Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and-5 markedly reduce IGF-I binding and alter their biologic actions
Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and-5 markedly reduce IGF-I binding and alter their biologic actions
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DOI：
10.1074/jbc.m000070200
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发表时间：
2000-06-16
影响因子：
4.8
通讯作者：
Clemmons, DR
中科院分区：
文献类型：
--
作者：
Imai, Y;Moralez, A;Clemmons, DR
胰岛素样生长因子结合蛋白-3和-5（IGFBP-3和-5）已显示以高亲和力结合胰岛素样生长因子-I和-II（IGF-I和-II）。珍贵的研究已经提出IGFBP-5的N-末端区域在残基49和74之间含有高亲和力结合所需的疏水补丁。进行这些研究以确定这些残基中的几个的诱变是否导致IGFBP-3和IGFBP-5对IGF-I的亲和力降低。取代IGFBP-5中的残基68、69、70、73和74（将一个带电残基Lys（68）变为中性残基，将四个疏水残基变为非疏水残基）导致IGFBP-5对IGF-I的亲和力降低约1000倍。IGFBP-3中同源残基的取代也导致亲和力降低>1000倍。这种降低的生理结果是IGFBP-5和IGFBP-5成为IGF-I刺激的细胞迁移和DNA合成的非常弱的抑制剂。同样，IGFBP-5抑制IGF-I刺激的受体磷酸化的能力减弱。这些变化似乎不是因为诱变诱导的蛋白质折叠的改变，因为IGFBP-5突变体对特异性IGFBP-5蛋白酶的蛋白水解切割完全敏感。总之，IGFBP-5中的残基68、69、70、73和74似乎对与IGF-I的高亲和力结合至关重要。IGFBP-3中的Homoprine残基也是必需的，这表明它们形成类似的结合口袋，并且对于两种蛋白质，这些残基形成核心结合位点的重要组分。这些突变体的可用性将使得有可能确定IGFBP-3和-5对分泌IGF-I的细胞类型中的细胞生理过程是否存在直接的、非IGF-I依赖性的作用。
Insulin-like growth factor-binding protein-3 and -5 (IGFBP-3 and -5) have been shown to bind insulin-like growth factor-I and -II (IGF-I and -II) with high affinity. Precious studies have proposed that the N-terminal region of IGFBP-5 contains a hydrophobic patch between residues 49 and 74 that is required for high affinity binding. These studies were undertaken to determine if mutagenesis of several of these residues resulted in a reduction of the affinity of IGFBP-3 and -5 for IGF-I. Substitutions for residues 68, 69, 70, 73, and 74 in IGFBP-5 (changing one charged residue, Lys(68), to a neutral one and the four hydrophobic residues to nonhydrophobic residues) resulted in an similar to 1000-fold reduction in the affinity of IGFBP-5 for IGF-I. Substitutions for homologous residues in IGFBP-3 also resulted in a >1000-fold reduction in affinity, The physiologic consequence of this reduction was that IGFBP-5 and -5 became very weak inhibitors of IGF-I-stimulated cell migration and DNA synthesis. Likewise, the ability of IGFBP-5 to inhibit IGF-I-stimulated receptor phosphorylation was attenuated. These changes did not appear to be because of alterations in protein folding induced by mutagenesis, because the IGFBP-5 mutant was fully susceptible to proteolytic cleavage by a specific IGFBP-5 protease, In summary, residues 68, 69, 70, 73, and 74 in IGFBP-5 appear to be critical for high affinity binding to IGF-I. Homologous residues in IGFBP-3 are also required, suggesting that they form a similar binding pocket and that for both proteins these residues form an important component of the core binding site. The availability of these mutants will make it possible to determine if there are direct, non-IGF-I-dependent effects of IGFBP-3 and -5 on cellular physiologic processes in cell types that secrete IGF-I.