The target site and role of NDP kinase in cellular signaling networks

本研究旨在探讨NDPK作为GTP供给分子在细胞信号系统(激素受体-G-蛋白-腺苷环化酶)中的功能作用。为此，我们首先利用显性阴性表达NDPK的PC12D细胞，观察突变的NDPK在细胞中过表达是否干扰激素反应。比较突变失活重组NDPK(rNDPKmα或mβ)转染的PC12 D细胞克隆和对照细胞克隆对PACAP刺激的cAMP水平的影响，发现与对照细胞相比，pNDPKα转染组细胞对PACAP刺激的cAMP水平趋于抑制。然而，这种抑制作用在不同的实验中是不同的。PC12D细胞的质膜含有NDPK活性，它可以从添加的GDP中形成GTP，导致PACAP和GDP共同激活AC。因此，我们随后研究了NDPKmα与质膜的混合是否能够影响内源性NDPK，从而改变PACAP刺激的cAMP的形成。结果表明，NDPKm-α对小鼠心脏功能无明显影响。同时，PACAP+GDP3对PC12D-rNDPKmPC12D-rNDPKmα细胞膜AC活性的刺激作用与对照细胞无明显差异，但PACAP刺激的膜AC活性完全依赖于GTP，而PACAP+GDDP作为NDPK的抑制剂可抑制其作用。由此可见，PC12D AC系统与NDPK密切相关，大鼠肝细胞膜PC12D AC系统与NDPK密切相关。质膜相关的NDPK是来源于胞质NDPK，还是一种有别于胞质NDPKs的新的膜特异性异构体，目前尚不清楚。

This study has been done to investigate the functional role of NDPK as a GTP-supplying molecule in cellular signaling system (hormone receptor-G-protein-adenylate cyclase). To this goal, we first utilized dominant negative NDPK-expressing PC12D cells, to see whether the mutant NDPK overexpressed in the cell interferes with the hormonal response. When we compared the PACAP-stimulated cAMP level between PC12D cell clone that was transfected with mutant inactive recombinant NDPK (rNDPKmα or mβ) and control cell clone, the stimulation by PACAP tended to be suppressed in the transfected cell with rNDPKα compared with the control cell. However, this suppression varied from experiment to experiment.The plasma membranes from PC12D cells contain NDPK activity which acts to form GTP from added GDP, leading to AC activation by PACAP plus GDP. Therefore, we then examined whether the NDPKmα mixed with the plasma membranes can affect the endogenous NDPK and, as a result, alter the cAMP formation stimulated by PACAP. The result showed no obvious effect by NDPKmα. Also, the AC activity in membranes from PC12D-rNDPKmα stimulated by PACAP plus GDP did not differ from that from the contol cells.As we demonstrated previously, however, the PACAP-stimulated AC activity of the purified membranes was completely dependent on GTP, and the effect of PACAP plus GDP was supressed by UDP as an inhibitor of NDPK. Therefore, it is clear that PC12D AC system is closely associated with NDPK as demonstrated for that of rat liver membrane. It remains to be studied whether the plasma membrane-associated NDPK is derived from the cytosolic one, or it is a novel membrane specific isoform distinct from the cytosolic NDPKs.