The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.
Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A) ؉ RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1-10 g of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.C RF is a 41-aa peptide best known for its indispensable role in initiating pituitary-adrenal responses to stress, an effect mediated by type 1 CRF receptors (1). In addition, CRF is widely distributed in brain and has been shown repeatedly to participate in the mobilization of complementary autonomic and behavioral adjustments to a variety of threatening circumstances (2, 3). This has fostered the widely held hypothesis that CRF plays an important role in the integration of adaptive responses to stress. Rigorous testing of this idea has been impeded by the fact that a number of the cell groups identified as sites of peptide action in eliciting stress-like autonomic and behavioral responses have been found to be lacking or impoverished in the expression of requisite ligand(s), receptor(s), or both (4, 5). This has kindled the search for additional CRF-related signaling molecules, which currently number two ligands, G protein-coupled receptors derived from two distinct genes (CRF-R1 and CRF-R2), and a binding protein, whose function remains incompletely understood (6, 7).A second mammalian CRF-related neuropeptide, urocortin (Ucn), was discovered recently by our group (8) and shown to be bound with high affinity by both known CRF receptor types, whereas CRF is bound in a highly preferential manner by CRF-R1. Centrally administered Ucn is more potent than CRF in suppressing appetite, but it is less so in generating acute anxiety-like effects and g...
Predictive methods, physicochemical measurements, and structure activity relationship studies suggest that corticotropin-releasing factor (CRF; corticoliberin), its family members, and competitive antagonists (resulting from N-terminal deletions) usually assume an a-helical conformation when interacting with the CRF receptor(s). To test this hypothesis further, we have scanned the whole sequence of the CRF antagonist Nle21'38]r/hCRF-(12-41) (r/hCRF, rat/human CRF; Nle, norleucine) with an i-(i + 3) bridge consisting of the Giu-Xaa-Xa:-Lys scaffold. We have found astressin {cyclo (30)(31)(32)(33) Nle2l,38,Glu30,Lys33] Corticotropin-releasing factor (CRF; corticoliberin) is a 41-residue peptide amide which stimulates the release of corticotropin (ACTH) (1, 2) and acts within the brain to mediate a wide range of stress responses (3). The actions of CRF are mediated through binding to CRF receptors, several of which have been characterized recently (4-10). These receptors, like those for growth hormone-releasing factor, calcitonin, and vasoactive intestinal peptide, are coupled via G proteins and have seven putative transmembrane domains. The actions of CRF can also be modulated by a 37-kDa CRF-binding protein (CRF-BP) (11). To probe the physiological role of CRF, we have developed competitive antagonists that are particularly potent when administered in the central nervous system; however, these same analogs bind pituitary receptors with lower affinity than does CRF, and their peripheral administration results in weak and short-lived effects in vivo (12). Synthetic CRF antagonists such as the a-helical CRF-(9-41)
The corticotropin releasing factor (CRF) family of ligands and their receptors coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play additional roles within the cardiovascular, gastrointestinal, and other systems. The actions of CRF and the related urocortins are mediated by activation of two receptors, CRF-R1 and CRF-R2, belonging to the B1 family of G protein-coupled receptors. The short-consensus-repeat fold (SCR) within the first extracellular domain (ECD1) of the CRF receptor(s) comprises the major ligand binding site and serves to dock a peptide ligand via its C-terminal segment, thus positioning the N-terminal segment to interact with the receptor's juxtamembrane domains to activate the receptor. Here we present the 3D NMR structure of ECD1 of CRF-R2 in complex with astressin, a peptide antagonist. In the structure of the complex the C-terminal segment of astressin forms an amphipathic helix, whose entire hydrophobic face interacts with the short-consensus-repeat motif, covering a large intermolecular interface. In addition, the complex is characterized by intermolecular hydrogen bonds and a salt bridge. These interactions are quantitatively weighted by an analysis of the effects on the full-length receptor affinities using an Ala scan of CRF. These structural studies identify the major determinants for CRF ligand specificity and selectivity and support a two-step model for receptor activation. Furthermore, because of a proposed conservation of the fold for both the ECD1s and ligands, this structure can serve as a model for ligand recognition for the entire B1 receptor family.3D structure ͉ astressin ͉ corticotropin releasing factor ͉ NMR T he ability of the body to adapt to stressful stimuli and the role of stress maladaptation in human diseases has been intensively investigated. Corticotropin releasing factor (CRF) (1), a 41-residue peptide, and its three paralogous peptides, urocortin (Ucn) 1, 2, and 3, play important and diverse roles in coordinating endocrine, autonomic, metabolic, and behavioral responses to stress (2, 3). CRF family peptides and their receptors are also implicated in the modulation of additional central nervous system functions including appetite, addiction, hearing, and neurogenesis and act peripherally within the endocrine, cardiovascular, reproductive, gastrointestinal, and immune systems (4, 5). CRF and related ligands initially act by binding to their G protein-coupled receptors (GPCRs). These belong to the peptide hormone B1 family (family B1 GPCRs), comprising receptors for growth hormone releasing factor, secretin, calcitonin, vasoactive intestinal peptide, glucagon, glucagon-like peptide-1, and parathyroid hormone. Two CRF receptors, CRF-R1 and CRF-R2, have been cloned in mammals (6, 7).Structure activity studies of CRF showed that the first eight N-terminal residues of the hormone are necessary for GPCR signaling (1, 8), whereas the C-terminal (Ϸ15) residues are important for binding (9,10). A two-domain behavior for ligand binding was a...
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