SMART MODE:

NORMAL GENOMIC

• Arai M, Assil IQ, Abou-Samra AB
• Characterization of three corticotropin-releasing factor receptors in catfish: a novel third receptor is predominantly expressed in pituitary and urophysis.
• Endocrinology. 2001; 142: 446-54
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• The present study reports the isolation of three complementary DNA (cDNA) clones encoding distinct subtypes of CRF receptors from the diploid catfish (cf) species, Ameiurus nebulosus. The first clone encodes a 446-amino acid protein (cfCRF-R1) that is highly homologous to mouse (m) CRF-R1 (93% identical). The cfCRF-R1 messenger RNA is highly expressed in the brain, and its distribution pattern correlates well with that of mammalian CRF-R1, except for weak expression in the pituitary. When transiently expressed in COS-7 cells, cfCRF-R1 bound CRF, urotensin I, and sauvagine with similar affinities. The second full-length cDNA, which was cloned from catfish heart, encodes a 406-amino acid protein that showed homology to murine CRF-R2 (88%) and when expressed in COS-7 cells preferentially bound sauvagine. The highest level of cfCRF-R2 expression was observed in the heart. The third full-length cDNA clone, which encodes a 428-amino acid protein, is structurally closer to cfCRF-R1 (85%) than to cfCRF-R2 (80%). This novel CRF receptor (cfCRF-R3) bound CRF with a 5-fold higher affinity than urotensin I and sauvagine and was expressed in the pituitary gland, urophysis, and brain. The presence of three different CRF receptors, each with distinct tissue distribution and ligand binding properties, suggests a complex CRF/urotensin I system.

• Perrin MH et al.
• Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor.
• J Biol Chem. 2001; 276: 31528-34
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• The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.

• Keller PA, Elfick L, Garner J, Morgan J, McCluskey A
• Corticotropin releasing hormone: therapeutic implications and medicinal chemistry developments.
• Bioorg Med Chem. 2000; 8: 1213-23
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• Corticotropin releasing hormone (CRH, sometimes known as CRF) is an endogenous 41 amino acid peptide that has been implicated in the onset of pregnancy, the 'fight or flight' response, in addition to a large number of physiological disorders. Recently, medicinal chemists have developed a number of potent and selective compounds that show promise in a vast array of therapeutic uses. Herein we review the current status of research.

• Baigent SM, Lowry PJ
• Urocortin is the principal ligand for the corticotrophin-releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide.
• J Mol Endocrinol. 2000; 24: 53-63
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• To purify novel ligands for the corticotrophin-releasing factor binding protein (CRF-BP) from ovine brain, whole brain was homogenised in methanol and the supernatant extracted on Sep-pak C18 cartridges followed by a preliminary HPLC step. Three peaks of ovine CRF-BP ligand activity were detected in the HPLC fractions, the first two of which were also detected by a specific corticotrophin-releasing factor two-site immunoradiometric assay, the third peak being detected by a human CRF-BP ligand assay, which will not detect ovine CRF. Human CRF-BP ligand-containing fractions were further purified by affinity chromatography on a human recombinant CRF-BP column with two additional HPLC steps. The human CRF-BP ligand was found to: (a) possess a molecular mass of 4707 Daltons, (b) have an N-terminal amino acid sequence (5 residues) identical to rat urocortin, (c) be detected by a specific urocortin radioimmunoassay, (d) have high affinity for both the human and ovine CRF-BPs and (e) be present in many regions of the ovine brain. Additionally, a 300 bp cDNA fragment sharing 83% homology with the rat urocortin gene was cloned from ovine brain, the product of which was predicted to have an identical amino acid sequence to that of rat urocortin. These pieces of information confirmed the identity of the human CRF-BP ligand as an ovine urocortin. The specially developed CRF-BP ligand assays showed that the rank orders of affinity of the CRF family members for human CRF-BP were: carp urotensin-1>>human CRF=rat/ovine urocortin>human urocortin>>frog sauvagine>>ovine CRF, and those for the ovine CRF-BP were: carp urotensin-1> human CRF=rat/ovine urocortin>human urocortin> frog sauvagine>>ovine CRF. This study describes a successful technique for the purification and detection of peptide ligands for the CRF-BP. We conclude that urocortin is the principal ligand for the CRF-BP in ovine brain and we could find no evidence for a centrally located mammalian sauvagine-like peptide.

• Dautzenberg FM, Huber G, Higelin J, Py-Lang G, Kilpatrick GJ
• Evidence for the abundant expression of arginine 185 containing human CRF(2alpha) receptors and the role of position 185 for receptor-ligand selectivity.
• Neuropharmacology. 2000; 39: 1368-76
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• The abundance of a histidine residue at position 185 (His(185)) of the human corticotropin-releasing factor (CRF) type 2 alpha receptor (hCRF(2alpha)) was investigated. His(185) has only been reported in hCRF(2); CRF(2) proteins from other species and all CRF(1) receptors encode an arginine (Arg(185)) at the corresponding position. Cloning of partial and full-length hCRF(2) cDNAs from a variety of neuronal and peripheral tissues revealed the existence of receptor molecules encoding Arg(185) only. Sequence analysis of the hCRF(2) gene verified the existence of Arg(185) also on genomic level. Full-length cDNAs encoding either the His(185) (R2H(185)) or the Arg(185) (R2R(185)) variants of hCRF(2alpha) were stably expressed in HEK293 cells and tested for ligand binding properties. In displacement studies R2H(185) and R2R(185) displayed a similar substrate specificity, human and rat urocortin, and the peptide antagonists astressin and alpha-helical CRF((9-41)) were bound with high affinity whereas human and ovine CRF were low-affinity ligands. Significant differences were observed for sauvagine and urotensin I, which bound with 3-fold (sauvagine) and 9-fold (urotensin I) higher affinity to R2R(185). These data indicate that hCRF(2), like all vertebrate CRF(1) and CRF(2) proteins encodes an arginine residue at the junction between extracellular domain 2 and transmembrane domain 3 and that this amino acid plays a role for the discrimination of some CRF peptide ligands.

