Regulation of Retinal cGMP Phosphodiesterases

描述（由申请人提供）：本研究计划的长期目标是阐明视杆细胞和视锥细胞cGMP-磷酸二酯酶（PDE 6）的功能和调节的分子机制。视杆细胞和视锥细胞PDE 6在脊椎动物视觉转导级联中作为关键效应酶。转导蛋白通过解除由两个γ-亚基（P γ）对PDE 6催化二聚体施加的抑制来激活PDE 6。活化的PDE 6以独特的高催化速率水解cGMP。为了研究PDE 6的结构和功能关系，我们开发了一种在昆虫细胞中表达PDE 6 α/PDE 5嵌合体的系统。通过将PDE 6序列逐步掺入现有的功能性PDE 6/PDE 5嵌合体中，将产生与PDE 6最大等效的嵌合PDE 6/PDE 5酶。将进行由PDE 6催化结构域模型指导的PDE 6样嵌合酶的突变分析，以鉴定对于cGMP的有效水解和酶对选择性竞争性抑制剂的敏感性至关重要的PDE 6结构元件。PDE 6催化亚基包含两个N末端GAF结构域（GAFA和GAFB），与环鸟苷酸的非催化结合有关。我们已经发现，P γ的聚阳离子区域结合PDE 6的GAFa结构域。我们推测，cGMP结合口袋的直接稳定Pgamma是已知的正协同Pgamma和非催化cGMP结合之间的机制。为了验证这个假设。将通过突变来鉴定PDE 6的GAFA结构域内的Pgamma接触残基。通过取代PDE 6潜在cGMP口袋内的选定残基，鉴定参与非催化cGMP结合的GAF结构域和残基。PDE 6 GAF结构域的模型将用于阐明cGMP和P γ结合位点之间相互关系的性质。杆状PDE 6催化α和β亚基形成不可解离的α-异二聚体，而锥状PDE 6 α '亚基形成α' α '同源二聚体。虽然认为PDE 6的GAF结构域参与二聚化，但尚未研究PDE 6亚基间界面。负责PDE 6特异性二聚化的位点和残基将通过分析嵌合和突变PDE之间的二聚体形成来鉴定。特异性PDE 6残基的作用将通过诱导PDE 6 α和β的突变GAF结构域之间的同源二聚化来确定。PDEalpha/beta-Pgamma相互作用的图谱表明了PDE 6组装的两种可能类型。P γ分子可以结合GAFa和来自催化二聚体的相同或不同亚基的催化位点。将使用交联方法检测PDE 6亚基的组装。这些研究将进一步加深我们对PDE 6的结构、功能和调控的理解，并有助于阐明PDE 6基因突变导致视网膜变性的机制。

DESCRIPTION (provided by applicant): The long-term goal of this research program as to elucidate molecular mechanisms of function and regulation of rod and cone cGMP-phosphodiesterases (PDE6) Rod and cone PDE6 serve as key effector enzymes in the vertebrate visual transduction cascade. Transducin activates PDE6 by relieving the inhibition imposed on the PDE6 catalytic dimer by two gamma-subunits (Pgamma). Activated PDE6 hydrolyzes cGMP with a uniquely high catalytic rate. To study the structure-and-function relationships of PDE6 we have developed a system for expression of PDE6alpha/PDE5 chimeras in insect cells. A chimeric PDE6/PDE5 enzyme maximally equivalent to PDE6 will be generated through a progressive incorporation of PDE6 sequence into existing functional PDE6/PDE5 chimeras. Mutational analysis of the PDE6-like chimeric enzymes guided by the model of PDE6 catalytic domain will be carried out to identify the structural elements of PDE6 critical for the efficient hydrolysis of cGMP and the enzyme sensitivity to selective competitive inhibitors. PDE6 catalytic subunits contain two N-terminal GAF domains, GAFa and GAFb, implicated in noncatalytic binding of cGMP. We have found that the polycationic region of Pgamma binds to the GAFa domains of PDE6. We hypothesize that a direct stabilization of the cGMP-binding pocket by Pgamma is the mechanism for known positive cooperativity between Pgamma and noncatalytic cGMP binding. To test this hypothesis. Pgamma contact residues within the GAFa domain of PDE6 will be identified by mutagenesis. The GAF domains and residues involved in the noncatalytic cGMP-binding will be identified by substituting selected residues within potential cGMP-pockets of PDE6. Models of PDE6 GAF domains will be utilized to elucidate the nature of the reciprocal relationship between the cGMP and Pgamma-binding sites. Rod PDE6 catalytic alpha and beta subunits form indissociable alphabeta heterodimers, whereas cone PDE6 alpha' subunit forms alpha' alpha' homodimers. Although the GAF domains of PDE6 are thought to be involved in the dimerization, the PDE6 intersubunit interface has not been investigated. Sites and residues responsible for the specific dimerization of PDE6 will be identified through the analysis of dimer formation between chimeric and mutant PDEs. The role of specific PDE6 residues will be established by inducing homodimerization between mutant GAF domains of PDE6 alpha and beta. The map of PDEalpha/beta-Pgamma interactions indicates two possible types of assembly of PDE6. A Pgamma molecule may bind to GAFa and the catalytic site from the same or different subunits of the catalytic dimer. The assembly of PDE6 subunits will be tested using a cross-linking approach. These studies will advance our understanding of the structure, function, and regulation of PDE6, and help to elucidate the mechanisms of retinal degeneration caused by mutations of PDE6 genes.