Human corticotropin releasing factor binding protein (CRF-BP) ligand structural requirements

Abstract

Human serum contains a 37 kDa CRF-BP which, by binding to CRF, blocks its ability to stimulate hypophysial ACTH release. This study examines requirements of recombinant CRF-BP for its ligands by testing the affinity of CRF-BP for synthetic analogs of CRF and peptides in the CRF family. CRF-BP binds hCRF and Urotensin I with high affinity, Sauvagine with moderate affinity, and oCRF with low affinity. In addition to naturally occurring CRFs, CRF-BP has a high affinity for a-helical CRF(9-41) and a low affinity for [DPhe12Nle21,38]hCRF(12-41), both of which are commonly used as CRF receptor antagonists. The relative affinities of CRF-BP for various CRF analogs indicate residues 6 through 33 are crucial for ligand binding. CRF-BP has little or no affinity (Ki>1000 nM) for amino (hCRF(1-20)) or carboxy (hCRF(21-41)) terminal CRF fragments, yet hCRF(6-33)OH is bound by CRF-BP with a Ki of 3.51 + 0.44 nM. A slightly shorter CRF fragment, hCRF(9-33)OH, is bound by CRF-BP with reduced but significant affinity and exhibits very low potency as a ACTH secretagogue in vitro. hCRF(9-33)OH could therefore be used to competitively inhibit the effects of CRF-BP on hCRF-induced ACTH secretion. A marked difference in the affinity of CRF-BP for oCRF (Ki = 1110 + 97 nM) as compared to hCRF (Ki = 0.166 + .008 nM), combined with the high degree of sequence homology in the critical central domain, suggests amino acids 22 through 25 are critical for binding. Altering oCRF residue 22, 23, or 25 to match hCRF greatly increases the affinity of CRF-BP for these ligands. Substitution of 2 ([Ala22Glu25]oCRF) or 3 ([Ala22Arg23Glu25]oCRF) of these central amino acids at once further increases the affinity of CRF-BP for these analogs, essentially matching that for hCRF.