Pharmacological characterization of recombinant rat corticotropin releasing factor binding protein using different sauvagine analogs

Jahn, Olaf; Eckart, Klaus; Sydow, Sabine; Hofmann, Bernhard; Spiess, Joachim

doi:10.1016/s0196-9781(00)00356-9

Cited by 29 publications

(45 citation statements)

References 39 publications

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“…Mouse UCNII, in contrast, has been reported to have a much greater affinity for CRF-BP than oCRF, with an IC50 in the 5nM range (Jahn et al, 2004, Isfort et al, 2006. Unfortunately, binding affinity for rat UCNII (employed in this study) has not been reported, and there is a Tyr (mouse) -> Asn (rat) substitution within a region known to be important for CRF-BP binding (Jahn et al, 2001). Regardless, it is likely that oCRF and rat UCNII have different affinities for CRF-BP.…”

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confidence: 75%

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Chronic low dose ovine corticotropin releasing factor or urocortin II into the rostral dorsal raphe alters exploratory behavior and serotonergic gene expression in specific subregions of the dorsal raphe

et al. 2007

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Corticotropin releasing factor (CRF) family peptides play key roles in integrating neural responses to stress. Both major CRF receptors have been pharmacologically identified in the dorsal raphe nucleus (DRN), a stress sensitive and internally heterogeneous nucleus supplying many forebrain regions with serotonergic input. Despite the involvement of chronic stress and serotonergic dysfunction in human mood and anxiety disorders, little is known about the effects of chronic CRF receptor activation on the DRN. We infused ovine CRF (1ng/hr), urocortin II (UCNII, 1ng/hr), or vehicle alone into rat DRN over 6 days. During infusion, animals were allowed to freely explore an open field for 15 minutes on each of two days, with the addition of a novel object on the second day. Following behavioral testing, 5-HT 1A , 5-HT 1B , serotonin transporter (SERT), and tryptophan hydroxylase-2 (Tph2) expression were examined through the DRN by in situ hybridization. Ovine CRF infusion resulted in significantly decreased novel object touches, climbs, as well as increased latency to first novel object contact. UCNII had a similar but less dramatic effect, decreasing only climbing behavior. Both ovine CRF and UCNII blunted the decrease in corner time expected on reexposure to the open field. Both peptides also produced regionally specific changes in gene expression: 5-HT 1A expression was increased 30% in the mid-rostral ventromedial DRN, while SERT was decreased by 30% in the mid-caudal shell dorsomedial DRN. There also appeared to be a shift in the relative level of Tph2 expression between the ventromedial and core dorsomedial DRN at the mid-rostral level. Changes in 5-HT 1A , SERT, and relative Tph2 mRNA abundance were correlated with novel object exploration. These findings suggest chronic intra-DRN administration of CRF agonists decreases exploratory behavior, while producing subregionally limited changes in serotonergic gene expression. These studies may be relevant to mechanisms underlying behavioral changes after chronic stress. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptDepression and anxiety disorders are among the leading causes of morbidity, mortality, and disability in the United States (Greenberg et al., 2003). They are often comorbid, and both are highly associated with chronic exposure to stress. Multiple lines of evidence suggest that chronic and inescapable stress produce long lasting effects on brain function and behavior that play a prominent role in the development, maintenance, and recurrence of both depression and a variety of anxiety disorders (Gulley and Nemeroff, 1993, Arborelius et al., 1999, Ressler and Nemeroff, 2000.Altered serotonin neurotransmission appears to be a central mechanism inducing both depressive (Maes and Meltzer, 1995) and anxiety disorders (Ressler and Nemeroff, 2000). The majority of serotonergic fibers to forebrain regions thought to be involved in mood and anxiety regulation arise from the dorsal raphe nucleus (DRN) and median raphe nucleus (MRN) ...

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confidence: 75%

“…oCRF and rat UCNII may have different affinities for CRF-BP. oCRF has been reported to have low affinity, with an IC50 in the 300nM range (Jahn et al, 2001). Mouse UCNII, in contrast, has been reported to have a much greater affinity for CRF-BP than oCRF, with an IC50 in the 5nM range (Jahn et al, 2004, Isfort et al, 2006.…”

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confidence: 99%

Chronic low dose ovine corticotropin releasing factor or urocortin II into the rostral dorsal raphe alters exploratory behavior and serotonergic gene expression in specific subregions of the dorsal raphe

et al. 2007

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“…Peptides were synthesized and purified as described recently, and characterized with a BioIon-20 plasma desorption mass spectrometer (BioIon Nordic, Uppsala, Sweden) (13,14).…”

Section: Methodsmentioning

confidence: 99%

“…rCRFBP was produced in HEK-293 cells stably transfected with cDNA coding for rCRFBP C-terminally fused with a His 6 sequence as described (13). rCRFR1 and mCRFR2␤ were obtained from membrane fractions prepared from HEK-293 cells stably transfected with cDNA coding for rCRFR1 and mCRFR2␤, respectively (14).…”

