effects (‘biased signaling’) are highlighted. While demonstrating the potential benefits of allosteric modulation, these studies significantly impact on the approaches and precautions that must be considered in the design, identification and development of allosteric modulators as therapeutics. In addition, these studies illustrate the importance of extracellular loop (ECL) 2 in the peptide-mediated activation transition of the GLP-1R. Collectively, this leads to enhanced understanding of the highly complex GLP-1R system, while also exemplifying the importance of extended pharmacological analysis of other GPCRs in order to gain a more comprehensive understanding of receptor systems

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. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-

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)NH(2) and GLP-1(7–36)NH(2)) 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 Ca(2+) 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 Met(149) receptor variant. At the Met(149) 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 Cys(333) variant, peptide responses were preserved but there was attenuated response to compound 2. Strikingly, the loss of peptide function at the Met(149) 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

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 oxynto-modulin 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