Mapping Sonic hedgehog-receptor interactions by steric interference
Mapping Sonic hedgehog-receptor interactions by steric interference
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DOI：
10.1074/jbc.275.15.10995
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发表时间：
2000-04-14
影响因子：
4.8
通讯作者：
Garber, EA
中科院分区：
文献类型：
--
作者：
Pepinsky, RB;Rayhorn, P;Garber, EA
我们已经定义了区域的Sonic刺猬（Shh）分子是重要的补丁（Ptc）受体结合靶向选定的表面氨基酸残基与不同大小和形状的探针，并评估这些修改功能的影响。围绕蛋白质表面的11个氨基酸残基被选择用于这些研究，并突变为半胱氨酸残基。然后用巯基特异性探针选择性地修饰这些半胱氨酸，并测试修饰的蛋白质的刺猬受体结合活性及其诱导C3 H10 T1/2细胞分化成成骨细胞的能力。基于这些分析，无论附着的大小，大约三分之一的Shh表面可以被修饰而不影响功能。这些位点位于C末端从蛋白质表面突出的位置附近。所有其他网站的修改敏感，表明Shh与其主要受体Ptc的相互作用是介导的Shh蛋白的大表面。对于位点Asn-50和Ser-156，用最小的测试探针丧失功能，表明这些残基非常接近Pt c结合位点。中和mAb 5E 1的表位定位于结构的接近但不同的区域。的结构活性数据提供了一个独特的视图之间的相互作用Shh和铂，这是不容易实现的常规映射策略。
We have defined regions in the Sonic hedgehog (Shh) molecule that are important for Patched (Ptc) receptor binding by targeting selected surface amino acid residues with probes of diverse sizes and shapes and assessing the effects of these modifications on function. Eleven amino acid residues that surround the surface of the protein were chosen for these studies and mutated to cysteine residues. These cysteines were then selectively modified with thiol-specific probes, and the modified proteins were tested for hedgehog receptor binding activity and their ability to induce differentiation of C3H10T1/2 cells into osteoblasts, Based on these analyses, approximately one-third of the Shh surface can be modified without effect on function regardless of the size of the attachment. These sites are located near to where the C terminus protrudes from the surface of the protein. All other sites were sensitive to modification, indicating that the interaction of Shh with its primary receptor Ptc is mediated over a large surface of the Shh protein. For sites Asn-50 and Ser-156, function was lost with the smallest of the probes tested, indicating that these residues are in close proximity to the Ptc-binding site. The epitope for the neutralizing mAb 5E1 mapped to a close but distinct region of the structure. The structure-activity data provide a unique view of the interactions between Shh and Ptc that is not readily attainable by conventional mapping strategies.