Section: Methodsmentioning

confidence: 99%

“…Interestingly, each residue of the ARAE sequence contributed to the high affinity of h͞rCRF, as was demonstrated by single residue exchanges (12). Although sauvagine (Svg), another peptide of the CRF peptide family, binds to CRFBP with only low affinity, the Svg analog containing the ARAE sequence, instead of Glu 21 -Lys 22 -Gln 23 -Glu 24 , binds with high affinity (13). However, it has not yet been investigated whether all residues of the ARAE motif are important.…”

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A single amino acid serves as an affinity switch between the receptor and the binding protein of corticotropin-releasing factor: Implications for the design of agonists and antagonists

Eckart

Jahn

Radulovic

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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In view of the observation that corticotropin-releasing factor (CRF) affects several brain functions through at least two subtypes of G protein-dependent receptors and a binding protein (CRFBP), we have developed synthetic strategies to provide enhanced binding specificity. Human͞rat CRF (h͞rCRF) and the CRF-like peptide sauvagine (Svg), differing in their affinities to CRFBP by two orders of magnitude, were used to identify the residues determining binding to CRFBP. By amino acid exchanges, it was found that Ala 22 of h͞rCRF was responsible for this peptide's high affinity to CRFBP, whereas Glu 21 located in the equivalent position of Svg prevented high affinity binding to CRFBP. Accordingly, [Glu 22 ]h͞rCRF was not bound with high affinity to CRFBP in contrast to [Ala 21 ]Svg, which exhibited such high affinity. Furthermore, the affinity of both peptides to either CRF receptor (CRFR) subtype was not reduced by these replacements, and their subtype preference was not changed. Thus, exchange of Ala and Glu and vice versa in positions 22 and 21 of h͞rCRF and Svg, respectively, serves as a switch discriminating between CRFBP and CRFR. On the basis of this switch function, development of new specific CRF agonists and antagonists is expected to be facilitated. One application was the modification of the CRF antagonist astressin (Ast), whose employment in animal experiments is limited by its low solubility in cerebrospinal fluid. Introduction of Glu residues into Ast generated with [Glu 11,16 ]Ast an acidic astressin, which efficiently antagonized in vivo the CRFR1-dependent reduction of locomotion induced by ovine CRF without detectable binding to CRFBP.

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Characterization of peptide?protein interactions using photoaffinity labeling and LC/MS

Jahn

Eckart

Tezval

et al. 2004

Analytical and Bioanalytical Chemistry

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The combination of photoaffinity labeling (PAL) with modern mass spectrometric techniques is a powerful approach for the characterization of peptide-protein interactions. Depending on the analytical strategy applied, a PAL experiment can provide different levels of information ranging from the identification of interaction partners to the structural characterization of ligand-binding sites. On the basis of LC/MS data generated in the framework of the identification of the binding site of the neuropeptide corticotropin-releasing factor (CRF) on its binding protein (CRFBP), the key role of LC/MS in the characterization of photoadducts on different structural levels was demonstrated. Covalent photoadducts of rat CRFBP (rCRFBP) were obtained by PAL with different mono- and bifunctional benzophenone photoprobes designed on the basis of the sequence of the synthetic CRF fragment human/rat CRF(6-33) which binds to CRFBP with high affinity. In view of the stoichiometry, LC/MS analysis revealed that the photoadducts consisted of one molecule of photoprobe and one molecule of rCRFBP. For a further characterization of the photoadducts on the oligopeptide level, enzymatic digests of unlabeled rCRFBP and of the respective photoadduct were compared by peptide mapping monitored with LC/MS. Thereby, it was found that the photoprobe that contained the photophore at its N-terminus labeled the amino acid sequence rCRFBP(34-38), whereas the photoprobe that contained the photophore at its C-terminus labeled rCRFBP(12-26). On the basis of the characterization of the photoadduct formed by rCRFBP and the bifunctional photoprobe that contained photophores on both termini, semiquantitative comparison of different enzymatic digests was accomplished by application of the mass-selective multiple ion chromatogram strategy.

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Pharmacological characterization of recombinant rat corticotropin releasing factor binding protein using different sauvagine analogs

Cited by 29 publications

References 39 publications

Chronic low dose ovine corticotropin releasing factor or urocortin II into the rostral dorsal raphe alters exploratory behavior and serotonergic gene expression in specific subregions of the dorsal raphe

Chronic low dose ovine corticotropin releasing factor or urocortin II into the rostral dorsal raphe alters exploratory behavior and serotonergic gene expression in specific subregions of the dorsal raphe

A single amino acid serves as an affinity switch between the receptor and the binding protein of corticotropin-releasing factor: Implications for the design of agonists and antagonists

Characterization of peptide?protein interactions using photoaffinity labeling and LC/MS

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