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

本研究旨在探讨NDPK作为gtp供应分子在细胞信号系统（激素受体- g蛋白腺苷酸环化酶）中的功能作用。为此，我们首先利用显性负表达NDPK的PC12D细胞，观察细胞中过表达的NDPK突变体是否会干扰激素反应。我们比较了转染突变型失活重组NDPK （rNDPKmα或mβ）的PC12D细胞克隆与对照细胞克隆之间PACAP刺激的cAMP水平，与对照细胞相比，转染rNDPKmα的细胞中PACAP的刺激倾向于被抑制。然而，这种抑制作用在不同的实验中有所不同。PC12D细胞的质膜含有NDPK活性，其作用是由增加的GDP形成GTP，导致PACAP和GDP激活AC。因此，我们随后研究了NDPKmα与质膜混合是否会影响内源性NDPK，从而改变PACAP刺激的cAMP形成。结果表明，NDPKmα对小鼠无明显影响。此外，PACAP + GDP刺激PC12D-rNDPKmα细胞膜的AC活性与对照细胞没有差异。然而，正如我们之前所证明的那样，纯化膜的PACAP刺激的AC活性完全依赖于GTP， PACAP加GDP的作用被UDP作为NDPK抑制剂抑制。由此可见，PC12D AC系统与大鼠肝膜的NDPK密切相关。质膜相关NDPK是否来源于胞质NDPK，或者它是一种不同于胞质NDPK的新型膜特异性异构体，还有待研究。

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.