Virtanen Heidi, Yang Jianmin, Bespalov Maxim M, Hiltunen Jukka O, Leppänen Veli-Matti, Kalkkinen Nisse, Goldman Adrian, Saarma Mart, Runeberg-Roos Pia
Institute of Biotechnology, Viikinkaari 9, P.O. Box 56, Viikki Biocenter, University of Helsinki, FIN-00014 Helsinki, Finland.
Biochem J. 2005 May 1;387(Pt 3):817-24. doi: 10.1042/BJ20041257.
The GDNF (glial cell line-derived neurotrophic factor)-binding receptor GFRalpha1 (GDNF family receptor alpha1) is attached to the membrane by a GPI (glycosylphosphatidylinositol) anchor and consists of three cysteine-rich domains. The region corresponding to the second and third domains has been shown previously to participate in ligand binding, and to interact with the transmembrane tyrosine kinase receptor RET. No function has so far been found for the N-terminal, first domain (D1). Here we show that the GPI-anchored full-length receptor binds 125I-GDNF two times more tightly than does a GPI-anchored truncated receptor lacking D1. Scintillation proximity assays with purified receptor proteins also show that the GDNF-binding capacity of the soluble full-length GFRalpha1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRalpha1. As RET stabilizes the binding of GDNF equally well to the full-length and truncated receptors, D1 seems not to be involved in the interaction between GFRalpha1 and RET. Moreover, soluble full-length GFRalpha1 mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRalpha1 protein. At low concentrations, the soluble fulllength receptor mediates the phosphorylation of RET more efficiently than the soluble truncated receptor. However, when the receptors are overexpressed on the cell surface as GPI-anchored proteins, or added to the growth medium at high concentrations as soluble proteins, full-length and truncated GFRalpha1 are indistinguishable in GDNF-dependent RET-phosphorylation assays. High levels of the receptors can thus mask a slightly impaired function in the phosphorylation assay. Based on assays with both GPI-anchored and soluble receptors, we therefore conclude that D1 contributes to the optimal function of GFRalpha1 by stabilizing the interaction between GFRalpha1 and GDNF.
胶质细胞系源性神经营养因子（GDNF）结合受体GFRα1（GDNF家族受体α1）通过糖基磷脂酰肌醇（GPI）锚定在细胞膜上，由三个富含半胱氨酸的结构域组成。先前已表明，对应于第二和第三结构域的区域参与配体结合，并与跨膜酪氨酸激酶受体RET相互作用。到目前为止，尚未发现N端的第一个结构域（D1）有任何功能。在此我们表明，GPI锚定的全长受体与125I-GDNF的结合比缺乏D1的GPI锚定截短受体紧密两倍。用纯化的受体蛋白进行的闪烁邻近分析也表明，可溶性全长GFRα1的GDNF结合能力比可溶性D1截短的GFRα1的GDNF结合能力高两倍。由于RET同样能很好地稳定GDNF与全长和截短受体的结合，D1似乎不参与GFRα1与RET之间的相互作用。此外，可溶性全长GFRα1比可溶性截短的GFRα1蛋白更有效地介导GDNF促进PC6-3细胞中的神经突生长。在低浓度下，可溶性全长受体比可溶性截短受体更有效地介导RET的磷酸化。然而，当受体作为GPI锚定蛋白在细胞表面过表达，或作为可溶性蛋白以高浓度添加到生长培养基中时，在GDNF依赖性RET磷酸化分析中，全长和截短的GFRα1没有区别。因此，高水平的受体可以掩盖磷酸化分析中略有受损的功能。基于对GPI锚定和可溶性受体的分析，我们因此得出结论，D1通过稳定GFRα1与GDNF之间的相互作用，有助于GFRα1的最佳功能。
