Corticotropin-releasing factor binding protein--a ligand trap?

The actions of the neuropeptide corticotropin-releasing factor (CRF) are modulated by a CRF binding protein (CRFBP). In view of the memory-enhancing effects of CRF, the release of endogenous CRF from CRFBP by CRFBP inhibitors has been suggested as a therapeutical strategy for the treatment of cognitive deficits. This mini-review will summarize recent advances in the field with a focus on the pharmaceutical potential of CRFBP inhibitors.

Three-amino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity.

Corticotropin-releasing factor (CRF) and the CRF-like peptide urocortin I (UcnI) exert their activity through two different CRF receptors, CRF1 and CRF2. Recently, UcnII and UcnIII have been discovered as potential endogenous agonists selective for CRF2 known to be involved in brain functions such as learning and anxiety, as well as in cardiovascular functions. A structure-affinity relationship study using chimeric peptides was designed to characterize mouse UcnII (mUcnII) and mUcnIII further and to investigate the structural basis of their receptor subtype selectivity. In the framework of this study, mUcnII (IC50 = 4.4 nM) but not mUcnIII was identified as high-affinity ligand for the rat CRF binding protein. Such affinity had previously not been observed for the human version of this protein. On the basis of secondary structure predictions, it was hypothesized that the amino acid motifs Pro-Ile-Gly of mUcnII and Pro-Thr-Asn of mUcnIII decrease alpha-helicity and thereby impair binding to CRF1. In support of this hypothesis, binding affinity to CRF1 of the chimeric peptides [Pro11Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/rCRF, and the corresponding rUcnI analogs was found to be decreased by three orders of magnitude, whereas binding affinity to CRF2 was much less affected. The dramatic decrease in binding affinity to CRF1 correlated with a decrease in alpha-helicity as indicated by the data of circular dichroism spectroscopy.

Cortagine, a specific agonist of corticotropin-releasing factor receptor subtype 1, is anxiogenic and antidepressive in the mouse model.

Two subtypes of the corticotropin-releasing factor (CRF) receptor, CRF(1) and CRF(2), differentially modulate brain functions such as anxiety and memory. To facilitate the analysis of their differential involvement, we developed a CRF(1)-specific peptidic agonist by synthesis of chimeric peptides derived from human/rat CRF, ovine CRF (oCRF), and sauvagine (Svg). High affinity to the CRF-binding protein was prevented by introduction of glutamic acid in the binding site of the ligand. The resulting chimeric peptide, [Glu(21),Ala(40)][Svg(1-12)]x[human/rat CRF(14-30)]x[Svg(30-40)], named cortagine, was analyzed pharmacologically in cell culture by using human embryonic kidney-293 cells transfected with cDNA coding for CRF(1) or CRF(2), in autoradiographic experiments, and in behavior experiments using male C57BL/6J mice for its modulatory action on anxiety- and depression-like behaviors with the elevated plus-maze test and the forced swim test (FST), respectively. We observed that cortagine was more selective than oCRF, frequently used as CRF(1)-specific agonist, in stimulating the transfected cells to release cAMP. Cortagine's specificity was demonstrated in autoradiographic experiments by its selective binding to CRF(1) of brain sections of the mouse. After injection into the brain ventricles, it enhanced anxiety-like behavior on the elevated plus-maze at a lower dose than oCRF. Whereas at high doses, oCRF injected into the lateral intermediate septum containing predominantly CRF(2) increased anxiety-like behavior as CRF(2)-specific agonists do, cortagine did not. In contrast to its anxiogenic actions, cortagine reduced significantly the immobility time in the FST as described for antidepressive drugs. Thus, cortagine combines anxiogenic properties with antidepressive effects in the FST.

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

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.

The binding protein of corticotropin-releasing factor: ligand-binding site and subunit structure.

