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

Little is known on the structural ligand requirements for corticotropin-rel easing factor binding protein (CRFBP) of the rat used as an important exper imental animal. To obtain such information recombinant rat CRFBP was produc ed in stably transfected HEK 293 cells. The primary structure and posttrans lational processing of purified rat CRFBP was established by peptide mappin g using HPLC combined with mass spectrometric analysis. Rat CRFBP was pharm acologically characterized employing a competition binding assay with triti um-labeled rat urocortin. The rank order of declining affinity of various C RF analogs was urotensin-I, human/rat CRF (h/rCRF), rat urocortin, sauvagin e (Svg), and ovine CRF in agreement with the rant, order found for human CR FBP. In contrast to astressin, the CRF receptor 2-selective antagonist anti -sauvagine-30 did not show any detectable specific binding to rat CRFBP. Th e significance of residues 10 to 12 and 21 to 24 of Svg for its low affinit y binding was established by changing these residues of Svg to those of h/r CRF. The corresponding residues 22 to 25 of h/rCRF represented the ARAE mot if determined to be crucial for binding in agreement with repot ted data on human CRFBP. Residues 11 to 13 of CRF introduced into Svg also enhanced th e affinity to rat CRFBP. (C) 2001 Elsevier Science Inc. All rights reserved .