• Gilligan PJ, Robertson DW, Zaczek R
• Corticotropin releasing factor (CRF) receptor modulators: progress and opportunities for new therapeutic agents.
• J Med Chem. 2000; 43: 1641-60

• Robinson BM et al.
• Cloning and characterization of corticotropin-releasing factor and urocortin in Syrian hamster (Mesocricetus auratus).
• Peptides. 1999; 20: 1177-85
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• Corticotropin-releasing factor and urocortin belong to a superfamily of neuropeptides that includes the urotensins-I in fishes and the insect diuretic peptides. Sequence analysis suggests that urocortin is the mammalian ortholog of urotensin-I, although the physiological role for this peptide in mammals is not known. Within the Rodentia, hamsters belong to a phylogenetically older lineage than that of mice and rats and possess significant differences in hypothalamic organization. We have, therefore, cloned the coding region of the Syrian hamster (Mesocricetus auratus) corticotropin-releasing factor and urocortin mature peptide by polymerase chain reaction. Hamster urocortin was prepared by solid-phase synthesis, and its pharmacological actions on human corticotropin-releasing factor R1 and R2 receptors were investigated. The deduced hamster corticotropin-releasing factor amino acid sequence and cleavage site is identical to that in rat, whereas the urocortin sequence is unique among the urocortin/urotensin-I/sauvagine family in possessing asparagine and alanine in positions 38 and 39, respectively. The hamster urocortin carboxy terminus sequence bears greater structural similarity to the insect diuretic peptide family, suggesting either retrogressive mutational changes within the mature peptide or convergent sequence evolution. Despite these changes, human and hamster urocortin are generally equipotent at cAMP activation, neuronal acidification rate, and R1/R2 receptor affinities.

• Radulovic J, Blank T, Eckart K, Radulovic M, Stiedl O, Spiess J
• CRF and CRF receptors.
• Results Probl Cell Differ. 1999; 26: 67-90

• Lovejoy DA, Balment RJ
• Evolution and physiology of the corticotropin-releasing factor (CRF) family of neuropeptides in vertebrates.
• Gen Comp Endocrinol. 1999; 115: 1-22
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• Corticotropin-releasing factor (CRF), urotensin-I, urocortin and sauvagine belong to a family of related neuropeptides found throughout chordate taxa and likely stem from an ancestral peptide precursor early in metazoan ancestry. In vertebrates, current evidence suggests that CRF on one hand, and urotensin-I, urocortin and sauvagine, on the other, form paralogous lineages. Urocortin and sauvagine appear to represent tetrapod orthologues of fish urotensin-I. Sauvagine's unique structure may reflect the distinctly derived evolutionary history of the anura and the amphibia in general. The physiological actions of these peptides are mediated by at least two receptor subtypes and a soluble binding protein. Although the earliest functions of these peptides may have been associated with osmoregulation and diuresis, a constellation of physiological effects associated with stress and anxiety, vasoregulation, thermoregulation, growth and metabolism, metamorphosis and reproduction have been identified in various vertebrate species. The elaboration of neural circuitry for each of the two paralogous neuropeptide systems appears to have followed distinct pathways in the actinopterygian and sarcopterygian lineages of vertebrates. A comparision of the functional differences between these two lineages predicts additional functions of these peptides.

• Ando H, Hasegawa M, Ando J, Urano A
• Expression of salmon corticotropin-releasing hormone precursor gene in the preoptic nucleus in stressed rainbow trout.
• Gen Comp Endocrinol. 1999; 113: 87-95
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• The behavior of genes encoding the corticotropin-releasing hormone (CRH) precursor in response to stress has not been extensively studied in teleosts. To clarify this problem, CRH cDNAs were isolated from a hypothalamic cDNA library of sockeye salmon, Oncorhynchus nerka, by screening with PCR products amplified from the hypothalamic mRNA with primers deduced from the sequence of the sucker CRH precursor. Two types of PCR products with a high degree of sequence homology were identified (CRH-I and CRH-II). A cDNA encompassing the entire coding sequence of the salmon CRH-I precursor was isolated. The salmon CRH-I cDNA encodes a 167-amino-acid precursor, which consists of a signal sequence, a cryptic peptide, and the carboxyl terminal 41-amino-acid sequence of CRH. The deduced amino acid sequence of salmon CRH peptide exhibits 66 to 80% homology with mammalian, Xenopus, and sucker CRHs, whereas it shows about 50% homology with sucker, carp, or sole urotensin I, a CRH-related neuropeptide in teleost fish. In situ hybridization histochemistry demonstrated CRH mRNA-positive perikarya in the preoptic nucleus in rainbow trout, Oncorhynchus mykiss, when the fish were stressed by confinement. Adjacent sections hybridized with probes for salmon vasotocin (VT) precursor showed many VT mRNA-positive neurons also in the preoptic nucleus, suggesting a colocalization of CRH and VT mRNAs in the same magnocellular neurons in the rainbow trout brain. The present results suggest that CRH may have important roles in the control of stress responses in salmonid fish.

