Anders J, Kjar S, Ibáñez C F
Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden.
J Biol Chem. 2001 Sep 21;276(38):35808-17. doi: 10.1074/jbc.M104968200. Epub 2001 Jul 9.
Using bioinformatic tools, mutagenesis, and binding studies, we have investigated the structural organization of the extracellular region of the RET receptor tyrosine kinase, a functional receptor for glial cell line-derived neurotrophic factor (GDNF). Multiple sequence alignments of seven vertebrate sequences and one invertebrate RET sequence delineated four distinct N-terminal domains, each of about 110 residues, containing many of the consensus motifs of the cadherin fold. Based on these alignments and the crystal structures of epithelial and neural cadherins, we have generated molecular models of each of the four cadherin-like domains in the extracellular region of human RET. The modeled structures represent realistic models from both energetic and geometrical points of view and are consistent with previous observations gathered from biochemical analyses of the effects of Hirschsprung's disease mutations affecting the folding and stability of the RET molecule, as well as our own site-directed mutagenesis studies of RET cadherin-like domain 1. We have also investigated the role of Ca(2+) in ligand binding by RET and found that Ca(2+) ions are required for RET binding to GDNF but not for GDNF binding to the GFRalpha1 co-receptor. In agreement with these results, RET, but not GFRalpha1, was found to bind Ca(2+) directly. Our results indicate that the overall architecture of the extracellular region of RET is more closely related to cadherins than previously thought. The models of the cadherin-like domains of human RET represent valuable tools with which to guide future site-directed mutagenesis studies aimed at identifying residues involved in ligand binding and receptor activation.
我们使用生物信息学工具、诱变技术和结合研究，对胶质细胞系源性神经营养因子（GDNF）的功能性受体RET受体酪氨酸激酶细胞外区域的结构组织进行了研究。对七个脊椎动物序列和一个无脊椎动物RET序列进行的多序列比对确定了四个不同的N端结构域，每个结构域约含110个残基，包含许多钙黏蛋白折叠的共有基序。基于这些比对以及上皮钙黏蛋白和神经钙黏蛋白的晶体结构，我们构建了人类RET细胞外区域四个类钙黏蛋白结构域各自的分子模型。从能量和几何角度来看，这些建模结构都代表了现实可行的模型，并且与先前从对影响RET分子折叠和稳定性的先天性巨结肠症突变效应的生化分析中收集到的观察结果一致，也与我们自己对RET类钙黏蛋白结构域1的定点诱变研究结果相符。我们还研究了Ca(2+)在RET配体结合中的作用，发现Ca(2+)离子是RET与GDNF结合所必需的，但不是GDNF与GFRalpha1共受体结合所必需的。与这些结果一致，发现RET能直接结合Ca(2+)，而GFRalpha1则不能。我们的结果表明，RET细胞外区域的整体结构与钙黏蛋白的关系比之前认为的更为密切。人类RET类钙黏蛋白结构域的模型是有价值的工具，可用于指导未来旨在确定参与配体结合和受体激活的残基的定点诱变研究。
