Duda T, Jankowska A, Venkataraman V, Nagele R G, Sharma R K
Unit of Regulatory and Molecular Biology, Department of Cell Biology, New Jersey Medical School and School of Osteopathic Medicine, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA.
Biochemistry. 2001 Oct 9;40(40):12067-77. doi: 10.1021/bi0108406.
This report defines the identity of a calcium-regulated membrane guanylate cyclase transduction system in the cilia of olfactory sensory neurons, which is the site of odorant transduction. The membrane fraction of the neuroepithelial layer of the rat exhibited Ca(2+)-dependent guanylate cyclase activity, which was eliminated by the addition of EGTA. This indicated that the cyclase did not represent a rod outer segment guanylate cyclase (ROS-GC), which is inhibited by free Ca(2+). This interpretation was supported by studies with the Ca(2+) binding proteins, GCAPs (guanylate cyclase activating proteins), which stimulate photoreceptor ROS-GC in the absence of Ca(2+). They did not stimulate the olfactory neuroepithelial membrane guanylate cyclase. The olfactory neuroepithelium contained a Ca(2+) binding protein, neurocalcin, which stimulated the cyclase in a Ca(2+)-dependent fashion. The cyclase was cloned from the neuroepithelium and was found to be identical in structure to that of the previously cloned cyclase termed GC-D. The cyclase was expressed in a heterologous cell system, and was reconstituted with its Ca(2+)-dependent activity in the presence of recombinant neurocalcin. The reconstituted cyclase mimicked the native enzyme. Immunocytochemical studies showed that the guanylate cyclase coexists with neurocalcin in the apical region of the cilia. Deletion analysis showed that the neurocalcin-regulated domain resides at the C-terminal region of the cyclase. The findings establish the biochemical, molecular, and functional identity of a novel Ca(2+)-dependent membrane guanylate cyclase transduction system in the cilia of the olfactory epithelium, suggesting a mechanism of the olfactory neuroepithelial guanylate cyclase regulation fundamentally distinct from the phototransduction-linked ROS-GC.
本报告确定了嗅觉感觉神经元纤毛中一种钙调节的膜鸟苷酸环化酶转导系统的特性，该纤毛是气味转导的部位。大鼠神经上皮层的膜部分表现出钙依赖性鸟苷酸环化酶活性，加入乙二醇双四乙酸（EGTA）后该活性消失。这表明该环化酶并非视杆外段鸟苷酸环化酶（ROS-GC），后者会被游离钙抑制。钙结合蛋白鸟苷酸环化酶激活蛋白（GCAPs）的研究支持了这一解释，GCAPs在无钙情况下刺激光感受器ROS-GC，却不刺激嗅觉神经上皮膜鸟苷酸环化酶。嗅觉神经上皮含有一种钙结合蛋白神经钙蛋白，它以钙依赖性方式刺激环化酶。从神经上皮克隆出该环化酶，发现其结构与先前克隆的称为GC-D的环化酶相同。该环化酶在异源细胞系统中表达，并在重组神经钙蛋白存在的情况下恢复其钙依赖性活性。重组环化酶模拟了天然酶。免疫细胞化学研究表明，鸟苷酸环化酶与神经钙蛋白共存于纤毛的顶端区域。缺失分析表明，神经钙蛋白调节结构域位于环化酶的C末端区域。这些发现确立了嗅觉上皮纤毛中一种新型钙依赖性膜鸟苷酸环化酶转导系统的生化、分子和功能特性，提示嗅觉神经上皮鸟苷酸环化酶的调节机制与光转导相关的ROS-GC根本不同。