• Bernier NJ, Lin X, Peter RE
• Differential expression of corticotropin-releasing factor (CRF) and urotensin I precursor genes, and evidence of CRF gene expression regulated by cortisol in goldfish brain.
• Gen Comp Endocrinol. 1999; 116: 461-77
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• Corticotropin-releasing factor (CRF) and urotensin I (UI) precursor cDNAs were cloned and sequenced from a goldfish brain cDNA library in order to investigate the distribution of CRF and UI mRNAs in goldfish brain and the regulation of CRF and UI gene expression. The CRF (966-bp) and UI (769-bp) cDNAs encode 163- and 146-amino acid precursors, respectively, and consist of a signal peptide sequence, a cryptic region, and a 41-amino acid mature peptide at the carboxy terminal. The deduced amino acid sequences of the CRF and UI peptides exhibit a sequence identity of 54%. Northern blot analysis revealed a single size of CRF (1.3 kb) and UI (2.0 kb) mRNAs, which are expressed in the telencephalon-preoptic, hypothalamic, optic tectum-thalamus, and posterior brain regions, but not in the pituitary. In addition, while the CRF gene is strongly expressed in the olfactory bulbs, the UI gene is not. In brain regions in which both genes are expressed, the mRNA levels of CRF were three- to sevenfold higher that those of UI. While the low expression levels of the UI gene prevented further analysis of its regulation, the regulation of CRF gene expression by cortisol was examined. In response to intraperitoneal implants of cortisol (300 microg/g BW) the level of CRF mRNA in the telencephalon-preoptic region decreased to 69% of control values at 6 and 24 h posttreatment. In sham-treated fish, in parallel with a transient injection stress-elicited increase in plasma cortisol, CRF mRNA levels declined to 72% of control value at 6 h postinjection and recovered after 24 h. Injection of the glucocorticoid antagonist, RU-486 (100 microg/g BW), prevented the reduction in CRF gene expression associated with the injection stress at 6 h and increased CRF mRNA levels to 145% of control value after 24 h. In contrast, the various implants had no effect on CRF mRNA levels in either the hypothalamus or the optic tectum-thalamus region. These results provide evidence of differential expression of the CRF and UI genes in hypothalamic and extrahypothalamic regions of goldfish brain. Furthermore, they demonstrate that stress levels of plasma cortisol can lead to a decrease in CRF gene expression that is mediated by glucocorticoid receptors in the telencephalon-preoptic region and give an indication of the regional specificity of the regulation of CRF gene expression by cortisol.

• Perrin MH, Vale WW
• Corticotropin releasing factor receptors and their ligand family.
• Ann N Y Acad Sci. 1999; 885: 312-28
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• The CRF receptors belong to the VIP/GRF/PTH family of G-protein coupled receptors whose actions are mediated through activation of adenylate cyclase. Two CRF receptors, encoded by distinct genes, CRF-R1 and CRF-R2, and that can exist in two alternatively spliced forms, have been cloned. The type-1 receptor is expressed in many areas of the rodent brain, as well as in the pituitary, gonads, and skin. In the rodent, one splice variant of the type-2 receptor, CRF-R2 alpha, is expressed mainly in the brain, whereas the other variant, CRF-R2 beta, is found not only in the CNS, but also in cardiac and skeletal muscle, epididymis, and the gastrointestinal tract. The poor correlation between the sites of expression of CRF-R2 and CRF, as well as the relatively low affinity of CRF for CRF-R2, suggested the presence of another ligand, whose existence was confirmed in our cloning of urocortin. This CRF-like peptide is found not only in brain, but also in peripheral sites, such as lymphocytes. The broad tissue distribution of CRF receptors and their ligands underscores the important role of this system in maintenance of homeostasis. Functional studies of the two receptor types reveal differences in the specificity for CRF and related ligands. On the basis of its greater affinity for urocortin, in comparison with CRF, as well as its brain distribution, CRF-R2 may be the cognate receptor for urocortin. Mutagenesis studies of CRF receptors directed toward understanding the basis for their specificity, provide insight into the structural determinants for hormone-receptor recognition and signal transduction.

• Eckart K et al.
• Actions of CRF and its analogs.
• Curr Med Chem. 1999; 6: 1035-53
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• Corticotropin-releasing factor (CRF), urocortin, sauvagine and urotensin I form the CRF family. These peptides bind with different affinities to two subtypes of CRF receptor (CRFR), CRFR1 and CRFR2. The latter exists as two splice variants, the neuronal CRFR2a and the peripheral CRFR2b. CRFR is a G protein-dependent receptor which acts mainly through Gs enhancing cAMP production. However, CRFR1 expressed in neutrophils of the spleen in response to immunologic stimulation and psychological stress does not seem to function through Gs, as indicated by the inability of CRF to stimulate the cAMP production of CRFR1+ neutrophils. Besides the two receptors, a 37 kD CRF binding protein (CRF-BP) binds several CRF peptides with high affinity. CRFR and CRF-BP do not share a common amino acid sequence representing the ligand binding site. In view of the unusually slow offrate of CRF-BP, it is proposed that CRF-BP provides an efficient uptake of free extracellular CRF. Thus, the time of exposure of CRFR to CRF or urocortin can be limited. At this time, the fate of the ligand CRF-BP complex is unclear. CRFR1 is not only involved in the hypophyseal stimulation of corticotropin release, but hippocampal CRFR1 mediates enhancement of stress-induced learning. CRFR1 may also be involved in basic anxiety. In contrast, at least in the mouse, CRFR2 of the lateral intermediate septum mediates tonic impairment of learning. In response to stressful stimuli or after local injection of high CRF doses, CRFR2 mediates anxiety. Effects requiring CRFR2 can be blocked specifically by the recently developed peptidic antagonist antisauvagine-30.

• Tellam DJ et al.
• Direct regulation of GnRH transcription by CRF-like peptides in an immortalized neuronal cell line.
• Neuroreport. 1998; 9: 3135-40
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• The existence of a CRF-dependent inhibition of GnRH transcription was investigated using a neuronal GnRH-expressing cell line (Gn11) stably transfected with mouse (-611 bp) or chicken (-3000 bp) GnRH promoter/luciferase reporter constructs. The presence of the CRF-R1 receptor was established using a specific CRF-R1 antiserum. After 7 h of incubation, urotensin-I and sauvagine increased the mouse GnRH-reporter bioluminescence by 1.3- and 1.2-fold, respectively, compared with control cells. Subsequently, CRF, urotensin-I and sauvagine decreased luciferase reporter activity to about 60% of the control values after 14 h. Similar trends occurred with the chicken GnRH promoter with UI increasing reporter gene activity 2.4-fold over the controls after 14 h incubation. These data provide additional evidence for the direct regulation of GnRH transcription by CRF-like peptides.

