Mammalian Phototransduction: Role of cGMP and Calcium

描述（由申请人提供）：视网膜光感受器中的视觉转导通过两种第二信使Ca 2+和cGMP的动态相互作用进行。光刺激cGMP水解，[cGMP]下降导致cGMP门控通道关闭和Ca 2+下降。参与调节cGMP和Ca 2+的两个关键酶系统是cGMP特异性磷酸二酯酶（PDE）和鸟苷酸环化酶（GC）。PDE是一种多亚基酶（PDE α-γ-δ 2），与外周膜相关，而GC是一种单亚基整合膜蛋白。光感受器GC的Ca 2+敏感性由称为GCAP（鸟苷酸环化酶激活蛋白）的Ca 2+结合蛋白介导。GCAPs对GC的调节，其复杂性因人视网膜中至少三种GCAPs和两种GC的存在而增强，目前还不完全清楚。本申请旨在调查有关PDE和GC/GCAP的监管过程中悬而未决的问题。具体目标1解决了PDE研究中未解决的问题，特别是鉴定在生物合成过程中新生多肽正确折叠所必需的分子伴侣，以及通过体外和体内表达阐明Pdelta（一个假定的第四亚基）的作用。在目标2中，我们提出解决机制，导致显性视锥细胞营养不良时，突变体GCAP在转基因小鼠的视网膜中表达。其他问题涉及GCAP 2和GC 2在光转导中的作用。GCAP 1，2双敲除揭示了一个表型的延迟在黑暗中的适应一致的激活剂的损失。我们发现（见初步结果/进展报告），转基因GCAP 1拯救GCAP 1/2双敲除表型，表明GCAP 1单独可以调节杆和锥光转导。阐明视网膜中的特定作用，特别是GCAP 2和GC 2，是特定目标3的一部分。总之，本研究将加深我们对cGMP和Ca 2+这两种重要的光传导第二信使的结构和功能的认识。

DESCRIPTION (provided by applicant): Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca2+ and cGMP. Light stimulates the hydrolysis of cGMP and the resulting fall in [cGMP] leads to closure of cGMP-gated channels and a drop in Ca2+. The two key enzyme systems involved in regulation of cGMP and Ca2+are a cGMP-specific phosphodiesterase (PDE) and guanylate cyclase (GC). PDE is a multisubunit enzyme (PDEalphabetagamma2delta2) and peripherally membrane associated, while GC is a single subunit integral membrane protein. The Ca2+ sensitivity of photoreceptor GC is mediated by Ca2+-binding proteins termed GCAPs (guanylate cyclase-activating proteins). The regulation of GCs by GCAPs, the complexity of which is enhanced by the presence of at least three GCAPs and two GCs in human retina, is incompletely understood. This application aims to investigate outstanding and unsolved questions in the regulatory processes regarding PDE and GC/GCAPs. Specific Aim 1 addresses unsolved problems in PDE research, particularly the identification of chaperones essential for the correct folding of nascent polypeptides during biosynthesis, and elucidation of the role of Pdelta, a putative fourth subunit, by in vitro and in-vivo expression. In Aim 2, we propose to address mechanisms leading to dominant cone dystrophy when mutant GCAPs are expressed in the retina of transgenic mice. Additional questions concern the roles of GCAP2 and GC2 in phototransduction. GCAP1,2 double knockouts revealed a phenotype of delay in dark adaptation consistent with loss of activators. We showed (see preliminary results/progress report) that transgenic GCAP1 rescues the GCAP1/2 double knockout phenotype, suggesting that GCAP1 alone may regulate both rod and cone phototransduction. Elucidation of specific roles, particularly for GCAP2 and GC2, in the retina is part of Specific Aim 3. Taken together, this research will enhance our knowledge of the structure and function of components involved in the regulation of cGMP and Ca 2+, two important secondary messengers of phototransduction.