Kjaer Svend, Ibáñez Carlos F
Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden.
J Biol Chem. 2003 Nov 28;278(48):47898-904. doi: 10.1074/jbc.M309772200. Epub 2003 Sep 26.
The RET receptor tyrosine kinase is activated by binding to a ligand complex formed by a member of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors bound to its cognate GDNF-family receptor-alpha (GFR alpha) glycosylphosphatidylinositol-linked co-receptor. Molecular modeling studies of the extracellular domain of RET (RETECD) have revealed the existence of four cadherin-like domains (CLD1-4) followed by a cysteine-rich domain. Cross-linking experiments have indicated that the RETECD makes direct contacts with both the GDNF ligand and GFR alpha 1 molecule in the complex, although it has low or no detectable affinity for either component alone. We have exploited sequence and functional divergences between the ectodomains of mammalian and amphibian RET molecules to map binding determinants in the human RETECD responsible for its interaction with the GDNF-GFR alpha 1 complex by homologue-scanning mutagenesis. We found that Xenopus RETECD was unable to bind to GDNF-GFR alpha-1 or neurturin (NTN)-GFR alpha-2 complexes of mammalian origin. However, a chimeric molecule containing CLD1, -2, and -3 from human RETECD, but neither domain alone, had similar binding activity as compared with wild type human RETECD, suggesting the existence of an extended ligand binding surface within the three N-terminal cadherin-like domains of human RETECD. Subsequent loss-of-function experiments at higher resolution identified three small subsets of residues, mapping on the same face of the molecular model of RET CLD1, that were required for the interaction of human RETECD with the GDNF-GFR alpha 1 complex. Additional experiments demonstrated that N-linked glycosylation of human RETECD was not required for ligand binding. Based on these observations, we propose a model for the assembly and architecture of the GDNF-GFR alpha 1-RET complex.
RET受体酪氨酸激酶通过与一种配体复合物结合而被激活，该配体复合物由神经胶质细胞系源性神经营养因子（GDNF）家族的一种神经营养因子与其同源的GDNF家族受体α（GFRα）糖基磷脂酰肌醇连接的共受体形成。对RET细胞外结构域（RETECD）的分子建模研究揭示了四个钙黏蛋白样结构域（CLD1 - 4），随后是一个富含半胱氨酸的结构域。交联实验表明，RETECD在复合物中与GDNF配体和GFRα1分子都有直接接触，尽管它对单独的任何一种成分的亲和力都很低或无法检测到。我们利用哺乳动物和两栖动物RET分子胞外结构域之间的序列和功能差异，通过同源扫描诱变来绘制人类RETECD中负责其与GDNF - GFRα1复合物相互作用的结合决定簇。我们发现非洲爪蟾RETECD无法与哺乳动物来源的GDNF - GFRα - 1或神经营养因子（NTN） - GFRα - 2复合物结合。然而，一个包含人类RETECD的CLD1、 - 2和 - 3但不单独包含任何一个结构域的嵌合分子，与野生型人类RETECD相比具有相似的结合活性，这表明在人类RETECD的三个N端钙黏蛋白样结构域内存在一个扩展的配体结合表面。随后在更高分辨率下进行的功能丧失实验确定了三个小的残基子集，它们位于RET CLD1分子模型的同一面上，是人类RETECD与GDNF - GFRα1复合物相互作用所必需的。额外的实验表明，人类RETECD的N - 连接糖基化对于配体结合不是必需的。基于这些观察结果，我们提出了一个GDNF - GFRα1 - RET复合物的组装和结构模型。
