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

Abstract

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.

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

“…The lack of the low-mass ion signals at m͞z ϭ 100, 112, and 129 indicated the labeling of the side chain of Arg-23 by [Bp 6,32 ]h͞ rCRF 6 -33 (25)(26)(27)(28)(29)(30)(31)(32)(33). In agreement with the presence of Pro-13 and Asn-20,24 in rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), the low-mass ion signals at m͞z ϭ 70 and 87 (25) were not absent, but their intensities were significantly decreased. The same observations held for the photoadduct fragment rCRFBP(12-26)ϫ[Bp 6,32 ]h͞rCRF 6 -33(25-33) obtained with the monofunctional photoprobe [Bp 32 ]h͞rCRF 6 -33 (data not shown).…”

“…4b) was assigned to rCRFBP(34-38) photolabeled by [Bp 6,32 ]h͞rCRF 6 -33 (6)(7)(8) (M calc ϭ 1,171.43; ⌬M ϭ 110 ppm). Thus, rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and rCRFBP(34-38) were identified as the binding sites of the C-and the N-terminal part of the bifunctional photoprobe, respectively. In agreement with this finding, the monofunctional photoprobes [Bp 32 ]h͞ rCRF 6 -33 and [Bp 6 ]h͞rCRF 6 -33 labeled rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and rCRFBP(34-38), respectively (data not shown).…”

“…Recombinant rCRFBP, containing a C-terminal His-tag, was produced in human embryonic kidney (HEK) 293 cells under serum-free conditions (16). Binding of peptides to rCRFBP was determined in PBS͞ 0.02% Nonidet P-40, using a scintillation proximity assay with [ 125 I-Tyr 0 ]h͞rCRF as radioligand (17).…”

“…The high-energy CID mass spectrum of the photoadduct fragment rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)ϫ[Bp 6,32 ]h͞rCRF 6 -33 (25)(26)(27)(28)(29)(30)(31)(32)(33) did not reveal any fragment ion signals that would be indicative of the cleavage of the peptide backbone of rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Therefore, the CID mass spectra of the photoadduct fragment and of synthetic rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(...…”

“…Therefore, the CID mass spectra of the photoadduct fragment and of synthetic rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) were compared with respect to their low-mass ion regions-as described above (data not shown). The lack of the low-mass ion signals at m͞z ϭ 100, 112, and 129 indicated the labeling of the side chain of Arg-23 by [Bp 6,32 ]h͞ rCRF 6 -33 (25)(26)(27)(28)(29)(30)(31)(32)(33).…”