Molecular Recognition of Corticotropin-releasing Factor by Its G-protein-coupled Receptor CRFR1

Abstract

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the ␣-␤-␤␣ fold observed for other class B GPCR ECDs, but the N-terminal ␣-helix is significantly shorter and does not contact CRF. CRF adopts a continuous ␣-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications. Corticotropin-releasing factor (CRF)3 is a 41-amino acid, C-terminally amidated neuropeptide originally isolated from sheep hypothalami based on its ability to stimulate secretion of adrenocorticotropin from pituitary cells (1). Several other CRFrelated peptides have since been identified, including the urocortins (Ucn) I, II, and III in mammals (2-5). Extensive studies over the last nearly 3 decades have highlighted the critical roles that CRF family peptides play in coordinating endocrine, autonomic, and behavioral responses to stress (reviewed in Refs. 6, 7). The CRF family of peptides exert their effects through the binding and activation of two paralogous cell surface G-protein-coupled receptors (GPCRs), CRFR1 (8) and CRFR2 (9 -11). CRF binds to both receptors but with higher affinity for CRFR1. UcnI binds equally well to both receptors, whereas UcnII and UcnIII are selective for CRFR2. CRF is the primary regulator of central stress responses; its binding to CRFR1 on the surface of pituitary corticotrope cells activates the hypothalamic-pituitary-adrenal axis. Consequently, there has been enormous interest in the therapeutic potential of CRFR1-selective antagonists for the treatment of anxiety, depression, and related disorders (reviewed in Refs. 7, 12).The CRF receptors belong to the class B/Secretin family of GPCRs (13), whose members include receptors for parathyroid hormone, calcitonin, glucagon, glucagon-like peptides, and other therapeutically important peptides. In addition to a 7-transmembrane helical domain common to all GPCRs, class B receptors have an N-terminal extracellular domain (...

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

“…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).…”

Molecular basis for negative regulation of the glucagon receptor

“…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 GPCRs, although currently undefined differences would ensure receptor specificity.…”

“…The GCGR ECD structure resembles the α-β-β-α fold common to other class B GPCR ECD structures (16)(17)(18)(19)(20)(21)(22)(23) and is most closely related to the glucagon-like peptide-1 receptor (GLP-1R). These receptors share 46% sequence identity within their ECDs, and their overall structures superimpose well, with an rmsd of 1.5 Å (Fig.…”

“…An individual homozygous for a P86S mutation has hallmarks of loss of glucagon action, and this receptor variant was unable to bind glucagon in vitro (10). Residues at the base of L4 adjacent to P86, including the invariant P82 and conserved Y84 and L85, form part of an extended hydrophobic surface in the canonical hormone-binding pocket (16)(17)(18)(19)(20)(21)(22)(23). Residues in L2 have also been shown to be critical for glucagon binding and/or receptor structure (24,25), including D63, which forms a salt bridge with the sidechains of K98 and R116 and is within H-bond distance of W68 and the backbone amide of S66 (Fig.…”

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

“…Insights into the structure of the predominant ligand binding domain, the amino terminus of the Family B GPCRs, have substantially advanced with the solution of NMR and crystal structures of the isolated ligand-bound amino terminus of the receptors for corticotrophin-releasing factor (11)(12)(13), pituitary adenylate cyclase-activating peptide (14), gastric inhibitory polypeptide (GIP) (15), glucagon-like peptide 1 (GLP1) (16), and parathyroid hormone (PTH) (17). Most of these ligands represented amino-terminally truncated hormones or analogues of these hormones.…”