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

描述（申请人提供）：RGS 14是一种新发现的效应蛋白，它整合了G蛋白和H-Ras/Raf 1/ERK信号通路. RGS 14是一种脑蛋白，其高度富集于海马区CA 2的神经元的树突和棘，并且其表达模式在很大程度上限于海马区CA 2的神经元的树突和棘。我们最近发现RGS 14作为CA 2神经元中突触可塑性（LTP）的天然抑制剂至关重要。此外，我们发现，RGS 14在CA 1神经元的RGS 14不表达块LTP有异位表达，这表明RGS 14从事共同的细胞信号通路突触可塑性的关键。与研究充分的CA 1区域不同，对那里的CA 2神经元或RGS 14知之甚少。CA 2与人类神经系统疾病有关，包括精神分裂症、自闭症/双相障碍谱和癫痫。缺乏RGS 14的小鼠（RGS 14-K 0）与野生型同窝小鼠相比，在空间学习和物体识别记忆方面表现出显著且出乎意料的增强，但在非露营依赖性行为方面没有表现出差异。RGS 14-KO小鼠还表现出令人惊讶的稳健的新生LTP，在CA 2中的突触能突触处具有增强的神经元兴奋性，对相邻CA 1神经元的可塑性没有影响。总之，这些发现突出了理解RGS 14调节神经元/突触可塑性的分子机制的重要性。在CA 2/CA 1神经元中，LTP的表达及其抑制是由于Ca++依赖性（CaM，CaMK II）和Ca++非依赖性（ERK，cAMP/PKA）机制。RGS 14结合失活的G？i1/3-GDP和活性的H-Ras-GTP，形成整合G蛋白和MAPK信号通路的异源三聚体信号复合物。RGS 14还以Ca++依赖性方式结合钙调蛋白（CaM）。这些发现表明RGS 14很好地调节宿主神经元的可塑性。与这一观点一致，RGS 14缺失后CA 2神经元中的新生LTP依赖于MEK/ERK信号传导。基于此，我的工作假设是，G <$i-GDP：RGS 14：H-Ras-GTP信号复合物整合了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剂。然而，RGS 14如何结合G蛋白、H-Ras和CaM以作为信号转导开关/整合剂的分子/结构基础是未知的。此外，天然RGS 14在其天然宿主CA 2神经元中的动态亚细胞定位和调节，以及G？i1：RGS 14：H-Ras信号传导复合物参与调节宿主CA 2/CA 1中突触可塑性的信号传导途径是完全未知的。具体目标是：目标1。确定RGS 14，G？i，H-Ras和CaM如何相互作用形成功能性信号复合物的结构基础和结构域间动力学。目标2：确定天然RGS 14的细胞特性以及RGS 14如何参与H-Ras/ERK信号通路以调节天然宿主CA 2神经元中的突触可塑性。目标3：确定CA 2或CA 1海马神经元中G？i：RGS 14：H-Ras复合物用于调节海马切片制备物中LTP的信号通路。

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.