Characterization of a sheep brain corticotropin releasing factor binding protein

Behan, Dominic P.; Cepoi, David; Fischer, Wolfgang; Park, Minkyu; Sutton, Steve W.; Lowry, P. J.; Vale, Wylie

doi:10.1016/0006-8993(95)01317-2

Cited by 25 publications

(14 citation statements)

References 25 publications

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“…The endogenous ovine brain CRF-BP, which shares 86% amino acid homology with the hCRF-BP (Behan et al 1996a), possesses different binding properties to its human counterpart, especially with respect to its greatly enhanced ability to bind sauvagine and oCRF. Sutton et al (1995) , and the substitution of these three key amino acids with the corresponding hCRF residues results in the formation of a high affinity hCRF-BP ligand.…”

Section: Discussionmentioning

confidence: 99%

“…Although these experiments alone do not allow us to compare the affinities of a particular ligand for the hCRF-BP with its affinity for the oCRF-BP, they do enable us to rank the CRF family members separately in order of affinity for the two CRF-BPs. Behan et al (1996a) previously demonstrated that hCRF has similar affinities for the human recombinant CRF-BP and native ovine CRF-BP and, therefore, the positions of the other peptide standard curves in relation to the hCRF standard curves will approximately reflect their relative affinities for the two CRF-BPs. The binding proteins share similar rank orders of potency in their abilities to bind the CRF family of peptides, the order for hCRF-BP being: carp urotensin> > hCRF=rat/ovine urocortin>human urocortin> > sauvagine> >oCRF, and for the oCRF-BP: carp urotensin>hCRF=rat/ovine urocortin>human urocortin>sauvagine> >oCRF.…”

Section: Human and Ovine Crf-bp Ligand Binding Curvesmentioning

confidence: 95%

“…In a variation of the above BP ligand assay, oCRF-BP isolated from ovine brain membranes (Behan et al 1996a) was used instead of recombinant hCRF-BP. Briefly, the oCRF-BP was isolated by homogenising 30 g ovine brain in 30 ml 10% sucrose containing 1% trasylol (Sigma) and 2 mM phenymethyl-sulphonyl-fluoride (PMSF) (Sigma).…”

Section: Immunoassaysmentioning

confidence: 99%

“…This placental CRF is believed to induce parturition (McLean et al 1995), with a liver-derived circulating CRF binding protein (CRF-BP) (Linton et al 1988, Behan et al 1989, Potter et al 1991 ensuring that any undesirable stress response to excess plasma CRF is inactivated during pregnancy (Linton et al 1990). Plasma CRF-BP circulates only in higher primates, although a membrane-bound form resides in the brain of all mammals studied (Potter et al 1991, Behan et al 1996a.…”

Section: Introductionmentioning

confidence: 99%

“…Brain was chosen as the starting material because in this tissue disparate distributions of CRF-BP mRNA and CRF mRNA imply the presence of additional CRF-BP ligands (Potter et al 1992). Ovine tissue was chosen because differences in affinities of the ovine (o) CRF-BP for oCRF and hCRF (Behan et al 1996a), might also indicate the existence of a novel preferred ligand for the CRF-BP in ovine brain. Additionally, the very low affinity of oCRF for the hCRF-BP (Linton et al 1988 meant that it would not be expected to interfere with the hCRF-BP ligand assay used to measure novel CRF-BP ligands, or with the affinity purification procedure involving use of a human recombinant CRF-BP column.…”

Section: Introductionmentioning

confidence: 99%

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Urocortin is the principal ligand for the corticotrophin-releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide

Baigent¹,

Lowry²

2000

Journal of Molecular Endocrinology

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To purify novel ligands for the corticotrophinreleasing 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 ligandcontaining 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.

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Section: Discussionmentioning

confidence: 99%

Section: Human and Ovine Crf-bp Ligand Binding Curvesmentioning

confidence: 95%

Section: Immunoassaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Urocortin is the principal ligand for the corticotrophin-releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide

Baigent¹,

Lowry²

2000

Journal of Molecular Endocrinology

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Characterization of native corticotropin-releasing factor receptor type 1 (cRFR1) in the rat and mouse central nervous system

1998

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Corticotropin releasing factor (CRF), the most important regulator of various responses to stress, acts through CRF receptors (CRFR). For their characterization in brain tissue of Sprague-Dawley rats and C57BL/6J mice, a recently described polyclonal antibody directed against the N-terminus of rat CRFR1 (rCRFR1) was used. The molecular weights of rat and mouse brain receptors were determined by Western blot analysis to be 80,000-76,000 and 83,000-79,000, respectively, whereas molecular weights of 72,000-59,000 were observed for CRFR1 from rat and mouse pituitary. Immunohistochemical analysis was performed with brain sections of naive rats and mice. Strong CRFR1 staining was detected in the cortex, cerebellum, mesencephalon and pons of both species, whereas weak staining was observed in amygdala and hippocampus. The striatum did not show immunoreactivity. The density of immunostaining was significantly lower in murine than in rat cortex. In contrast, in the pons and mesencephalon of mice, higher density of immunostaining was observed than in the same brain structures of rats. On the basis of the observed differences, it is suggested that CRFR1 is differentially processed in rats and mice. In addition, the density of CRFR1 staining differed between both species.

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Urocortin interaction with corticotropin-releasing factor (CRF) binding protein (CRF-BP) : a novel mechanism for elevating ‘free’ CRF levels in human brain

et al. 1996

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Characterization of a sheep brain corticotropin releasing factor binding protein

Cited by 25 publications

References 25 publications

Urocortin is the principal ligand for the corticotrophin-releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide

Urocortin is the principal ligand for the corticotrophin-releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide

Characterization of native corticotropin-releasing factor receptor type 1 (cRFR1) in the rat and mouse central nervous system

Urocortin interaction with corticotropin-releasing factor (CRF) binding protein (CRF-BP) : a novel mechanism for elevating ‘free’ CRF levels in human brain

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