TRAFFICKING OF THE PLASMA MEMBRANE CA2+-ATPASE TO RAFTS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。神经元筏是具有独特脂质和蛋白质组成的质膜隔室；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Ca2+信号事件组织的局部位点，但没有关于介导Ca2+信号的特定Ca2+调节蛋白的信息存在。我们最近发现质膜Ca2+- atp酶（PMCA），一种Ca2+转运蛋白在从大鼠脑突触质膜（SPMs）分离的筏中富集。完整海马神经元的共聚焦显微镜进一步证实了PMCA在筏结构域的共定位，表明这种关联可能发生在体内。免疫印迹分析显示，PMCA的所有4种同工型，包括它们的¿a´和¿b´剪接变体，都在筏中定位。正如我们最初的假设所提出的那样，存在“a”和“b”变体，而不仅仅是PMCA 2b，这表明筏结构域包含Ca2+信号微调所需的PMCA亚型的全部补体。对筏区与非筏区PMCA活性的测量表明，筏区相关的PMCA具有更高的特异性活性，这表明该PMCA池可能对神经元的整体PMCA功能有更重要的贡献。为了确定PMCA与筏相关的性质，我们耗尽了细胞胆固醇，这是维持筏结构的主要脂质。细胞胆固醇的慢性和急性消耗都没有破坏PMCA与筏的关联，这表明它们之间的相互作用非常紧密。为了确定可能将PMCA锚定在筏上的潜在蛋白质伙伴，进行了共免疫沉淀研究。结果表明，筏PMCA与钙调蛋白、NAP-22和GAP-43相关，这些蛋白已知用于组织神经元筏结构域。未来的目标是确定招募PMCA进入和离开筏结构域的分子机制。

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.