RETINAL PHOTORECEPTOR CYCLIC GMP PHOSPHODIESTERASE

光感受器杆状外段cGMP磷酸二酯酶(PDE， Alphabetagamma2)将细胞质中的3‘，5’-cGMP转化为5‘-GMP。 依赖方式，在视觉的放大级联中起到纽带的作用 转导。在小鼠中，未加工的阿尔法亚基由859组成 和856个残基的β亚基。阿尔法和贝塔有一个重复的 结构域结构和ARE，由位于其C- 终末，翻译后异戊二烯，一种修饰，提供 膜缔合的分子锚。缺陷(截断在 编码β亚基的基因中的残基347)被推定为禁用 PDE的催化活性，并导致RD的视网膜变性 老鼠。因为阿尔法亚单位看起来表达正常 (尽管降低了水平)，我们得出结论，阿尔法和贝塔都 亚基是形成功能性酶所必需的。为了证明这一点 假设，我们希望在特定的目标1中先完整地表达，然后再表达 转基因RD小鼠视杆感光细胞中突变的β亚基。 转基因的表达被预测为提供完整的大小 补充阿尔法和伽马以形成功能PDE，从而允许 正常杆外节段的发展。我们还建议引入 β亚基C-末端CAAX盒的突变(C853S) 研究膜结合缺陷的生物学后果。 在第二个特定目标中，我们希望在功能上表达两个字母 和单细胞表达系统中的β亚基(细菌和昆虫 感染重组杆状病毒的细胞)。这项研究的目的是 是在体外重建一种有活性的酶并确定影响因素 对重建来说是必不可少的。在《特定目标2》的最后部分，我们 建议在CAAX框中引入特定的点突变 建议的催化结构域来研究它们对膜结合的影响， 重组和催化活性。这项研究，利用基因 在细菌、昆虫细胞和转基因小鼠中表达，将产生 有关PDE备注的结构和功能的详细信息 到目前为止可用。

The photoreceptor rod outer segment cGMP phosphodiesterase (PDE, alphabetagamma2) converts cytoplasmic 3',5'-cGMP to 5'-GMP in a light- dependent manner, acting as a link in the amplification cascade of visual transduction. In mouse, the non-processed alpha subunit consists of 859 and the beta subunit of 856 residues. Alpha and beta have a duplicated domain structure and are, signalled by a CAAX box motif at their C- termini, posttranslationally isoprenylated, a modification that provides the molecular anchor for membrane association. A defect (truncation at residue 347) in the gene encoding the beta subunit is presumed to disable PDE catalytic activity and to cause a retinal degeneration in the rd mouse. Since the alpha subunit appears to be expressed normally (although at a reduced level), we conclude that both alpha and beta subunits are necessary to form a functional enzyme. To prove this hypothesis, we wish in specific aim 1 to express first intact, later mutated beta subunits in rod photoreceptors of a transgenic rd mouse. Expression of the transgene is predicted to provide a full size complement for alpha and gamma to form a functional PDE, thus allowing development of normal rod outer segments. We also propose to introduce a mutation (C853S) in the C-terminal CAAX box of the beta subunit to study the biological consequences of a defect in membrane association. In the second specific aim we wish to functionally express both the alpha and beta subunits in unicellular expression systems (bacteria and insect cells infected with recombinant baculovirus). The goal of this research is to reconstitute an active enzyme in vitro and to determine the factors essential for reconstitution. In the last part of specific aim 2, we propose to introduce specific point mutations in the CAAX box and the proposed catalytic domain to study their effects upon membrane binding, reconstitution, and catalytic activity. This research, using gene expression in bacteria, insect cells, and transgenic mice, will yield detailed information about the structure and function of PDE not available to date.