Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.
Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia; Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Moscow 119991, Russia.
Cell Calcium. 2018 Jul;73:55-69. doi: 10.1016/j.ceca.2018.04.003. Epub 2018 Apr 14.
Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1-101), or its short scaffolding domain (81-101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca-signals thereby maintaining efficient phototransduction recovery and light adaptation.
视杆细胞膜含有富含胆固醇的去污剂抗性膜 (DRM) 筏，其聚集视觉级联蛋白以及参与光转导调节的蛋白质，如视紫红质激酶和鸟苷酸环化酶。窖蛋白-1 是 DRM 的主要完整成分，对各种信号蛋白具有支架和调节作用。在这项研究中，属于神经元钙传感器 (NCS) 家族的光感受器 Ca 结合蛋白 recoverin、NCS1、GCAP1 和 GCAP2 被识别为新型窖蛋白-1 相互作用伙伴。所有四种 NCS 蛋白都与从光照的牛视杆外段分离的 DRM 中的窖蛋白-1 共分馏。根据下拉测定、表面等离子体共振光谱和等温热滴定法数据，它们能够通过新的结构位点与窖蛋白-1 的 N 端片段 (1-101) 或其短支架结构域 (81-101) 进行高亲和力结合。在 recoverin 中，该位点位于靠近第三个 EF 手模体的 C 端结构域内，由 NCS 蛋白之间保守的芳香族氨基酸组成。值得注意的是，NCS 蛋白与窖蛋白-1 的结合仅在没有钙的情况下发生，这与它们的 Ca 游离形式中窖蛋白-1 结合位点的更高可及性一致。一致地，窖蛋白-1 的存在对 Ca 饱和 recoverin 或 NCS1 对视紫红质激酶的调节活性没有影响，但上调了 GCAP2，GCAP2 增强了鸟苷酸环化酶活性，处于 Ca 游离构象。此外，与窖蛋白-1 的相互作用降低了 recoverin 对 Ca2+结合的协同性并增加了其亲和力，这显然是通过促进其豆蔻酰基的暴露来实现的。我们认为，在低钙条件下，NCS 蛋白通过与窖蛋白-1 结合而区室化在光感受器筏中，这可能增强它们的活性或确保它们对 Ca 信号更快的反应，从而维持有效的光转导恢复和光适应。
