Recently, the concept of ligand-directed signaling-the ability of different ligands of an individual receptor to promote distinct patterns of cellular response-has gained much traction in the field of drug discovery, with the potential to sculpt biological response to favor therapeutically beneficial signaling pathways over those leading to harmful effects. However, there is limited understanding of the mechanistic basis underlying biased signaling. The glucagon-like peptide-1 receptor is a major target for treatment of type-2 diabetes and is subject to ligand-directed signaling. Here, we demonstrate the importance of polar transmembrane residues conserved within family B G protein-coupled receptors, not only for protein folding and expression, but also in controlling activation transition, ligand-biased, and pathway-biased signaling. Distinct clusters of polar residues were important for receptor activation and signal preference, globally changing the profile of receptor response to distinct peptide ligands, including endogenous ligands glucagon-like peptide-1, oxyntomodulin, and the clinically used mimetic exendin-4.

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 Ca 2ϩ 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.

GPCRs exhibit a common architecture of seven transmembrane helices (TMs) linked by intracellular loops and extracellular loops (ECLs). Given their peripheral location to the site of G-protein interaction, it might be assumed that ECL segments merely link the important TMs within the helical bundle of the receptor. However, compelling evidence has emerged in recent years revealing a critical role for ECLs in many fundamental aspects of GPCR function, which supported by recent GPCR crystal structures has provided mechanistic insights. This review will present current understanding of the key roles of ECLs in ligand binding, activation and regulation of both family A and family B GPCRs. LINKED ARTICLESThis article is part of a themed section on the Molecular Pharmacology of G Protein-Coupled Receptors (GPCRs). To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-6. To view the 2010 themed section on the same topic visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2010.159.issue-5/issuetoc Abbreviations A2AR, A2A adenosine receptor; AT1R, angiotensin II type 1 receptor; b1AR, b1-adrenergic receptor; b2AR, b2-adrenergic receptor; C5aR, complement factor 5a receptor; CAM, constitutively activating mutation; CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; CRF, corticotropin-releasing factor; D3R (D2R), D3 (D2) dopamine receptor; GLP-1, glucagon-like peptide-1; ECL, extracellular loop; H1R, histamine H1 receptor; HIV-1, human immunodeficiency virus type 1; M2R (M4R), M2 (M4) muscarinic acetylcholine receptor; NDI, nephrogenic diabetes insipidus; PACAP, pituitary adenylyl cyclase-activating peptide; PTH, parathyroid hormone; TM, transmembrane helix; V1aR, V1a vasopressin receptor; V2R, V2 vasopressin receptor IntroductionGPCRs form the largest class of membrane proteins in the human genome, with >800 unique receptors. They are central to cell signalling and are of great commercial value to the pharmaceutical industry worldwide, with~50% of clinically marketed drugs and~25% of top-selling drugs targeting this receptor family (Lagerström and Schiöth, 2008). GPCRs are activated by a wide variety of agonists which differ with respect to chemical class, physical properties and size -from photons and small biogenic amines to peptides and large glycoproteins (Hill, 2006).Historically, it was envisaged that binding any agonist induced the 'on' conformation that activated a single G-protein type to initiate an intracellular signal. It is now recognized that GPCR signalling is much more complex than this. Individual GPCRs can activate multiple types of G-protein, not just one type, and signalling can be G-protein independent, such as b-arrestin-dependent GPCR activation of MAPK (Azzi et al., 2003). Furthermore, there is compelling evidence to indicate that the classification of an individual ligand can be dictated by the signalling system being observed. For example, the peptide ligand SP-G is an antagonist for V 1a vasopressin receptor (V1aR) inositol phos...

SUMMARY Association of receptor activity-modifying proteins (RAMP1–3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR: RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.

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.

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 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 2ϩ mobilization); ligand binding and cell surface receptor expression were also determined. We demonstrate both ligandand 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.

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