RETINAL PHOTORECEPTOR CYCLIC GMP PHOSPHODIESTERASE

光感受器杆外节cGMP磷酸二酯酶（PDE， alphabetagamma 2）在轻- 依赖的方式，作为一个链接，在放大级联的视觉 转导 在小鼠中，未加工的α亚基由859个 和856个残基的β亚基。 阿尔法和贝塔有一个复制的 结构域结构，并通过其C- 末端，后异戊二烯化，提供 膜结合的分子锚。 缺陷（截断） 推测编码β亚基的基因中的一个残基（第347位残基 PDE的催化活性和引起视网膜变性的rd 老鼠. 由于α亚基的表达正常， （虽然在降低的水平），我们得出结论，无论是阿尔法和贝塔 亚基是形成功能酶所必需的。 为了证明这一点 假设，我们希望在具体目标1中首先完整地表达，然后 转基因RD小鼠视杆细胞中突变的β亚基。 预计转基因的表达提供完整大小的 α和γ的互补以形成功能性PDE，从而允许 正常视杆外节的发育。 我们还建议引入 β亚基的C-末端CAAX盒中的突变（C853 S）， 研究膜结合缺陷的生物学后果。 在第二个具体目标中，我们希望在功能上表达α和α。 和β亚基在单细胞表达系统（细菌和昆虫 用重组杆状病毒感染的细胞）。 本研究的目的 是在体外重建一种活性酶， 对于重建至关重要。 在具体目标2的最后一部分，我们 建议在CAAX盒中引入特定的点突变， 提出了催化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， PDE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.