Molecular basis for negative regulation of the glucagon receptor

Abstract

Members of the class B family of G protein-coupled receptors (GPCRs) bind peptide hormones and have causal roles in many diseases, ranging from diabetes and osteoporosis to anxiety. Although peptide, small-molecule, and antibody inhibitors of these GPCRs have been identified, structure-based descriptions of receptor antagonism are scarce. Here we report the mechanisms of glucagon receptor inhibition by blocking antibodies targeting the receptor's extracellular domain (ECD). These studies uncovered a role for the ECD as an intrinsic negative regulator of receptor activity. The crystal structure of the ECD in complex with the Fab fragment of one antibody, mAb1, reveals that this antibody inhibits glucagon receptor by occluding a surface extending across the entire hormone-binding cleft. A second antibody, mAb23, blocks glucagon binding and inhibits basal receptor activity, indicating that it is an inverse agonist and that the ECD can negatively regulate receptor activity independent of ligand binding. Biochemical analyses of receptor mutants in the context of a high-resolution ECD structure show that this previously unrecognized inhibitory activity of the ECD involves an interaction with the third extracellular loop of the receptor and suggest that glucagon-mediated structural changes in the ECD accompany receptor activation. These studies have implications for the design of drugs to treat class B GPCR-related diseases, including the potential for developing novel allosteric regulators that target the ECDs of these receptors.T he glucagon receptor (GCGR) is a member of the class B G protein-coupled receptor (GPCR) family (1) that mediates the activity of glucagon, a pancreatic islet-derived peptide hormone that plays a central role in the pathophysiology of diabetes (2). Several GCGR antagonists that improve glycemic control in animal models of diabetes and diabetic patients have been described (3-8). Although biochemical studies of glucagon and GCGR mutants have facilitated the mapping of some elements that contribute to glucagon binding (4, 9-12), the molecular mechanisms of GCGR activation and inhibition remain largely unknown because there are currently no high-resolution structures of GCGR. The current model for activation class B GPCRs proposes a tethering mechanism whereby the C-terminal half of the peptide ligand first binds a large extracellular domain (ECD), thereby enabling a high-affinity interaction of the N-terminal half of the ligand with a cleft formed by the transmembrane α-helical bundle (13,14), termed the juxtamembrane (JM) domain. This interaction induces a structural change in the transmembrane and intracellular face of the receptor that enables G protein coupling, likely similar to that described for the activated form of the β-adrenergic receptor (15). Recent structural studies of several class B GPCR ECDs and ECD-ligand complexes support this model (16)(17)(18)(19)(20)(21). Glucagon likely interacts with GCGR in a similar fashion to the interaction of other peptide ligands with class B GPC...

Structure of the full-length glucagon class B G-protein-coupled receptor

“…The TMD in the GCGR-FL structure features the canonical seven-transmembrane helical bundle (helices I–VII), which shares a similar conformation compared to the previously solved crystal structures of the GCGR-TMD 8,9 with Cα root-mean-square deviation (RMSD) of 1.2 Å (PDB ID: 4L6R) and 0.8 Å (PDB ID: 5EE7). The antibody mAb1 interacts with the αA helix and loops L2, L4 and L5 of GCGR-ECD as previously reported 6 . Additionally, it makes close contact with ECL1 of the receptor (Fig.…”

“…Since the recovery of 20% of the glucagon-binding ability corresponds to the effect of one of the single-site mutations Q113C, these data support the molecular contact between the ECD and ECL1 as observed in the MD simulations, and suggest that the interaction between these regions may play important roles in constraining the conformation of the apo receptor in an inactive state. Previous MD simulations and disulfide cross-linking studies indicated that the ECD of apo GCGR interacts with ECL1 and can be cross-linked to ECL3 7 , whereas chimera studies suggested that the ECD of GCGR stabilizes the receptor in an inactive state through interactions with ECL3 6 . These data combined with our studies suggest the existence of a dynamical interface between the ECD and TMD of GCGR.…”

“…1a). The ECD comprises the common α-β-β-α fold as observed in the ECD structures of GCGR and other class B GPCRs 6,12,13 with Cα RMSD of 1.3 Å compared to the same domain in the crystal structure of the GCGR-ECD bound to mAb1 6 (PDB ID: 4ERS). Four asparagine residues, N46, N59, N74 and N78 within the ECD are glycosylated by N-acetyl-D-glucosamines (NAGs).…”

“…The antibodies mAb1 and mAb23 have been studied for their effects on GCGR activation 6 . The investigations showed that mAb1 behaves as an antagonist by blocking ligand access through direct interactions with the residues in the ECD required for glucagon binding, while mAb23 not only blocks glucagon binding but also reduces basal receptor activity, acting as an inverse agonist.…”

“…These receptors consist of an ECD and a TMD, both of which are required for binding to their endogenous peptide ligands and regulation of cell signal transduction 2,5 . Previous studies suggested that tertiary interactions between the ECD and TMD play a critical role in regulating receptor activity of class B GPCRs 6,7 . Structures of the ECDs of several class B GPCRs have been solved 2 , and recently, the crystal structures of the TMDs of three class B GPCRs, the human GCGR 8,9 , corticotrophin-releasing factor receptor 1 (CRF 1 R) 10 and glucagon-like peptide-1 receptor (GLP-1R) 11 , have been determined, providing insights into ligand recognition and selectivity of these physiologically important receptors.…”