Mizutani Akihiro, Kuroda Yukiko, Futatsugi Akira, Furuichi Teiichi, Mikoshiba Katsuhiko
Laboratory for Developmental Neurobiology and Laboratory for Molecular Neurogenesis, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan.
J Neurosci. 2008 May 14;28(20):5369-82. doi: 10.1523/JNEUROSCI.4738-07.2008.
Homer proteins are components of postsynaptic density (PSD) and play a crucial role in coupling diverse target molecules. However, the regulatory aspect of Homer-mediated coupling has been addressed only about a dominant-negative effect of Homer1a, which requires de novo gene expression. Here, we present evidence that Homer-mediated coupling is regulated by its phosphorylation state. We found that Homer3, the predominant isoform in Purkinje cells, is phosphorylated by calcium/calmodulin-dependent protein kinase II (CaMKII) both in vitro and in vivo. Biochemical fractionation with phosphor-specific antibodies revealed the presence of phosphorylated Homer3 in the cytosolic fraction in contrast to high levels of nonphosphorylated Homer3 in PSD. In P/Q-type voltage-gated-Ca2+ channel knock-out mice, in which CaMKII activation was reduced, the levels of Homer3 phosphorylation and the soluble form of Homer 3 were markedly lower. Furthermore, both robust phosphorylation of Homer3 and its dissociation from metabotropic glutamate receptor 1alpha (mGluR1alpha) were triggered by depolarization in primary cultured Purkinje cells, and these events were inhibited by CaMKII inhibitor. An in vitro binding kinetic analysis revealed that these phosphorylation-dependent events were attributable to a decrease in the affinity of phosphorylated Homer3 for its ligand. In a heterologous system, the Ca2+ signaling pattern induced by mGluR1alpha activation was modulated by the Homer3 phosphorylation state. Together, these findings suggested that Homer3 in Purkinje cells might function as a reversible coupler regulated by CaMKII phosphorylation and that the phosphorylation is capable of regulating the postsynaptic molecular architecture in response to synaptic activity.
荷马蛋白是突触后致密区（PSD）的组成部分，在连接多种靶分子中起关键作用。然而，关于荷马介导的连接的调控方面，仅涉及荷马1a的显性负效应，这需要从头基因表达。在此，我们提供证据表明，荷马介导的连接受其磷酸化状态调控。我们发现，浦肯野细胞中主要的异构体荷马3在体外和体内均被钙/钙调蛋白依赖性蛋白激酶II（CaMKII）磷酸化。用磷酸特异性抗体进行生化分级分离显示，与PSD中高水平的非磷酸化荷马3相比，胞质部分存在磷酸化的荷马3。在P/Q型电压门控Ca2+通道敲除小鼠中，CaMKII激活减少，荷马3的磷酸化水平和可溶性荷马3形式明显降低。此外，在原代培养的浦肯野细胞中，去极化触发了荷马3的强烈磷酸化及其与代谢型谷氨酸受体1α（mGluR1α）的解离，而这些事件被CaMKII抑制剂抑制。体外结合动力学分析表明，这些磷酸化依赖性事件归因于磷酸化的荷马3对其配体亲和力的降低。在异源系统中，mGluR1α激活诱导的Ca2+信号模式受荷马3磷酸化状态的调节。总之，这些发现表明，浦肯野细胞中的荷马3可能作为一种受CaMKII磷酸化调节的可逆连接物发挥作用，并且这种磷酸化能够响应突触活动调节突触后分子结构。