• Sakai K, Yamada M, Horiba N, Wakui M, Demura H, Suda T
• The genomic organization of the human corticotropin-releasing factor type-1 receptor.
• Gene. 1998; 219: 125-30
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• We determined the genomic organization of human CRF type-1 receptor (hCRF-R1). The gene coding for hCRF-R1 consists of at least 14 exons and spans over 20 kilobases. hCRF-R1's three reported isoforms originate from the same gene by alternative splicing. The first hCRF-R1, which binds to CRF with the highest affinity and transduces the most sensitive cAMP accumulation in response to CRF, is encoded in a total of 13 exons, the only one excluded being exon 6. The second isoform contains an additional 29-amino acid sequence which corresponds to exon 6. Unlike the first isoform, the third lacks a 40-amino acid sequence, corresponding to exon 3. Exon-intron boundaries are the same as that of the consensus sequence. Locations of introns in the coding sequence are similar to human CRF-R1, rat CRF-R1, human CRF-R2alpha and others belonging to the human glucagon receptor family.

• Mimmack ML, Parrott RF, Vellucci SV
• Rapid communication: molecular cloning of the porcine corticotropin-releasing factor gene.
• J Anim Sci. 1998; 76: 2205-6

• Yasuda N, Nakamura K
• Heterogeneity of corticotropin-releasing factor (CRF).
• Jpn J Physiol. 1997; 47: 147-59
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• Although CRH is the major contributor to the physiological regulation of ACTH secretion, it must be recognized that a considerable degree of heterogeneity exists regarding the chemistry and biological activity of various CRF-active substances, some of which still remain to be fully clarified. The widespread extrahypothalamic sources and diverse extrapituitary actions of CRFs suggest that multiple CRF components must act in concert for the organism to cope with the environmental changes and stresses.

• Valdenaire O, Giller T, Breu V, Gottowik J, Kilpatrick G
• A new functional isoform of the human CRF2 receptor for corticotropin-releasing factor.
• Biochim Biophys Acta. 1997; 1352: 129-32
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• We have identified the human counterpart of the corticotropin-releasing factor receptor subtype 2beta. Its functional response to human urocortin was demonstrated after stable expression in HEK-293 cells. The receptor was also shown to bind sauvagine, corticotropin-releasing factor and urocortin. In contrast to rodents, the human CRF(2beta) receptor is only weakly expressed in heart and skeletal tissues, where the CRF(2alpha) isoform is predominant. Moreover, we have identified additional mRNAs of the CRF(2beta) type which are probably a consequence of aberrant splicing events.

• Turnbull AV, Rivier C
• Corticotropin-releasing factor (CRF) and endocrine responses to stress: CRF receptors, binding protein, and related peptides.
• Proc Soc Exp Biol Med. 1997; 215: 1-10
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• Corticotropin-releasing factor (CRF) is a 41-amino acid neuropeptide, which is recognized as a critical mediator of complimentary, stress-related endocrine, autonomic, and behavioral responses in mammalian species. CRF belongs to a family of structurally related peptides including frogskin sauvagine and fish urotensin I. The effects of CRF and related peptides are mediated by two distinct receptors, which differ in their anatomical distribution, as well as in their pharmacological characteristics. In addition, CRF is bound with high affinity by a CRF binding protein (CRF-BP), which is a putative inhibitor of CRF action. CRF is probably not the sole endogenous ligand for CRF receptors or the CRF-BP, since a second mammalian member of the CRF family, urocortin, has recently been identified. This article describes recent findings with respect to CRF, its receptors, binding protein, and CRF-related peptides, which provide further insights into the role and mechanisms of CRF action in stress responses.

• Rothemund S, Krause E, Beyermann M, Bienert M
• Hydrophobically induced conformation in ovine corticotropin-releasing hormone.
• J Pept Res. 1997; 50: 184-92
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• Multiple peptide synthesis has been applied for the simultaneous synthesis of systematic replacement sets of model peptides which varied in length from 18 to 36 residues and ovine corticotropin-releasing hormone (oCRH), a 41-residue receptor-binding peptide. The peptides were utilized to analyze the capability of the stationary phase during RP-HPLC to induce secondary structure in long-chain linear peptides. Double D-amino acid replacement studies demonstrate that nonamphipathic helical domains can be recognized, even in the presence of highly amphipathic domains. On the other hand, systematic alteration of hydrophobicity at each residue along the sequence by methionine and methionine sulfoxide replacements results in characteristic pattern of HPLC retention-time differences, which is shown to provide a useful method to probe hydrophobic surface regions in helical peptides. Both amino acid replacement strategies were successfully applied to characterize the hydrophobically induced structure of oCRH. Although an alpha-helix is formed from residues 6 to 32, the N-terminal residues 1-5 and the C-terminal region 33-41 do not show any regular structure. The helical domain from residues 12 to 20 is highly amphipathic.

• Yu J, Xie LY, Abou-Samra AB
• Molecular cloning of a type A chicken corticotropin-releasing factor receptor with high affinity for urotensin I.
• Endocrinology. 1996; 137: 192-7
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• The hypothalamic-pituitary-adrenal axis is an essential physiological system in many species. CRF, the major neuropeptide regulating ACTH secretion, is highly conserved in its primary sequence. Evolutionary conservation of the CRF sequence suggests that the CRF receptor (CRF-R) complementary DNA and examined its properties. The avian CRF-R complementary DNA encodes a 420-amino acid protein that is 87-88% identical to those of human, rat, and mouse. Most sequence divergence occurs in the putative signal peptide and the extracellular amino-terminus of the receptor. Five additional amino acids are inserted in the amino-terminus of the cCRF-R. When expressed in COS-7 cells, the cCRF-R binds the CRF and urotensin I radioligands with high affinities. Urotensin I competes for binding to the chicken CRF-R, expressed in COS-7 cells, with an apparent affinity 20 times higher than that of CRF. Both urotensin I and sauvagine were more effective in stimulating cAMP accumulation in COS-7 cells transfected with the cCRF-R than CRF. The effects of CRF and urotensin I on inositol phosphate accumulation were also tested. Urotensin I was an effective as CRF in stimulating inositol phosphate accumulation in COS-7 cells transfected with the cCRF-R. These data suggest that the sequence of the CRF-R is highly conserved from avian to mammalian species and that, despite its high sequence homology to the type A mammalian CRF-R, the ligand binding properties of cCRF-R are similar to those of the type B CRF-R i.e. a higher affinity for urotensin I than for CRF.

• Donaldson CJ et al.
• Cloning and characterization of human urocortin.
• Endocrinology. 1996; 137: 2167-70
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• Urocortin, a new member of the CRF peptide family which also includes urotensin I and sauvagine, was recently cloned from the rat midbrain. The synthetic replicate of urocortin was found to bind with high affinity to type 1 and type 2 CRF receptors and, based upon its anatomic localization within the brain, was proposed to be a natural ligand for the type 2 CRF receptors. Using a genomic library, we have cloned the human counterpart of rat urocortin and localized it to human chromosome 2. Human and rat urocortin share 95% identity within the mature peptide region. Synthetic human urocortin binds with high affinity to CRF receptor types 1, 2 alpha, and 2 beta, stimulates cAMP accumulation from cells stably transfected with these receptors, and acts in vitro to release ACTH from dispersed rat anterior pituitary cells. In addition, the CRF-binding protein binds human urocortin with high affinity and can prevent urocortin-stimulated ACTH secretion in vitro. The inhibitory effect of the CRF-binding protein on human urocortin can be blocked by biologically inactive CRF fragments, such as CRF(9-33).

• Behan DP et al.
• Characterization of a sheep brain corticotropin releasing factor binding protein (Brain Research 709 (1996) 265-274) (BRES 11959).
• Brain Res. 1996; 732: 267-267

• Lovejoy DA
• Peptide hormone evolution: functional heterogeneity within GnRH and CRF families.
• Biochem Cell Biol. 1996; 74: 1-7
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• Recent investigations indicate that the gonadotropin-releasing hormone (GnRH) and corticotropin-releasing factor (CRF) family of peptides are each composed of at least two functionally discrete paralogous lineages. [His5Trp7Tyr8]GnRH (chicken GnRH-II) is associated with brain neuromodulatory and possibly peripheral endocrine activity, whereas [Arg8]GnRH (mammal GnRH) and its orthologues play major roles as hypothalamic releasing factors. Similarly, CRF appears to be the primary vertebrate ACTH-releasing peptide, whereas the paralogous lineage of urotensin-I-sauvagine has been associated with a variety of diverse peripheral activities. In phylogenetically older species, representatives of both GnRH and CRF family lineages have been characterized. Structural and functional conservation of these peptide systems in vertebrates suggest that additional GnRH-like and CRF-like peptides will be found in the mammal brain.

• Behan DP, De Souza EB, Potter E, Sawchenko P, Lowry PJ, Vale WW
• Modulatory actions of corticotropin-releasing factor-binding protein.
• Ann N Y Acad Sci. 1996; 780: 81-95

• Vaughan J et al.
• Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor.
• Nature. 1995; 378: 287-92
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• 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.

• Behan DP, De Souza EB, Lowry PJ, Potter E, Sawchenko P, Vale WW
• Corticotropin releasing factor (CRF) binding protein: a novel regulator of CRF and related peptides.
• Front Neuroendocrinol. 1995; 16: 362-82
• Display abstract

• A 37-kDa corticotropin releasing factor (CRF) binding protein (CRF-BP) was purified from human plasma by repeated affinity purification and subsequently sequenced and cloned. The human and rat CRF-BP cDNAs encode proteins of 322 amino acids with one putative signal sequence, one N-glycosylation site, and 10 conserved cysteines. Human CRF-BP binds human CRF with high affinity but has low affinity for the ovine peptide. In contrast, sheep CRF-BP binds human and ovine CRF with high affinity. The CRF-BP gene consists of seven exons and six introns and is located on chromosome 13 and loci 5q of the mouse and human genomes, respectively. CRF-BP inhibits the adrenocorticotrophic hormone (ACTH) releasing properties of CRF in vitro. CRF-BP dimerizes after binding CRF and clears the peptide from blood. This clearance mechanism protects the maternal pituitary gland from elevated plasma CRF levels found during the third trimester of human pregnancy. CRF-BP is expressed in the brains of all species so far tested but is uniquely expressed in human liver and placenta. In brain, CRF-BP is membrane associated and is predominantly expressed in the cerebral cortex and subcortical limbic structures. In some brain areas CRF-BP colocalizes with CRF and CRF receptors. The protein is also present in pituitary corticotropes, where it is under positive glucocorticoid control, and is likely to locally modulate CRF-induced ACTH secretion. The ligand requirements of the CRF receptor and the CRF-BP can be distinguished in that central human CRF fragments, such as CRF (6-33) and CRF (9-33), have high affinity for CRF-BP but low affinity for the CRF receptor. The binding protein's ability to inhibit CRF-induced ACTH secretion can be reversed by CRF (6-33) and CRF (9-33), suggesting that ligand inhibitors may have utility in elevating free CRF levels in disease states associated with decreased CRF. Thus, by controlling the amount of free CRF which activates CRF receptors, it is likely that the CRF-BP is an important modulator of CRF both in the CNS and in the periphery.

