LIGAND REQUIREMENTS OF THE HUMAN CORTICOTROPIN-RELEASING FACTOR-BINDING PROTEIN

CRF-binding protein (CRF-BP), identified as a 37-kilodalton human seru m protein, binds human (h) CRF (k(d) = 0.17 +/- nM) and blocks hCRF's ability to stimulate ACTH release by pituitary cells in vitro. The pre sent study examines ligand requirements of CRF-BP by testing the affin ity of recombinant CRF-BP for synthetic analogs of CRF and peptides in the CRF family. The relative affinities of various fragments of hCRF or related peptides for CRF-BP indicate that residues 9-28 are crucial for ligand binding. CRF-BP binds human/ rat CRF and urotensin-I with high affinity, sauvagine with moderate affinity, and ovine (o) CRF wit h low affinity. The marked difference in the affinity of CRF-BP for oC RF (K-i = 1100 +/- 97 nM) compared to hCRF (K-i = 0.17 +/- 0.01 nM), w hen considered with the importance of the central domain, suggests tha t amino acids 22, 23, and/or 25 are critical for binding. Altering oCR F residues 22, 23, or 25 individually or collectively to match those o f hCRF increases the affinity of CRF-BP for these ligands; [Ala(22),Ar g(23),Glu(25)]oCRF, in which all three of these central amino acids ar e substituted by their hCRF counterparts, binds CRF-BP with an affinit y equal to that of hCRF. CRF-BP has differential affinities for CRF re ceptor antagonists, binding alpha-helical CRF-(9-41) with high affinit y and [D-Phe(12),Nle(21,38)]hCRF-(12-41) with low affinity. Thus, the structural requirements for binding to CRF-BP can clearly be distingui shed from those for CRF receptor recognition of both agonists and anta gonists. Peptides such as hCRF-(9-33), with low biological activity bu t which retain high affinity for the binding protein, can competitivel y override the effects of CRF-BP to block CRF-induced ACTH secretion, raising the possibility that whereas endogenous CRF-BP serves to limit the distribution or duration of action of CRF, specific pharmacologic al inhibitors of the ligand-binding protein interaction might be used to therapeutically elevate free CRF levels.