REGULATION AND INTRACELLULAR LOCALISATION OF PHOSPHOIN OSITIDE-BINDING PROTEINS.

配体识别，细胞内定位和磷肌苷结合蛋白的生理重要性进行了研究。研究发现，pleckstrin同源结构域（PH）中的某些碱性氨基酸对特定配体识别很重要，因此，它们负责含PH蛋白与质膜的相互作用。磷脂酶C-δ1 （plc -δ1）尤其如此。我们证明，渗透胁迫导致荧光从表达gfp标记的PLCδ1或其PH结构域的肾上皮细胞的质膜上快速解离。当细胞骨架破坏试剂处理后，解离率增加，表明PtdIns(4,5)P2水解活性受膜旁结构调节。释放的gfp - plc - δ1转移到核周区域，可能是内质网，但其靶向机制可能与PLA2（和PKC）不同，其C2结构域可能起作用。虽然在正常条件下细胞核中很少存在，但PLCδ1确实在细胞质溶胶（加上质膜）和细胞核之间穿梭。我们已经证明PLCδ1有一个功能性的核输出信号序列，破坏它或抑制crm1/输出依赖的核输出可以让酶留在细胞核中。由于肌醇多磷酸及其激酶的新作用已被假设，这一发现将为核磷酸肌醇信号传导提供进一步的见解。然后，我们以一个自由生活的变形虫为模型，探索了PtdIns(4,5)P2/PLC在细胞运动中的作用。当用PLCδ1- ph或PLCδ1处理变形虫膜片时，肌动蛋白丝的网状结构逐渐消失。此外，一种特定的PLC抑制剂在体内可逆地阻断了假足的形成和细胞的运动，这表明细胞骨架的重塑是由Ca^<2+>-敏感的PLC调节的，它可以抑制PtdIns(4,5)P2。

Ligand recognition, intracellular localization and physiological importance of phosphoinositide-binding proteins have been examined. It was found that certain basic amino acids in the pleckstrin homology (PH) domain are important for specific ligand recognition, and therefore, responsible for interaction of PH-containing proteins with the plasma membrane. This was especially the case for phospholipase C-δ1 (PLCδ1). We demonstrated that osmotic stress caused a rapid dissociation of the fluorescence from the plasma membrane of renal epithelial cells expressing GFP-tagged PLCδ1 or its PH domain. The dissociation rate increased when cells were treated with cytoskeleton-disrupting reagents, suggesting the PtdIns(4,5)P2 hydrolysing actuvity is regulated by juxtamembrane structures. Released GFP-PLCδ1 translocated to perinuclear regions, presumably ER, but the mechanism of targeting may be different from those of PLA2 (and PKC) whose C2 domain may play a role. Although little is present in the nucleus under normal conditions, PLCδ1 does shuttle between the cytosol (plus the plasma membrane) and the nucleus. We have shown that PLCδ1 has a functional nuclear export signal sequence, and disruption of it or inhibition of the crm1/exportin-dependent nuclear exp ort could let the enzyme stay in the nucleus. This finding would provide further insights into the nuclear phosphoinositide signalling, since new roles of inositol polyphosphates and their kinases have been postulated. We then explored roles of PtdIns(4,5)P2/PLC in cell movements using a free living amoeba as a model. When membrane sheets prepared from amoebae were treated with PLCδ1-PH or PLCδ1, a mesh work structure of actin filaments gradually disappeared. Furthermore, a specific inhibitor of PLC reversibly blocked the pseudopod formation and the cell movement in vivo, suggesting that remodelling of cytoskeleton is regulated by Ca^<2+>-sensitive PLC that hy droly ses PtdIns(4,5)P2.