Molecular, Biochemical, and Electrophysiological Characterization of Drosophila norpA Mutants (*)
Molecular, Biochemical, and Electrophysiological Characterization of Drosophila norpA Mutants (*)
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DOI：
10.1074/jbc.271.9.4937
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发表时间：
1996-03
期刊：
影响因子：
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通讯作者：
Michael T. Pearn;L. Randall;R. Shortridge;M. Burg;W. Pak
中科院分区：
文献类型：
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作者：
Michael T. Pearn;L. Randall;R. Shortridge;M. Burg;W. Pak
肌醇磷酸信号转导已经涉及各种各样的真核细胞过程。在果蝇中，光转导级联是由norpA基因编码的磷酸肌醇特异性磷脂酶C（PLC）介导的。我们的特点是8 norpA突变视网膜电图（ERG），西方，分子和体外PLC活性分析。突变体的ERG反应显示等位基因依赖性的振幅降低和动力学延迟。突变体还表现出等位基因依赖性降低体外PLC活性水平和大大降低或不可检测的NorpA蛋白水平。三个携带错义突变，五个携带norpA编码序列内的无义突变。在错义突变体中，氨基酸取代发生在PLC之间高度保守的残基处。这些取代降低了NorpA蛋白和PLC活性两者的水平，其中PLC活性的降低大于可以简单地由蛋白质的降低所解释的。突变对蛋白质的量和活性的影响远大于它们对ERG的影响，表明在效应子（NorpA）蛋白质水平上的转导信号的放大。转基因果蝇产生的空norpA突变体的种系转化使用的P-元件构建体含有野生型norpA cDNA驱动的ninaE启动子。转化的果蝇在R1-R6光感受器中显示出电生理表型的拯救，但在R7或R8中没有。R1-R6光感受器的变性表型也得到了挽救。
Inositol phosphate signaling has been implicated in a wide variety of eukaryotic cellular processes. In Drosophila, the phototransduction cascade is mediated by a phosphoinositide-specific phospholipase C (PLC) encoded by the norpA gene. We have characterized eight norpA mutants by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued.