Corticotropin-releasing factor (CRF), a peptide first isolated from mammalian brain, is critical in the regulation of the pituitary-adrenal axis, and in complementary stress-related endocrine, autonomic and behavioural responses. Fish urotensin I and amphibian sauvagine were considered to be homologues of CRF until peptides even more closely related to CRF were identified in these same vertebrate classes. We have characterized another mammalian member of the CRF family and have localized its urotensin-like immunoreactivity to, and cloned related complementary DNAs from, a discrete rat midbrain region. The deduced protein encodes a peptide that we name urocortin, which is related to urotensin (63% sequence identity) and CRF (45% sequence identity). Synthetic urocortin evokes secretion of adrenocorticotropic hormone (ACTH) both in vitro and in vivo and binds and activates transfected type-1 CRF receptors, the subtype expressed by pituitary corticotropes. The coincidence of urotensin-like immunoreactivity with type-2 CRF receptors in brain, and our observation that urocortin is more potent than CRF at binding and activating type-2 CRF receptors, as well as at inducing c-Fos (an index of cellular activation) in regions enriched in type-2 CRF receptors, indicate that this new peptide could be an endogenous ligand for type-2 CRF receptors.

Amyloids are highly organized cross β-sheet-rich protein or peptide aggregates that are associated with pathological conditions including Alzheimer's disease and type II diabetes. However, amyloids may also have a normal biological function as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. Here, we show that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross β-sheet-rich conformation. Thus, in contrast to the original association of amyloids with diseases, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology.Cells transport newly synthesized secretory proteins and peptides in vesicles via the endoplasmic reticulum (ER) and Golgi for release into the extracellular space (1,2). Some secretory cells, such as neuroendocrine cells and exocrine cells, store secretory proteins and peptides for extended time periods in a highly concentrated form in membrane-enclosed electron-dense cores termed "secretory granules" (1,3,4), which are derived from the Golgi complex. The dense cores of these granules are made up of large, insoluble secretory protein and peptide aggregates that are formed by self-association (4-6). The granules are not amorphous, but possess a distinct molecular organization, possibly of crystalline structures (7) or large intermolecular aggregates (5,8).Amyloid fibrils are cross-β-sheet structures that are primarily associated with several neurodegenerative diseases including Alzheimer's disease. However, amyloid fibril formation also provides biologically functional entities termed functional amyloids (9) and are present in Escherichia coli (10), silkworm (11), fungi (12), and mammalian skin (13). The cross-β-sheet motif is composed of intermolecular β-sheets along the fibril axis with the β-strands aligned perpendicularly to the fibril axis. An amyloid-like structure of peptide and protein hormones in secretory granules could explain most of their properties.To address the question whether peptide and protein hormones are stored in secretory granules in an amyloid-like aggregation state, we first asked if a diverse set of peptide and protein hormones could form amyloids in vitro at granule-relevant pH 5.5. 42 peptide and protein hormones from multiple species and organs were selected randomly, some linear and some cyclic, with a variety of different three dimensional structures (Table S2). This set of hormones was assayed for a capacity to form amyloids by the amyloid-specific dyes thioflavin T (Thio T), congo red (CR), luminescent conjugated polyelectrolyte probes (LCP), by the conformational transition into β-sheet-rich structure measured by circular dichroism (CD), and by the presence of fibrils in electron microscopy (EM) images. Furthermore, x-ray fiber diffraction was measured for a subset of hormones (Table S1). Only 10 hormones out of the 42 showed significant formation of...

Two G protein-coupled receptors have been identified that bind corticotropin-releasing factor (CRF) and urocortin (UCN) with high affinity. Hybridization histochemical methods were used to shed light on controversies concerning their localization in rat brain, and to provide normative distributional data in mouse, the standard model for genetic manipulation in mammals. The distribution of CRF-R1 mRNA in mouse was found to be fundamentally similar to that in rat, with expression predominating in the cerebral cortex, sensory relay nuclei, and in the cerebellum and its major afferents. Pronounced species differences in distribution were few, although more subtle variations in the relative strength of R1 expression were seen in several forebrain regions. CRF-R2 mRNA displayed comparable expression in rat and mouse brain, distinct from, and more restricted than that of CRF-R1. Major neuronal sites of CRF-R2 expression included aspects of the olfactory bulb, lateral septal nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, medial and posterior cortical nuclei of the amygdala, ventral hippocampus, mesencephalic raphe nuclei, and novel localizations in the nucleus of the solitary tract and area postrema. Several sites of expression in the limbic forebrain were found to overlap partially with ones of androgen receptor expression. In pituitary, rat and mouse displayed CRF-R1 mRNA signal continuously over the intermediate lobe and over a subset of cells in the anterior lobe, whereas CRF-R2 transcripts were expressed mainly in the posterior lobe. The distinctive expression pattern of CRF-R2 mRNA identifies additional putative central sites of action for CRF and/or UCN. Constitutive expression of CRF-R2 mRNA in the nucleus of the solitary tract, and stress-inducible expression of CRF-R1 transcripts in the paraventricular nucleus may provide a basis for understanding documented effects of CRF-related peptides at a loci shown previously to lack a capacity for CRF-R expression or CRF binding. Other such "mismatches" remain to be reconciled.

