TRAFFICKING OF THE PLASMA MEMBRANE CA2+-ATPASE TO RAFTS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 神经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，作为组织的钙离子信号事件，但没有信息存在特定的钙离子调节蛋白介导的钙离子信号的神经筏。我们最近发现，质膜Ca ~（2+）-ATP酶（PMCA）是一种Ca ~（2+）转运蛋白，它在大鼠脑突触质膜（SPM）的筏中富集。完整的海马神经元的共聚焦显微镜进一步证实了共同定位的PMCA筏域，这表明这种协会在体内发生的可能性。免疫印迹分析表明，所有4种异构体的PMCA，包括它们的'a'和'B'剪接变体，定位于筏。正如我们最初的假设所提出的，“a”和“B”变体的存在，而不仅仅是PMCA 2 B，表明筏结构域包含微调Ca 2+信号所需的PMCA亚型的完整互补。在筏与非筏域的PMCA活性的测量表明，筏相关的PMCA具有更高的比活性，这表明该池的PMCA可能是一个更显着的贡献者整体PMCA功能的神经元。为了确定PMCA与筏的关联的性质，我们耗尽了细胞胆固醇，已知在体内维持筏结构的主要脂质。无论是慢性和急性细胞胆固醇耗竭没有破坏协会的PMCA与筏表明一个非常紧密的相互作用。为了确定可能将PMCA锚在筏上的潜在蛋白质伴侣，进行了免疫共沉淀研究。结果表明，筏PMCA与钙调蛋白，NAP-22和GAP-43，已知组织神经元筏域的蛋白质。未来的目标是确定招募PMCA进出筏域的分子机制。

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.