Structural basis by which alternative splicing modulates the organizer activity of FGF8 in the brain
Structural basis by which alternative splicing modulates the organizer activity of FGF8 in the brain
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DOI：
10.1101/gad.1365406
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发表时间：
2006-01-15
影响因子：
10.5
通讯作者：
Mohammadi, M
中科院分区：
文献类型：
--
作者：
Olsen, SK;Li, JYH;Mohammadi, M
四种人类FGF8剪接异构体中的两种，FGF8a和FGF8b，在发育过程中在中后脑区域表达。虽然这些异构体之间的唯一区别是在FGF8b的N端存在另外11个氨基酸，但这些异构体对中脑和后脑前部的模式形成能力明显不同。为了揭示选择性剪接调控FGF8组织活性的结构基础，我们将FGF8b的晶体结构与成纤维细胞生长因子受体2(FGFR2c)的“c”剪接异构体(FGFR2c)结合起来。利用表面等离子体共振(SPR)技术，我们还研究了FGF17(FGF17b)的b亚型FGF8a和FGF8b以及FGF18的受体结合特异性。FGF8b-FGFR2c结构表明，替代剪接允许FGF8b的苯丙氨酸32(F32)与受体Ig结构域3内的疏水凹槽之间的单个额外接触，该凹槽也存在于FGFR1c、FGFR3c和FGFR4中。与结构一致的是，F32突变为丙氨酸将FGF8b对所有这些受体的亲和力降低到Fgf8a特有的水平。更重要的是，对FGF8b(F32a)突变体在鸡胚胎和小鼠中脑外植体的中后脑构型能力的分析表明，该突变在功能上将FGF8b转化为Fgf8a。此外，我们的数据表明，相对于FGF8a和FGF8b，FGF17b和FGF18与受体结合的中间亲和力也解释了这两个配体不同的构图能力。我们还表明，FGF8受体结合的特异性模式不同于其他FGFs，并为FGF8b-FGFR1c在中后脑发育过程中的生理性相互作用提供了第一个生化证据。与FGF8在胚胎发育中不可或缺的作用一致，我们发现FGF8受体结合模式早在线虫中就出现了，并在整个进化过程中得到了保存。
Two of the four human FGF8 splice isoforms, FGF8a and FGF8b, are expressed in the mid-hindbrain region during development. Although the only difference between these isoforms is the presence of an additional 11 amino acids at the N terminus of FGF8b, these isoforms possess remarkably different abilities to pattern the midbrain and anterior hindbrain. To reveal the structural basis by which alternative splicing modulates the organizing activity of FGF8, we solved the crystal structure of FGF8b in complex with the "c" splice isoform of FGF receptor 2 (FGFR2c). Using surface plasmon resonance (SPR), we also characterized the receptor-binding specificity of FGF8a and FGF8b, the "b" isoform of FGF17 (FGF17b), and FGF18. The FGF8b-FGFR2c structure shows that alternative splicing permits a single additional contact between phenylalanine 32 (F32) of FGF8b and a hydrophobic groove within Ig domain 3 of the receptor that is also present in FGFR1c, FGFR3c, and FGFR4. Consistent with the structure, mutation of F32 to alanine reduces the affinity of FGF8b toward all these receptors to levels characteristic of FGF8a. More importantly, analysis of the mid-hindbrain patterning ability of the FGF8b(F32A) mutant in chick embryos and murine midbrain explants shows that this mutation functionally converts FGF8b to FGF8a. Moreover, our data suggest that the intermediate receptor-binding affinities of FGF17b and FGF18, relative to FGF8a and FGF8b, also account for the distinct patterning abilities of these two ligands. We also show that the mode of FGF8 receptor-binding specificity is distinct from that of other FGFs and provide the first biochemical evidence for a physiological FGF8b-FGFR1c interaction during mid-hindbrain development. Consistent with the indispensable role of FGF8 in embryonic development, we show that the FGF8 mode of receptor binding appeared as early as in nematodes and has been preserved throughout evolution.