• Kishimoto T, Pearse RV 2nd, Lin CR, Rosenfeld MG
• A sauvagine/corticotropin-releasing factor receptor expressed in heart and skeletal muscle.
• Proc Natl Acad Sci U S A. 1995; 92: 1108-12
• Display abstract

• Corticotropin-releasing factor (CRF) mediates many critical aspects of the physiological response to stress. These effects are elicited by binding to specific high-affinity receptors, which are coupled to guanine nucleotide stimulatory factor (Gs)-response pathways. Recently, a gene encoding a receptor for CRF, expressed in pituitary and the central nervous system (PC-CRF receptor), was isolated and characterized. Here we report the identification and characterization of a second, distinct CRF receptor that is expressed primarily in heart and skeletal muscle and exhibits a specific ligand preference and antagonist sensitivity compared with the PC-CRF receptor. We refer to this second receptor as the heart/muscle (HM)-CRF receptor.

• Lightman SL
• Corticotropin-releasing factor. From stress to cognition.
• Nature. 1995; 378: 233-4

• Mol JA, van Wolferen M, Kwant M, Meloen R
• Predicted primary and antigenic structure of canine corticotropin releasing hormone.
• Neuropeptides. 1994; 27: 7-13
• Display abstract

• Although the dog has been recognized as a useful model for the study of the cerebrospinal and peripheral actions of corticotropin releasing hormone (CRH) the exact amino acid composition of canine CRH is still unknown. In the present study the structure of canine CRH was predicted from the partial sequence of the gene encoding canine CRH. The CRH gene was amplified from genomic DNA obtained from white blood cells by a polymerase chain reaction and subsequently sequenced using the dideoxy method. The likely structure of canine CRH is: SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-NH2, which is identical to the structure of human, rat and equine CRH. PEPSCAN analysis of 3 different CRH antisera predicted an antiserum raised against a conjugate of human CRH and CNBr -activated thyroglobulin to be the antiserum of choice for the measurement of CRH in the dog. Preliminary data confirmed the existence of the highest cross-reactivity of a canine hypothalamus extract, known to have a high content of CRH, with antisera directed against human, rat CRH. As a result of the present study immunological tools for CRH estimations are characterized. Also, a homologous DNA probe for in situ hybridizations has become available for further investigations.

• Behan DP, Potter E, Sutton S, Fischer W, Lowry PJ, Vale WW
• Corticotropin-releasing factor-binding protein. A putative peripheral and central modulator of the CRF family of neuropeptides.
• Ann N Y Acad Sci. 1993; 697: 1-8

• Grossman A, Costa A, Navarra P, Tsagarakis S
• The regulation of hypothalamic corticotropin-releasing factor release: in vitro studies.
• Ciba Found Symp. 1993; 172: 129-43
• Display abstract

• Although there are various ways in which the regulation of hypothalamic corticotropin-releasing factor (CRF) may be investigated, the most direct is by the study of CRF secretion from rat hypothalami incubated in vitro. Using this technique, we have found stimulation of secretion by noradrenaline, acetylcholine, serotonin, neuropeptide Y, and interleukins 1 and 6; inhibitory modulation was shown by GABA, substance P, atrial natriuretic peptide, opioid peptides and precursors of nitric oxide. Studies of these interactions demonstrated certain non-linear characteristics which may allow appropriate mathematical models to be devised; this may aid in our understanding of clinical disorders associated with CRF excess.

• Stenzel-Poore MP, Heldwein KA, Stenzel P, Lee S, Vale WW
• Characterization of the genomic corticotropin-releasing factor (CRF) gene from Xenopus laevis: two members of the CRF family exist in amphibians.
• Mol Endocrinol. 1992; 6: 1716-24
• Display abstract

• In mammals, the release of pituitary ACTH is stimulated by CRF. Two related peptides exist in nonmammalian vertebrates, sauvagine from frog skin and urotensin-I from the urophysis of teleost fish. Their related structures (approximately 50%) and capacity to stimulate the release of ACTH from mammalian and fish pituitaries has led to the proposal that sauvagine and urotensin-I are homologs of mammalian CRF. However, sauvagine does not appear to stimulate ACTH release in amphibians, although mammalian CRF (ovine) induces a potent response from amphibian pituitaries. This could indicate that the main function of sauvagine does not involve ACTH regulation and suggests that an additional CRF-like peptide exists in Amphibia. We report here the isolation of two highly homologous CRF-like genes from the frog, Xenopus laevis. Analysis of the expression pattern of these CRF-like genes revealed mRNA in splenic tissue and in the preoptic nucleus and paraventricular organ of the brain. The amino acid sequence of the mature peptide regions (1-41) of both X. laevis genes is strikingly conserved, sharing more than 93% homology with mammalian CRFs, yet only 50% homology with sauvagine. In view of the fact that these new amphibian CRF-like genes share far greater homology with mammalian CRF than that exhibited by sauvagine, we propose that the new Xenopus CRF-like genes are the amphibian counterparts to mammalian CRF. Thus, two members of the CRF family have now been identified in the Amphibia, namely CRF and sauvagine.

• Morley SD, Schonrock C, Richter D, Okawara Y, Lederis K
• Corticotropin-releasing factor (CRF) gene family in the brain of the teleost fish Catostomus commersoni (white sucker): molecular analysis predicts distinct precursors for two CRFs and one urotensin I peptide.
• Mol Mar Biol Biotechnol. 1991; 1: 48-57
• Display abstract

• Molecular cloning experiments indicate the presence of two distinct CRF genes in the sucker genome encoding independent 162-amino-acid precursors, which both consist of a signal sequence, succeeded by a cryptic peptide and subsequently by the hormone moiety. The two 41-amino-acid CRF peptides differ by an Ala-->Val substitution at amino acid position 28. CRF transcripts are primarily found in the sucker pre-optic nucleus (PON), to a much lesser extent in the lateral tuberal nucleus (LTN). In contrast, urotensin I (U I) encoding mRNA is equally present in both tissues. In urophysectomized fish, U I mRNA is elevated especially in LTN tissue, while CRF mRNA levels remain more or less constant in the PON and LTN regions.

