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

In view of the observation that corticotropin-releasing factor (CRF) affect s several brain functions through at least two subtypes of G protein-depend ent receptors and a binding protein (CRFBP), we have developed synthetic st rategies to provide enhanced binding specificity. Human/rat CRF (h/rCRF) an d the CRF-like peptide sauvagine (Svg), differing in their affinities to CR FBP by two orders of magnitude, were used to identify the residues determin ing binding to CRFBP. By amino acid exchanges, it was found that Ala(22) of h/rCRF was responsible for this peptide's high affinity to CRFBP, whereas Glu(21) located in the equivalent position of Svg prevented high affinity b inding to CRFBP. Accordingly, [Glu(22)]h/rCRF was not bound with high affin ity to CRFBP in contrast to [Ala(21)]Svg, which exhibited such high affinit y. Furthermore, the affinity of both peptides to either CRF receptor (CRFR) subtype was not reduced by these replacements, and their subtype preferenc e was not changed. Thus, exchange of Ala and Glu and vice versa in position s 22 and 21 of h/rCRF and Svg, respectively, serves as a switch discriminat ing between CRFBP and CRFR. On the basis of this switch function, developme nt of new specific CRF agonists and antagonists is expected to be facilitat ed. One application was the modification of the CRF antagonist astressin (A st), whose employment in animal experiments is limited by its low solubilit y in cerebrospinal fluid. Introduction of Glu residues into Ast generated w ith [GIu(11,16)]Ast an acidic astressin, which efficiently antagonized in v ivo the CRFR1-dependent reduction of locomotion induced by ovine CRF withou t detectable binding to CRFBP.