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Ligand requirements of the human corticotropin-releasing factor-binding protein
S W Sutton1, D P Behan, S L Lahrichi
1Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92138.
Insights
CRF-binding protein (CRF-BP) binds human corticotropin-releasing factor (CRF) and blocks its action. Ligand analysis reveals specific amino acid residues critical for CRF-BP binding, distinguishing it from CRF receptor interactions.
Area of Science:
- Endocrinology
- Molecular Biology
- Biochemistry
Background:
- CRF-binding protein (CRF-BP) is a serum protein that binds human CRF (hCRF) and inhibits its stimulatory effect on ACTH release.
- Understanding the molecular basis of CRF-BP ligand interactions is crucial for deciphering its physiological role and therapeutic potential.
Purpose of the Study:
- To investigate the ligand binding requirements of CRF-BP.
- To differentiate the binding specificities of CRF-BP from those of the CRF receptor.
Main Methods:
- Recombinant CRF-BP was used to test the binding affinity for synthetic analogs of CRF and related peptides.
- Affinity measurements (Kd and Ki) were performed using various CRF fragments and modified peptides.
Main Results:
- CRF-BP exhibits high affinity for human/rat CRF and urotensin-I, moderate affinity for sauvagine, and low affinity for ovine CRF.
- Residues 9-28 of hCRF are crucial for CRF-BP binding, with specific amino acids (22, 23, 25) in the central domain being critical.
- CRF-BP displays differential binding to CRF receptor antagonists, indicating distinct structural requirements compared to receptor recognition.
Conclusions:
- The structural basis for CRF-BP ligand recognition is distinct from CRF receptor binding.
- Pharmacological modulation of CRF-BP activity could offer therapeutic strategies for conditions involving CRF dysregulation by altering free CRF levels.
Abstract:
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.