• Vamvakopoulos NC et al.
• Structural analysis of the regulatory region of the human corticotropin releasing hormone gene.
• FEBS Lett. 1990; 267: 1-5
• Display abstract

• A DNA fragment containing the human corticotropin releasing hormone (CRH) gene, along with 9 kb of upstream and 4 kb of downstream sequences, was isolated from a human genomic DNA library. Nucleotide sequence analysis of the proximal 918 nucleotides 5' flanking the putative major mRNA start site of the human gene and comparison to the 866 nucleotide long homologous ovine sequence, revealed that this region of the CRH gene consists of two distinct areas with different degrees of homology, varying from 72% to 94%. The putative functional features of the human sequence were identified. Many, but not all, features were conserved in the ovine sequence. The highly conserved nature of the regulatory region of this gene makes it a good candidate for tracing possible related genetic defects of the hypothalamic-pituitary-adrenal (HPA) axis.

• Tanaka K, Itoh S, Shimizu N
• [Structure and regulation of expression of corticotropin-releasing hormone (CRH) gene and processing of CRH-precursor]
• Nippon Rinsho. 1989; 47: 2146-51

• Fukuoka SI, Scheele G
• Complementary nucleotide sequence for monitor peptide, a novel cholecystokinin-releasing peptide in the rat.
• Nucleic Acids Res. 1989; 17: 10111-10111

• Thompson RC, Seasholtz AF, Herbert E
• Rat corticotropin-releasing hormone gene: sequence and tissue-specific expression.
• Mol Endocrinol. 1987; 1: 363-70
• Display abstract

• The rat corticotropin releasing hormone (CRH) gene has been isolated and characterized by DNA sequence analysis. The gene exhibits a structural organization similar to that of the human CRH gene. The nucleotide sequence encoding the entire rat CRH precursor is located on the second exon, while exon I encodes the 5'-untranslated region of the mRNA. Analysis of the nucleotide sequence homology between the human and rat CRH genes reveals several highly conserved regions including the CRH peptide-encoding sequence and the 5'-flanking sequence. RNA blot analysis demonstrates that CRH mRNA can be observed in numerous regions of the rat brain as well as the spinal cord, adrenal gland, pituitary, and testis.

• Patthy M, Schlesinger DH, Horvath J, Mason-Garcia M, Szoke B, Schally AV
• Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami.
• Proc Natl Acad Sci U S A. 1986; 83: 2969-73
• Display abstract

• Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin-releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin-releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation.

• Kumahara Y, Tsuchiya H
• [Methods for the analysis of the corticotropin releasing factor]
• Nippon Rinsho. 1986; 44: 497-507

• Yanaihara N, Yanaihara C, Mochizuki T, Iguchi K
• [Chemical structure, biosynthesis and body distribution of the corticotropin releasing factor]
• Nippon Rinsho. 1986; 44: 486-91

• Ishida I, Ichikawa T, Deguchi T
• Cloning and sequence analysis of cDNA encoding urotensin I precursor.
• Proc Natl Acad Sci U S A. 1986; 83: 308-12
• Display abstract

• The primary structure of the precursor of urotensin I, a neuropeptide hormone from the caudal neurosecretory system of the carp Cyprinus carpio, has been determined by analyzing the nucleotide sequence of cloned DNA complementary to the mRNA encoding it. The precursor consists of 145 amino acid residues and the carboxyl terminus represents the 41-amino acid sequence of urotensin I, preceded by Lys-Arg and followed by Gly-Lys. Sequence homology as well as similar organization of the precursors of urotensin I and mammalian corticotropin-releasing factors suggest that they are evolutionarily related. RNA transfer blot analysis indicates that mRNA encoding the precursor of urotensin I is present only in the spinal cord and not in the brain, intestine, liver, or kidney of the carp.

• Patthy M, Horvath J, Mason-Garcia M, Szoke B, Schlesinger DH, Schally AV
• Isolation and amino acid sequence of corticotropin-releasing factor from pig hypothalami.
• Proc Natl Acad Sci U S A. 1985; 82: 8762-6
• Display abstract

• A polypeptide was isolated from acid extracts of porcine hypothalami on the basis of its high ability to stimulate the release of corticotropin from superfused rat pituitary cells. After an initial separation by gel filtration on Sephadex G-25, further purification was carried out by reversed-phase HPLC. The isolated material was homogeneous chromatographically and by N-terminal sequencing. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptide and on carboxypeptidase Y digestion, the primary structure of this 41-residue polypeptide was determined to be Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2. Porcine corticotropin-releasing factor (CRF) shares a common amino acid sequence (residues 1-39) with rat and human CRF and differs from these only in positions 40 and 41. However, isoleucine was also present at position 40 in porcine CRF, but in a smaller percentage than asparagine. The sequence of porcine CRF shows 83% homology with ovine CRF. Porcine CRF markedly stimulated the release of corticotropin from superfused rat and pig pituitary cells. The biological activity and close structural relationship to CRFs of other species indicate that the peptide isolated represents porcine CRF.

