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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

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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 53 Urocortin is the principal ligand for the corticotrophin- releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide S M Baigent and P J Lowry School of Animal and Microbial Sciences, University of Reading, Reading, Berkshire RG6 6AJ, UK (Requests for offprints should be addressed to S M Baigent; Email: [email protected]) ABSTRACT To purify novel ligands for the corticotrophin- Additionally, a 300 bp cDNA fragment sharing 83% releasing factor binding protein (CRF-BP) from homology with the rat urocortin gene was cloned ovine brain, whole brain was homogenised in from ovine brain, the product of which was methanol and the supernatant extracted on Sep-pak predicted to have an identical amino acid sequence C18 cartridges followed by a preliminary HPLC to that of rat urocortin. These pieces of information step. Three peaks of ovine CRF-BP ligand activity confirmed the identity of the human CRF-BP were detected in the HPLC fractions, the first two ligand as an ovine urocortin. The specially of which were also detected by a specific developed CRF-BP ligand assays showed that corticotrophin-releasing factor two-site immuno- the rank orders of affinity of the CRF family radiometric assay, the third peak being detected by members for human CRF-BP were: carp urotensin- a human CRF-BP ligand assay, which will not 1>>human CRF=rat/ovine urocortin>human detect ovine CRF. Human CRF-BP ligand- urocortin>>frog sauvagine>>ovine CRF, and those containing fractions were further purified by affinity for the ovine CRF-BP were: carp urotensin-1> chromatography on a human recombinant CRF-BP human CRF=rat/ovine urocortin>human urocortin> column with two additional HPLC steps. The frog sauvagine>>ovine CRF. This study describes human CRF-BP ligand was found to: (a) possess a a successful technique for the purification and molecular mass of 4707 Daltons, (b) have an detection of peptide ligands for the CRF-BP. We N-terminal amino acid sequence (5 residues) conclude that urocortin is the principal ligand for identical to rat urocortin, (c) be detected by a the CRF-BP in ovine brain and we could find specific urocortin radioimmunoassay, (d) have high no evidence for a centrally located mammalian affinity for both the human and ovine CRF-BPs and sauvagine-like peptide. (e) be present in many regions of the ovine brain. Journal of Molecular Endocrinology (2000) 24, 53–63 INTRODUCTION circulation, and the plasma levels rise progressively to reach a peak at term (Campbell et al. 1987, Sasaki Corticotrophin-releasing factor (CRF) is a 41 amino et al. 1987). This placental CRF is believed to acid neuropeptide which was first isolated from induce parturition (McLean et al. 1995), with a ovine hypothalami in 1981 (Vale et al. 1981). CRF liver-derived circulating CRF binding protein is released from the hypothalamus in response (CRF-BP) (Linton et al. 1988, Behan et al. 1989, to stressful stimuli and its secretion results in Potter et al. 1991) ensuring that any undesirable activation of the hypothalamo–pituitary–adrenal stress response to excess plasma CRF is inactivated axis (Linton et al. 1985), alterations in behavioural during pregnancy (Linton et al. 1990). Plasma characteristics (Mele et al. 1987, Johnson et al. CRF-BP circulates only in higher primates, 1994) and changes in autonomic functions (Gunion although a membrane-bound form resides in the & Taché 1987, Nink et al. 1994). In higher brain of all mammals studied (Potter et al. 1991, primates, peripheral CRF concentrations are Behan et al. 1996a). normally low, but during pregnancy the peptide Anomalies in the relationship between the CRF is released by the placenta into the maternal family members and the CRF-BP led to the belief Journal of Molecular Endocrinology (2000) 24, 53–63 Online version via http://www.endocrinology.org 0952–5041/00/024–053 2000 Society for Endocrinology Printed in Great Britain 54 and · CRF-BP ligands in ovine brain that additional ligands for the CRF-BP may exist in bovine serum albumin, wt/vol, 0·1% sodium azide, mammals. For example, CRF and CRF-BP mRNA wt/vol). do not always co-localise in regions of the brain The h/o CRF two-site immunoradiometric assay (Potter et al. 1992). Additionally, peripheral was a modification of that described by Linton et al. CRF-BP circulates in males and non-pregnant (1987) using an unlabelled rabbit anti-hCRF(1–20) females (Linton et al. 1988) in the absence of ligand antibody in conjunction with a radioiodinated sheep (Linton et al. 1987). The higher affinity of the CRF anti-hCRF(21–41) antibody; oCRF was used as homologue, fish urotensin-1, for the human (h) standard. Complexes