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...

Corticotropin-releasing factor (CRF) is the principal neuroregulator of the hypothalamic-pituitaryadrenocortical axis and plays an important role In coordinating the endocrine, autonomic, and behavioral responses to stress and immune chaLenge. We report here the cloning of a cDNA coding for a CRF receptor from a human corticotropic tumor library. The Here we present the expression cloning and functional expression of a cDNA encoding a CRF receptor from a human Cushing corticotropic cell tumor. We also report a variant of the receptor produced by alternative splicing.t MATERIALS AND METHODSLibrary Construction. A human corticotropic adenoma removed from a patient with Cushing disease was generously provided by Mary Lee Vance and the neurosurgery unit headed by Edward Laws (University of Virginia School of Medicine). Total RNA was prepared by guanidine extraction and poly(A)+ RNA was obtained with oligotex-dt (Qiagen). The corresponding cDNA was ligated into pcDNA1 (Invitrogen), yielding a library of =1.5 x 106 primary recombinants, 80%6 of which contained inserts. A AZAP II (Stratagene) library was synthesized from the same human Cushing tumor cDNA by using Not I/EcoRI adapters.Expression Cloning. Expression screening of the pcDNAl library was carried out as reported (19). Binding to transfected COS-M6 cells was assessed by incubation with 1 x 106 cpm of [125I]iodotyrosyl ovine CRF (Peninsula Laboratories) [iodinated as described (16)] in 0.7 ml ofbinding buffer [0.1% ovalbumin in HDB (25 mM Hepes/137 mM NaCl/5 mM KCI/0.7 mM Na2HPO4, pH 7.5)] for 90 min at 21°C.Phage Library Screening. A 1.2-kb Pst I fraginent in the coding region of hct-0 was used to screen the AZAP II library by standard methods. Double-stranded sequencing was performed by the dideoxy chain-termination method with the Sequenase kit (United States Biochemical). Radioreceptor Assay of Cloned Receptor. Two days after transfection with 10-20 ug of plasmid DNA, the COS-M6 cells were washed with HDB and detached by incubation with 0.5 mM EDTA in HDB for 15 min at 21°C. The cells were washed twice with HDB and homogenized in 5% sucrose. The homogenate was centrifuged at 600 x g for 5 min, after which the supernatant was centrifuged at 40,000 x g for 20 min. The resulting membrane homogenate, P2, was resuspended at 1-4 mg/ml in 10%o sucrose and used in the binding assay as described (16). Dissociation constants were calculated from relative potencies by using the ALLFIT program (20) and determined from competitive displacement assays with rat/human CRF (r/hCRF) as the standard.cAMP Accumulation Assay. COS-M6 cells were transfected as described (21). At least 2 hr before treatments, the medium was changed to contain 0.1% serum. After a 30-min preincubation in medium with or without 0.1 mM 3-isobutyl-1-

