Sutton S W, Behan D P, Lahrichi S L, Kaiser R, Corrigan A, Lowry P, Potter E, Perrin M H, Rivier J, Vale W W
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92138.
Endocrinology. 1995 Mar;136(3):1097-102. doi: 10.1210/endo.136.3.7867564.
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.
促肾上腺皮质激素释放因子结合蛋白（CRF-BP）被鉴定为一种37千道尔顿的人血清蛋白，它能与人促肾上腺皮质激素释放因子（hCRF）结合（解离常数Kd = 0.17±0.01 nM），并在体外阻断hCRF刺激垂体细胞释放促肾上腺皮质激素（ACTH）的能力。本研究通过检测重组CRF-BP对CRF合成类似物及CRF家族中肽段的亲和力，来研究CRF-BP的配体需求。hCRF或相关肽段的不同片段对CRF-BP的相对亲和力表明，第9至28位氨基酸残基对于配体结合至关重要。CRF-BP与人/大鼠CRF及尾加压素-I具有高亲和力，与蛙皮素具有中等亲和力，与羊促肾上腺皮质激素释放因子（oCRF）具有低亲和力。考虑到中央结构域的重要性，CRF-BP对oCRF（抑制常数Ki = 1100±97 nM）与hCRF（Ki = 0.17±0.01 nM）的亲和力存在显著差异，这表明第22、23和/或25位氨基酸对于结合至关重要。将oCRF的第22、23或25位氨基酸残基单独或共同替换为hCRF的相应残基，可增加CRF-BP对这些配体的亲和力；[丙氨酸22、精氨酸23、谷氨酸25]oCRF中，这三个中央氨基酸均被hCRF的对应氨基酸取代，其与CRF-BP的结合亲和力与hCRF相当。CRF-BP对CRF受体拮抗剂具有不同的亲和力，与α-螺旋CRF-(9-41)具有高亲和力，与[D-苯丙氨酸12、亮氨酸21,38]hCRF-(12-41)具有低亲和力。因此，与CRF-BP结合的结构要求可明显区别于CRF受体对激动剂和拮抗剂的识别要求。诸如hCRF-(9-:33)这类生物活性低但对结合蛋白仍保持高亲和力的肽段，可竞争性地克服CRF-BP阻断CRF诱导的ACTH分泌的作用，这增加了一种可能性，即内源性CRF-BP用于限制CRF的分布或作用持续时间，而配体-结合蛋白相互作用的特异性药理抑制剂或许可用于治疗性提高游离CRF水平。
