TRAFFICKING OF THE PLASMA MEMBRANE CA2+-ATPASE TO RAFTS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。神经元筏是一种质膜隔膜，具有独特的脂质和蛋白质组成；它们富含胆固醇、鞘脂和参与信号转导的蛋白质。有令人信服的证据表明，神经元筏是组织钙信号事件的局部部位，但目前还没有关于介导钙信号的特定钙调节蛋白的信息。我们最近发现，大鼠脑突触质膜(SPM)中的RAFT富含质膜钙-ATPase(PMCA)，它是一种钙离子转运体。对完整的海马神经元的共聚焦显微镜进一步证实了PMCA在RAFTS区域的共同定位，表明这种关联可能发生在体内。免疫印迹分析表明，PMCA的4种异构体，包括它们的a和b剪接变异体，都定位于筏中。A和b变体的存在，而不是我们最初假设中提出的PMCA 2b的存在，表明RAFT结构域包含微调钙信号所需的PMCA亚型的全部补充。对RAFT结构域和非RAFT结构域PMCA活性的测量表明，RAFT相关的PMCA具有更高的比活性，这表明这个PMCA池可能对神经元的PMCA整体功能有更重要的贡献。为了确定PMCA与RAFT的关系的性质，我们消耗了细胞胆固醇，这是已知在体内维持RAFT结构的主要脂类。细胞胆固醇的慢性和急性耗竭都没有破坏PMCA与RAFT的关联，这表明两者之间存在非常紧密的相互作用。为了确定可能将PMCA锚定在筏子上的潜在蛋白质伙伴，进行了免疫共沉淀研究。结果表明，RAFT PMCA与钙调蛋白、NAP-22和GAP-43相关，这些蛋白是组织神经元RAFT结构域的蛋白质。未来的目标是确定招募PMCA进入和离开RAFT结构域的分子机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Neuronal rafts are plasma membrane compartments with a unique lipid and protein composition; they are enriched in cholesterol, sphingolipids, and proteins involved in signal transduction. There is compelling evidence to implicate neuronal rafts as being local sites for the organization of Ca2+ signaling events but no information exists on specific Ca2+ regulatory proteins that mediate the Ca2+ signal. We have recently discovered that the plasma membrane Ca2+-ATPase (PMCA), a Ca2+ transporter is enriched in rafts isolated from rat brain synaptic plasma membranes (SPMs). Confocal microscopy of intact hippocampal neurons further confirmed the co-localization of PMCA in rafts domains, suggesting the possibility of this association occurring in vivo. Immunoblot analysis showed that all 4 isoforms of PMCA, including their ¿a¿ and ¿b¿ splice variants, were localized in rafts. The presence of both ¿a¿ and ¿b¿ variants and not exclusively PMCA 2b, as proposed in our original hypothesis suggests that raft domains contain the full complement of PMCA subtypes necessary for fine-tuning of the Ca2+ signal. Measurement of PMCA activity in raft vs non-raft domains showed that the raft-associated PMCA had higher specific activity suggesting that this pool of PMCA may be a more significant contributor to overall PMCA function in neurons. To determine the nature of the association of PMCA with rafts, we depleted cellular cholesterol, the major lipid known to maintain raft structure in vivo. Both chronic and acute depletion of cellular cholesterol did not disrupt the association of PMCA with rafts suggesting a very tight interaction. In efforts to determine the potential protein partners that may anchor PMCA to rafts, co-immunoprecipitation studies were performed. The results showed that raft PMCA was associated with calmodulin, NAP-22, and GAP-43, proteins known to organize neuronal raft domains. Future goals are to determine molecular mechanisms that recruit PMCA into and out of raft domains.