PDF(Pubmed)
Abstract：
During nervous system development, Sonic hedgehog (Shh) guides developing commissural axons toward the floor plate of the spinal cord. To guide axons, Shh binds to its receptor Boc and activates downstream effectors such as Smoothened (Smo) and Src family kinases (SFKs). SFK activation requires Smo activity and is also required for Shh-mediated axon guidance. Here we report that β-arrestin1 and β-arrestin2 (β-arrestins) serve as scaffolding proteins that link Smo and SFKs in Shh-mediated axon guidance. We found that β-arrestins are expressed in rat commissural neurons. We also found that Smo, β-arrestins, and SFKs form a tripartite complex, with the complex formation dependent on β-arrestins. β-arrestin knockdown blocked the Shh-mediated increase in Src phosphorylation, demonstrating that β-arrestins are required to activate Src kinase downstream of Shh. β-arrestin knockdown also led to the loss of Shh-mediated attraction of rat commissural axons in axon turning assays. Expression of two different dominant-negative β-arrestins, β-arrestin1 V53D which blocks the internalization of Smo and β-arrestin1 P91G-P121E which blocks its interaction with SFKs, also led to the loss of Shh-mediated attraction of commissural axons. In vivo, the expression of these dominant-negative β-arrestins caused defects in commissural axon guidance in the spinal cord of chick embryos of mixed sexes. Thus we show that β-arrestins are essential scaffolding proteins that connect Smo to SFKs and are required for Shh-mediated axon guidance.
摘要：
在神经系统发育过程中,Sonichedgehog（Shh）引导向脊髓底板发展连合轴突。为了引导轴突，Shh结合其受体Boc并激活下游效应物，例如Smoothened（Smo）和Src家族激酶（SFK）。SFK激活需要Smo活性,并且也是Shh介导的轴突引导所需要的。在这里，我们报道了β-arrestin1和β-arrestin2（β-arrestins）作为支架蛋白，在Shh介导的轴突指导中连接Smo和SFK。我们发现β-arrestin在大鼠连合神经元中表达。我们还发现Smo,β-抑制素和SFK形成三方复合物，复合物的形成依赖于β-抑制素。β抑制蛋白敲除阻断Shh介导的Src磷酸化增加，证明需要β-抑制素激活Shh下游的Src激酶。β-抑制蛋白敲除还导致轴突转向测定中Shh介导的大鼠连合轴突的吸引力丧失。两种不同显性阴性β-抑制素的表达，β-arrestin1V53D阻断Smo和β-arrestin1P91G-P121E的内化，阻断其与SFK的相互作用，也导致Shh介导的连合轴突吸引力的丧失。在体内,这些显性阴性β-arrestin的表达导致混合性别的鸡胚脊髓连合轴突指导缺陷。因此，我们表明β-arrestins是将Smo连接到SFK的必需支架蛋白，并且是Shh介导的轴突指导所必需的。意义陈述轴突的正确引导对于神经系统的形成很重要。Sonichedgehog（Shh）介导的轴突导向依赖于非典型G蛋白偶联受体（GPCR）Smoothened（Smo）下游Src家族激酶（SFK）的激活。SFK如何在Smo下游被激活是未知的。在这项研究中,我们发现β-arrestin1和2（β-arrestins）充当Smo和SFK之间的支架蛋白。我们还发现β-抑制素是激活SFK所必需的。敲除β-抑制素或表达显性负β-抑制素导致Shh介导的连合轴突吸引力丧失。在体内,显性阴性β-arrestins的表达导致连合轴突引导缺陷。我们的工作首次确定了β-arrestins在轴突指导中的作用。
