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 (...

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic ␣-helix to the same hydrophobic groove in the ECD as occupied by PTH, but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally "unwound." The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECDbinding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics. The parathyroid hormone receptor (PTH1R)3 is a class B G protein-coupled receptor (GPCR) that transduces signals from two related signaling molecules that have distinct functions in biology: parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) (Ref. 1; reviewed in Ref. 2). PTH is an 84-amino acid polypeptide endocrine hormone that is produced by the parathyroid glands and secreted into the circulation in response to low calcium levels (reviewed in Refs. 3-5), to act on bone and kidney cells and thus restore blood calcium to normal levels. In bone, PTH directly stimulates osteoblasts, resulting in bone formation (reviewed in Ref. 6), which in turn activate osteoclasts to induce bone resorption. In the kidney, PTH stimulates the reabsorption of filtered calcium, inhibits the reabsorption of phosphate, and stimulates the synthesis of 1,25-dihydroxyvitamin D3. The paradoxical anabolic/ catabolic actions of PTH on bone can be modulated by exogenous PTH, and provide the molecular basis for the clinical use of PTH as an anabolic therapy for osteoporosis (7). Anabolic PTH therapy requires intermittent administration to minimize bone-resorptive effects, which predominate with sustained administration of PTH. PTHrP is a 141-amino acid polypeptide that was originally isolated as the factor responsible for humoral hyperca...

Norrin is a cysteine-rich growth factor that is required for angiogenesis in the eye, ear, brain, and female reproductive organs. It functions as an atypical Wnt ligand by specifically binding to the Frizzled 4 (Fz4) receptor. Here we report the crystal structure of Norrin, which reveals a unique dimeric structure with each monomer adopting a conserved cystine knot fold. Functional studies demonstrate that the novel Norrin dimer interface is required for Fz4 activation. Furthermore, we demonstrate that Norrin contains separate binding sites for Fz4 and for the Wnt ligand coreceptor Lrp5 (low-density lipoprotein-related protein 5) or Lrp6. Instead of inducing Fz4 dimerization, Norrin induces the formation of a ternary complex with Fz4 and Lrp5/6 by binding to their respective extracellular domains. These results provide crucial insights into the assembly and activation of the Norrin-Fz4-Lrp5/6 signaling complex.[Keywords: Norrin structure; cystine knot growth factor; Wnt/b-catenin signaling; Frizzled 4; low-density lipoprotein receptor-related protein 5/6; tetraspanin 12] Supplemental material is available for this article.

The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an ␣-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.G protein-coupled receptors (GPCRs) 3 constitute the largest family of cell-surface signal transduction proteins in the human genome. They function as plasma membrane-embedded receptors for ligands such as biogenic amines, peptides, and protein hormones. The receptor transduces the hormone binding signals across the membrane to heterotrimeric G proteins. The oligomeric state of the receptor and the stoichiometry of the receptor-G protein complex is a topic of considerable debate (1-4). Many GPCRs from each of three major classes (A, B, and C) have been shown to homo-oligomerize, and hetero-oligomerization of structurally related members of the same class has also been demonstrated (2,3,5). In many cases the specific oligomeric state of the receptor is unclear. It often is assumed to be the dimer, although evidence for higher order arrays of receptors also exists (6 -8). Ligand effects on oligomerization can vary from no effect (9) to ligand-induced oligomerization (10) or ligand-induced disruption of oligomerization (11). The receptor domains responsible for oligomerization can vary from individual helices of the 7-transmembrane (7-TM) helical domain (12) to extracellular (13) or intracellular domains (14). The most convincing argument for a functional role for oligomerization comes from studies of the class C ␥-aminobutyric acid, type B receptor (14 -16) and the sweet and umami taste receptors (17), which function as obligate heterodimers. In addition, the class A leukotriene B4 and dopamine D2 receptors have been shown to couple to G protein as dimers (18,19). These studies suggested that the pentameric complex of a GPCR dimer and a single G protein heterotrimer is the minimal functional signaling unit. However, several rigorous biochemical studies have shown that the ␤2-adrenergic receptor and rhodopsin, both class A receptors, are capable of functionally coupling to G proteins as monomers (20 -23) even though oligomerization of these receptors is well documented (6,8,24). Perhaps the only ...