Structure and Function of the G-alpha-i1:RGS14:H-Ras signaling complex

描述(申请人提供)：RGS14是一种新发现的效应蛋白，它整合了G蛋白和H-RAS/RAF1/ERK信号通路。RGS14是一种脑内蛋白，其表达模式主要局限于海马区CA2区神经元的树突和棘细胞。我们最近发现RGS14在CA2神经元中作为突触可塑性(LTP)的天然抑制因子是至关重要的。此外，我们发现RGS14在不表达RGS14的CA1神经元中的异位表达阻断了那里的LTP，这表明RGS14参与了对突触可塑性至关重要的共同细胞信号通路。与研究充分的CA1区不同，人们对那里的CA2神经元或RGS14知之甚少。CA2与人类神经疾病有关，包括精神分裂症、自闭症/双相情感障碍和癫痫。与野生型小鼠相比，缺乏RGS14(RGS14-KO)的小鼠在空间学习和目标识别记忆方面表现出显著的意外增强，但在非海马区依赖行为方面没有差异。RGS14-KO小鼠还表现出令人惊讶的强健的新生LTP，CA2中谷氨酸能突触的神经元兴奋性增强，而对邻近CA1神经元的可塑性没有影响。总之，这些发现强调了理解RGS14调节神经元/突触可塑性的分子机制的重要性(S)。在CA2/CA1神经元中，LTP的表达及其抑制是由于钙依赖(CaM，CaMKII)和非钙非依赖性(ERK，cAMP/PKA)机制所致。RGS14与失活的G-�i1/3-GTP和活性H-RAS-GTP结合，形成一个整合G蛋白和MAPK信号通路的异源三聚体信号复合体。RGS14还以钙依赖的方式与钙调蛋白(CaM)结合。这些发现表明，RGS14在调节宿主神经元的可塑性方面处于有利地位。与这一观点一致的是，RGS14丢失后CA2神经元中新生的LTP依赖于MEK/ERK信号。基于此，我的工作假设是G-�I-GDp：RGS14：H-Ras-GTP信号复合体整合了G蛋白、MAPK和Ca++/CaM信号通路，作为宿主神经元突触可塑性的天然抑制因子。然而，RGS14如何结合G蛋白、H-RAS和CaM作为信号开关/整合子的分子/结构基础尚不清楚。此外，天然RGS14在宿主CA2神经元中的动态亚细胞定位和调节，以及G�i1：RGS14：H-RAS信号复合体参与调节宿主CA2/CA1突触可塑性的信号通路是完全未知的。具体目标是：目的1.确定RGS14、G�I、H-RAS和CaM如何相互作用形成功能信号复合体的结构基础和域间动力学。目的：确定天然RGS14的细胞特性以及RGS14如何参与H-RAS/ERK信号通路调节自然宿主CA2神经元突触可塑性。目的：研究海马神经元G�I：RGS14：H-RAS复合体调节海马片制备LTP的信号转导途径。

DESCRIPTION (provided by applicant): RGS14 is a newly appreciated effector protein that integrates G protein and H- Ras/Raf1/ERK signaling pathways. RGS14 is a brain protein that is highly enriched in and largely restricted in its expression pattern to dendrites and spines of neurons of hippocampal region CA2. We recently discovered that RGS14 is critically important as a natural suppressor of synaptic plasticity (LTP) in CA2 neurons. Furthermore, we show that ectopic expression of RGS14 in CA1 neurons where RGS14 is not expressed blocks LTP there, suggesting that RGS14 engages common cell signaling pathways critical for synaptic plasticity. Unlike the well-studied CA1 region, very little is known about CA2 neurons or RGS14 there. The CA2 is implicated in human neurological diseases including schizophrenia, the autism/bipolar spectrum of disorders, and epilepsy. Mice lacking RGS14 (RGS14-KO) exhibit a marked and unexpected enhancement in spatial learning and object recognition memory compared with wild type littermates, but show no differences in non- hippocampal-dependent behaviors. RGS14-KO mice also exhibit a surprisingly robust nascent LTP with enhanced neuronal excitability at glutamatergic synapses in CA2, with no impact on plasticity in adjacent CA1 neurons. Together, these findings highlight the importance of understanding the molecular mechanism(s) whereby RGS14 regulates neuronal/synaptic plasticity. Within CA2/CA1 neurons, LTP expression and its suppression is due to both Ca++-dependent (CaM, CaMKII) and Ca++-independent (ERK, cAMP/PKA) mechanisms. RGS14 binds inactive G�i1/3-GDP and active H-Ras-GTP to form a heterotrimeric signaling complex that integrates G protein and MAPK signaling pathways. RGS14 also binds calmodulin (CaM) in a Ca++-dependent manner. These findings suggest RGS14 is well positioned to regulate plasticity in host neurons. Consistent with this idea, the nascent LTP in CA2 neurons following loss of RGS14 is dependent on MEK/ERK signaling. Based on this, my working hypothesis is that the G�i-GDP:RGS14:H-Ras-GTP signaling complex integrates G protein, MAPK and Ca++/CaM signaling pathways to serve as a natural suppressor of synaptic plasticity in host neurons. However, the molecular/structural basis for how RGS14 binds G proteins, H-Ras and CaM to operate as a signaling switch/integrator is unknown. Furthermore, the dynamic subcellular localization and regulation of native RGS14 in its natural host CA2 neurons, and the signaling pathways that the G�i1:RGS14: H-Ras signaling complex engages to regulate synaptic plasticity in host CA2/CA1 is entirely unknown. The Specific Aims are: AIM 1. Determine the structural basis and interdomain dynamics for how RGS14, G�i, H-Ras and CaM interact to form a functional signaling complex. AIM 2: Determine the cellular properties of native RGS14 and how RGS14 engages the H-Ras/ERK signaling pathway to regulate synaptic plastic in natural host CA2 neurons. AIM 3: Determine the signaling pathways used by the G�i:RGS14: H- Ras complex in CA2 or CA1 hippocampal neurons to regulate LTP in hippocampal slice preparations.