Ligand requirements of the human corticotropin-releasing factor-binding protein.
CRF-binding protein (CRF-BP), identified as a 37-kilodalton human serum protein, binds human (h) CRF (Kd = 0.17 +/- 0.01 nM) and blocks hCRF's ability to stimulate ACTH release by pituitary cells in vitro. The present study examines ligand requirements of CRF-BP by testing the affinity 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 with low affinity. The marked difference in the affinity of CRF-BP for oCRF (Ki = 1100 +/- 97 nM) compared to hCRF (Ki = 0.17 +/- 0.01 nM), when considered with the importance of the central domain, suggests that amino acids 22, 23, and/or 25 are critical for binding. Altering oCRF residues 22, 23, or 25 individually or collectively to match those of hCRF increases the affinity of CRF-BP for these ligands; [Ala22, Arg23, Glu25]oCRF, in which all three of these central amino acids are substituted by their hCRF counterparts, binds CRF-BP with an affinity equal to that of hCRF. CRF-BP has differential affinities for CRF receptor antagonists, binding alpha-helical CRF-(9-41) with high affinity and [D-Phe12, Nle21,38]hCRF-(12-41) with low affinity. Thus, the structural requirements for binding to CRF-BP can clearly be distinguished from those for CRF receptor recognition of both agonists and antagonists. Peptides such as hCRF-(9-33), with low biological activity but which retain high affinity for the binding protein, can competitively 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 pharmacological inhibitors of the ligand-binding protein interaction might be used to therapeutically elevate free CRF levels.
Sutton
S W
SW
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92138.
Behan
D P
DP
Lahrichi
S L
SL
Kaiser
R
R
Corrigan
A
A
Lowry
P
P
Potter
E
E
Perrin
M H
MH
Rivier
J
J
Vale
W W
WW
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003346:Corticotropin-Releasing Hormone / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism; D011994:Recombinant Proteins
D002352:Carrier Proteins; D008024:Ligands; D010446:Peptide Fragments; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; C067133:corticotropin releasing factor-binding protein; D003346:Corticotropin-Releasing Hormone