Corticotropin-releasing factor (CRF) is a major hypophysiotropic peptide regulating pituitary-adrenal response to stress, and it Is also widely expressed in the central nervous system. (2), with some major areas of mismatch (14). One potential basis for this discrepancy derives from the recent identification of a distinct CRF-binding protein (CRF-BP), which is prominently expressed in brain, including subsets of CRF-containing pathways and/or their targets (15). The identification of this distinct high-affinity CRF-binding moiety, which is in a position to modify central and pituitary actions of CRF by autocrine or paracrine mechanisms, could limit the ability to draw inferences concerning CRF-R distributions on the basis of ligand-binding patterns alone. The present study employed hybridization histochemical methods to provide an initial overview ofthe cellular sites ofexpression ofthe CRF-R in rat brain and pituitary. Dual staining approaches were used to directly compare loci of CRF-R mRNA expression with those of ACTH in the pituitary and of CRF-BP in brain. MATERIALS AND METHODSIn Situ Hybridization. Male (n = 7) and female (n = 3) adult Sprague-Dawley albino rats (200-300 g) were perfused transcardially with 4% paraformaldehyde in 0.1 M pH 9.5 sodium borate buffer, and regularly spaced series of 20-to 30-,umthick frozen sections through brain and pituitary were taken as described (16). Radiolabeled antisense and sense (control) complementary RNA (cRNA) copies were synthesized from a full-length or N-terminal 344 bp fragment of the rat CRF cDNA subcloned in pBluescript KS vector (Stratagene). (a-33P]UTP was used for probe synthesis, and in situ hybridization was performed as previously described (16,17). cRNA probes to full-length rat CRF-R coding region were labeled to 40-60% total incorporation, and hybridization was carried out under high stringency [50%6 (vol/vol) formamide with final washes carried out in 0.2x SSC at 700C (lx SSC is 0.15 M NaCl/0.015 M sodium citrate, pH 7.0)].Combined in Situ Hybridization and Immun emis try. Concurrent localization of ACTH immunoreactivity -ir) and CRF-R mRNA in pituitaries was performed by using a modification of a procedure described by Watts and Swanson (18). This involved first applying a conventional biotinavidin-immunoperoxidase protocol, to localize primary antibodies raised in rabbit against the rat corticotropin fragment ACTH-(23-39). Rat ACTH-(23-39) was coupled to human a-globulins with bisdiazotized benzidine, and antisera were

Corticotropin-releasing factor (CRF; corticoliberin) regulates the secretion of corticotropin (ACTH) and 18-endorphin and has a broad range of effects on the nervous, endocrine, reproductive, cardiovascular, gastrointestinal, and immune systems. Recently, human, rat, and mouse CRF receptors (CRF-R) have been cloned and functionally and anatomically characterized. We report here the cloning of a second CRF-R cDNA (CRF-RB), which encodes a protein of431 amino acids, which is 16 amino acids longer and 68% similar to the previously cloned CRF-R, CRF-RA. When transiently expressed in COS-M6 cells, CRF-RB binds CRF with high affinity [Kd = 1.2 (0.57-2.5) nM] and transduces the CRI-stimulated signal of the accumulation of intracellular cAMP, which is inhibited by a CRF antagonist. Comparison of the amino acid sequences of CRF-RB and the previously cloned receptor reveals major differences in the N-terminal domain and in the extracellular loops, whereas the sequences of the intracellular loops are nearly identical. CRF-RB and related transcripts are expressed in the heart, as well as in other tissues, including the gastrointestinal tract, epididymis, and brain.Corticotrophin-releasing-factor (CRF; corticoliberin), the 41-amino acid peptide originally isolated from the hypothalamus (1) as the major regulator of corticotropin (ACTH) and f3-endorphin secretion by the anterior pituitary, has been shown to be widely distributed in, and to have multiple effects on, a wide variety of tissues (2, 3). Consistent with the broad range of roles proposed for CRF, high-affinity binding sites have been found in pituitary (4), brain (5, 6), adrenals (7), spleen (8), and monocytes (9). Recently, our group (10, 11) and others (12, 13) reported the cloning of CRF receptors (CRF-R), which we now refer to as CRF-RA, from pituitary and brain. These receptors belong to the seven transmembrane domain (TMD) calcitonin/vasoactive intestinal peptide/ growth hormone-releasing hormone receptor family. The distribution (14) and functionality of CRF-RA indicated that it satisfied many criteria for a physiologic CRF receptor. In a human Cushing disease tumor cDNA library, we also observed the presence of a splice variant, CRF-RA2 (10), in which 29 amino acids are inserted into the first intracellular loop.During the course of the characterization of the mouse gene encoding the CRF-R, we obtained evidence for a related gene, CRF-RB, which we partially sequenced. RNase protection analysis indicated high expression of this gene in the heart. We report here the cloning and characterization of a cDNA from a mouse heart cDNA library encoding a second CRF-R. § MATERIALS AND METHODS [a-32P]dCTP and the following primers: sense strand, 5'-CTGCATCACCACCATCTTCAACT-3'; and antisense strand, 5'-AGCCACTTGCGCAGGTGCTC-3'. The template used in generating the probe was plasmid DNA corresponding to one exon of CRF-RB extending from amino acid 206 to 246 (see Fig. 1). PCR amplification was carried out for 30 cycles (denaturation at 94°C for 1 min, annealing at 5...