Corticotropin-releasing factor (CRF), recognized as an important stress factor, binds to a CRF receptor and a CRF-binding protein (CRFBP) that represents a reservoir of endogenous CRF. Although CRFBP was observed to dimerize, at least in part, the ligand was found to be exclusively bound to the monomer-as indicated by photoaffinity labeling. We localized the CRF binding site by using photoaffinity labeling in combination with different mass spectrometric techniques. The amino acid residues Arg-23 and Arg-36 of CRFBP were identified as the sites of photoincorporation of monofunctional and bifunctional photoprobes designed on the basis of the amino acid sequence of human/rat CRF(6-33). It was, therefore, concluded that the sequence of amino acid residues 23-36 of CRFBP is involved in ligand binding. Our data are in support of an antiparallel alignment of the photoprobe with the amino acid residues 23-36 of the CRFBP monomer.

Priming of long-term potentiation in mouse hippocampus by corticotropin-releasing factor and acute stress: implications for hippocampus-dependent learning.

In the present experiments, we characterized the action of human/rat corticotropin-releasing factor (h/rCRF) and acute stress (1 hr of immobilization) on hippocampus-dependent learning and on synaptic plasticity in the mouse hippocampus. We first showed that h/rCRF application and acute stress facilitated (primed) long-term potentiation of population spikes (PS-LTP) in the mouse hippocampus and enhanced context-dependent fear conditioning. Both the priming of PS-LTP and the improvement of context-dependent fear conditioning were prevented by the CRF receptor antagonist [Glu(11,16)]astressin. PS-LTP priming and improved learning were also reduced by the protein kinase C inhibitor bisindolylmaleimide I. Acute stress induced the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) 2 hr after the end of the stress session. The CaMKII inhibitor KN-62 antagonized the stress-mediated learning enhancement, however, with no effect on PS-LTP persistence. Thus, long-lasting increased neuronal excitability as reflected in PS-LTP priming appeared to be essential for the enhancement of learning in view of the observation that inhibition of PS-LTP priming was associated with impaired learning. Conversely, it was demonstrated that inhibition of CaMKII activity reduced contextual fear conditioning without affecting PS-LTP priming. This observation suggests that priming of PS-LTP and activation of CaMKII represent two essential mechanisms that may contribute independently to long-term memory.

Synthesis and Properties of Diuridine Phosphate Analogues Containing Thio and Amino Modifications.

Several analogues of diuridine phosphate (UpU) were synthesized in order to investigate why replacing the 2'-hydroxyl with a 2'-amino group prevents hydrolysis. These analogues were designed to investigate what influence the 2'-substituent and 5'-leaving group have upon the rate of hydrolysis. All the analogues were considerably more labile than UpU toward acid-base-catalyzed hydrolysis. In the pH region from 6 to 9, the rate of hydrolysis of uridylyl (3'-5') 5'-thio-5'-deoxyuridine (UpsU) hydrolysis rose, in a log linear fashion, from a value of 5 x 10(-)(6) s(-)(1) at pH 6 to 3200 x 10(-)(6) s(-)(1) at pH 9, indicating that attack on the phosphorus by the 2'-oxo anion is rate-limiting in the hydrolysis mechanism. In contrast, the rate of uridylyl (3'-5') 5'-amino-5'-deoxyuridine (UpnU) hydrolysis fell from a value of 1802 x 10(-)(6) s(-)(1) at pH 5 to 140 x 10(-)(6) s(-)(1) at pH 7.5, where it remained constant up to pH 11.5, thus indicating an acid-catalyzed reaction. The analogue 2'-amino-2'-deoxyuridylyl (3'-5') 5'-thio-5'-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stability of amUpT, with rates between 85 x 10(-)(6) s(-)(1) and 138 x 10(-)(6) s(-)(1). The hydrolysis of 2'-amino-2'-deoxyuridylyl (3'-5') 5'-amino-5'-deoxythymidine (amUpnT) rose from 17 x 10(-)(6) s(-)(1) at pH 11.5 to 11 685 x 10(-)(6) s(-)(1) at pH 7.0, indicating an acid-catalyzed reaction, where protonation of the 5'-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg(2+), Zn(2+), and Cd(2+) ions, but a correlation with interaction between metal ion and leaving group could only be demonstrated for amUpsU. UpsU and UpnU are also substrates for RNase A with UpsU having similar Michaelis-Menten parameters to UpU. In contrast, UpnU is more rapidly degraded with an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of k(cat).