were precipitated with a sheep CRF-BP compared with that of hCRF also raises anti-rabbit antibody. This assay is specific for doubts as to whether CRF is the principal ligand hCRF and oCRF. All antibodies were raised, for its binding protein (Lederis et al. 1982, 1983, purified and iodinated in our laboratory. Sutton et al. 1995). The gene encoding a urotensin- For the hCRF-BP ligand assay, hCRF standard like peptide, urocortin, has been cloned from rat (range 0·4–100 ng/ml) or sample was incubated with and human brain regions (Vaughan et al. 1995, 2·5 ng recombinant hCRF-BP (50 µl) (Linton et al. Donaldson et al. 1996). Urocortin possesses a 1993) containing 0·1% Nonidet P-40 (vol/vol) similar affinity to hCRF for the hCRF-BP (Sigma, Poole, Dorset, UK) at room temperature (Vaughan et al. 1995), yet its distribution and for 3 h. After incubation with 125I-hCRF abundance has been reported to be limited both in (35 000 c.p.m./22·7 nCi per tube) overnight at 4 C, the brain (Vaughan et al. 1995, Kozicz et al. 1998) complexes were precipitated with a sheep antibody and in the periphery of rats (Oki et al. 1998). Our raised against the hCRF-BP and also with a donkey experiments help to establish that the addition of anti-sheep antibody. This assay detects high affinity urocortin to the CRF family accounts for the molar ligands for the hCRF-BP including fish urotensin- excesses of CRF-BP over its ligands in the 1, human and rat urocortin and hCRF. The mammalian brain. amphibian homologue, sauvagine, is less readily The aim of this study was to isolate, purify and detected and oCRF is undetectable. All components characterise novel high affinity mammalian ligands for this assay were made in our laboratory except for for the hCRF-BP from ovine brain tissue using the donkey anti-sheep antibody, which was obtained classical biochemical purification techniques and from IGi (Gateshead, Tyne and Wear, UK) and the Edman Degradation amino acid sequencing, fol- standard peptides were kindly donated by Jean lowed by a molecular biological approach to isolate Rivier and co-workers at the Salk Institute, CA, the genes coding for such peptides. Brain was USA. chosen as the starting material because in this tissue In a variation of the above BP ligand assay, disparate distributions of CRF-BP mRNA and oCRF-BP isolated from ovine brain membranes CRF mRNA imply the presence of additional (Behan et al. 1996a) was used instead of recom- CRF-BP ligands (Potter et al. 1992). Ovine tissue binant hCRF-BP. Briefly, the oCRF-BP was was chosen because differences in affinities of the isolated by homogenising 30 g ovine brain in 30 ml ovine (o) CRF-BP for oCRF and hCRF (Behan 10% sucrose containing 1% trasylol (Sigma) and et al. 1996a), might also indicate the existence of a 2 mM phenymethyl-sulphonyl-fluoride (PMSF) novel preferred ligand for the CRF-BP in ovine (Sigma). The homogenate was centrifuged at 3000 brain. Additionally, the very low affinity of oCRF g for 5 min at 4 C to pellet nuclei and particulate for the hCRF-BP (Linton et al. 1988, Sutton et al. matter. The supernatant was centrifuged for 30 min 1995) meant that it would not be expected to at 28 000g at 4 C. The resulting membrane pel- interfere with the hCRF-BP ligand assay used to lets were resuspended in 4 ml 10% sucrose and stored measure novel CRF-BP ligands, or with the affinity at 20 C. Just prior to use 1% Nonidet P-40 was purification procedure involving use of a human added to the thawed membrane preparation and the recombinant CRF-BP column. sample vortexed to dissociate the oCRF-BP from the membranes. The assay was performed with both oCRF and hCRF being used as separate standards. The oCRF-BP ligand assay was better than the MATERIALS AND METHODS hCRF-BP ligand assay at detecting oCRF and sauvagine, as well as the high affinity hCRF- Immunoassays BP ligands. This ‘universal’ assay detected all At each stage of purification, fractions were assayed characterised CRF family members. for both CRF immunoreactivity and hCRF-BP A specific urocortin radioimmunoassay became ligand activity. All reagents were diluted in assay available soon after we had reached the end of the buffer (0·05 M phosphate buffer containing 0·5% purification procedure. Standard urocortin (Salk Journal of Molecular Endocrinology (2000) 24, 53–63 www.endocrinology.org CRF-BP ligands in ovine brain · and 55 Institute, USA) or sample was incubated with a using acetonitrile gradients as described previously, sheep anti-human urocortin antibody (1:64 dilution, and flow rates of 1 ml/min and 250 µl/min respect- 0·1% Nonidet P-40, donated by Roger Smith of ively. Approximately 50 ng purified hCRF-BP the John Hunter Hospital, Newcastle, Australia) at ligand were subjected to electrospray mass spec- 4 C overnight.
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