• Swanson LW
• Alzheimer's disease and corticotropin-releasing factor.
• JAMA. 1985; 254: 3085-6

• Fujii N et al.
• Studies on peptides. CXXIV. Solution synthesis of the hentetracontapeptide amide corresponding to the entire amino acid sequence of human corticotropin releasing factor (hCRF).
• Chem Pharm Bull (Tokyo). 1984; 32: 4797-805

• Fujii N, Nomizu M, Akaji K, Shimokura M, Katakura S, Yajima H
• Studies on peptides. CXXIII. Preparations of nine peptide fragments for the synthesis of human corticotropin releasing factor (hCRF).
• Chem Pharm Bull (Tokyo). 1984; 32: 4786-96

• Rivier J, Rivier C, Vale W
• Synthetic competitive antagonists of corticotropin-releasing factor: effect on ACTH secretion in the rat.
• Science. 1984; 224: 889-91
• Display abstract

• Polypeptide analogs of the known members of the corticotropin-releasing factor (CRF) family were synthesized and tested in vitro and in vivo for enhanced potency or competitive antagonism. Predictive methods and physicochemical measurements had suggested an internal secondary alpha-helical conformation spanning about 25 residues for at least three members of the CRF family. Maximization of alpha-helix-forming potential by amino acid substitutions from the native known sequences (rat/human and ovine CRF, sauvagine, and carp and sucker urotensin 1) led to the synthesis of an analog that was found to be more than twice as potent as either of the parent peptides in vitro. In contrast, certain amino-terminally shortened fragments, such as alpha-helical CRF or ovine CRF residues 8 to 41, 9 to 41, and 10 to 41, were found to be competitive inhibitors in vitro. Selected antagonists were examined and also found to be active in vivo.

• Fujino M, Kobayashi S
• [Trends in the studies of natural peptides]
• Nippon Rinsho. 1984; 42: 217-27

• Spiess J, Rivier J, Vale W
• Sequence analysis of rat hypothalamic corticotropin-releasing factor with the o-phthalaldehyde strategy.
• Biochemistry. 1983; 22: 4341-6
• Display abstract

• Sequence analysis was performed on a 41-residue polypeptide that has been identified as the predominant form of high intrinsic corticotropin-releasing activity of rat hypothalamus. The sequence of residues 1-39 of this corticotropin-releasing factor (CRF) was determined by Edman degradation of a partially purified peptide in a highly sensitive spinning cup sequencer after selective blocking of CRF or its main contaminant with o-phthalaldehyde. This approach was validated by peptide mapping of CRF of a highly purified preparation. Peptide mapping was accomplished with reverse-phase high-pressure liquid chromatography of CRF fragments obtained by digestion with clostripain. The identities of the fragments cleaved from CRF were established by chromatographic comparison with synthetic peptides, amino acid analysis, and Edman degradation. On the basis of these experiments, the primary structure of rat hypothalamic CRF was established to be H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Se r-Asn - Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. It is expected that the o-phthalaldehyde strategy will facilitate the sequence analysis of partially purified peptides containing proline residues.

• Rivier J, Spiess J, Vale W
• Characterization of rat hypothalamic corticotropin-releasing factor.
• Proc Natl Acad Sci U S A. 1983; 80: 4851-5
• Display abstract

• A polypeptide was purified from rat hypothalamic extracts on the basis of its high intrinsic activity to release corticotropin (ACTH) from cultured rat anterior pituitary cells and its immunoactivity in a radioimmunoassay directed against the NH2 terminus (residues 4-20) of ovine hypothalamic corticotropin-releasing factor (CRF). Based on Edman degradation, peptide mapping, and amino acid analysis, the primary structure of this rat CRF was established to be: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val- Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Le u-Met-Glu-Ile-Ile-NH2. The hypophysiotropic potency of synthetic rat CRF did not deviate significantly from the potencies of the isolated native peptide or of synthetic ovine CRF. The close structural relationship between rat and ovine hypothalamic CRF is indicated by an 83% sequence homology.

• Shibahara S et al.
• Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.
• EMBO J. 1983; 2: 775-9
• Display abstract

• A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.

• Miller RJ
• Corticotropin releasing factor arrives at last.
• Med Biol. 1983; 61: 191-5

• Schulte HM, Chrousos GP, Chatterji DC, Gold PW, Oldfield EH, Loriaux DL
• Safety of corticotropin-releasing factor.
• Lancet. 1983; 1: 1222-1222

• Yasuda N, Greer MA, Aizawa T
• Corticotropin-releasing factor.
• Endocr Rev. 1982; 3: 123-40

• Spiess J, Rivier J, Rivier C, Vale W
• Primary structure of corticotropin-releasing factor from ovine hypothalamus.
• Proc Natl Acad Sci U S A. 1981; 78: 6517-21
• Display abstract

• Sequence analysis was performed of an ovine hypothalamic 41-residue polypeptide that had been postulated to be a putative corticotropin-releasing factor (CRF) because of its high intrinsic corticotropin releasing activity. The NH2-terminal 39 residues of CRF were determined by Edman degradation of 0.6-3.5 nmol of peptide in a Wittmann-Liebold modified Beckman 890C spinning cup sequencer with reverse-phase high-pressure liquid chromatography for the identification of amino acid phenylthiohydantoins (direct micro-sequence analysis). Evidence for residue 40 (isoleucine) was provided by direct micro-sequence analysis of 2.0 nmol of acetylated CRF selectively cleaved at its arginine residues by trypsin prior to analysis. The thermolytic COOH-terminal fragment isoleucyl-alanineamide was characterized as its dansyl derivative. Based on the analytical data, the following primary structure is proposed for ovine hypothalamic CRF: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val- Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Le u-Leu-Asp -Ile-Ala-NH2. In agreement with this proposal, the synthetic replicate of CRF is highly potent in stimulating secretion of both corticotropin and beta-endorphin-like immunoactivities.

• Fink G
• Has corticotropin-releasing factor finally been found?
• Nature. 1981; 294: 511-2

• Hiroshige T
• [Variation of hypothalamic content of corticotropin-releasing factor (CRF) in the regulation of ACTH secretion]
• Nippon Naibunpi Gakkai Zasshi. 1970; 46: 988-9

• Smelik PG, Hedge GA
• Dissociation between production and storage of corticotrophin-releasing factor.
• J Endocrinol. 1969; 43: 0-0

• Hiroshige T, Ito S
• [CRF (corticotropin-releasing factor)]
• Nippon Rinsho. 1969; 27: 1364-73

• Hiroshige T
• [Corticotropin releasing factor (CRF)]
• Nippon Ishikai Zasshi. 1968; 60: 539-51

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