Nemoto Y, Ikeda J, Katoh K, Koshimoto H, Yoshihara Y, Mori K
Department of Neuroscience, Osaka Bioscience Institute, Japan.
J Cell Biol. 1993 Nov;123(4):963-76. doi: 10.1083/jcb.123.4.963.
R2D5 is a mouse monoclonal antibody that labels rabbit olfactory receptor neurons. Immunoblot analysis showed that mAb R2D5 recognizes a 22-kD protein with apparent pI of 4.8, which is abundantly contained in the olfactory epithelium and the olfactory bulb. We isolated cDNA for R2D5 antigen and confirmed by Northern analysis and neuronal depletion technique that R2D5 antigen is expressed predominantly, but not exclusively, in olfactory receptor neurons. Analysis of the deduced primary structure revealed that R2D5 antigen consists of 189 amino acids with calculated M(r) of 20,864 and pI of 4.74, has three calcium-binding EF hands, and has possible phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and cAMP-dependent protein kinase (A kinase). Using the bacterially expressed protein, we directly examined the biochemical properties of R2D5 antigen. R2D5 antigen binds Ca2+ and undergoes a conformational change in a manner similar to calmodulin. R2D5 antigen is phosphorylated in vitro by CaM kinase II and A kinase at different sites, and 1.81 and 0.80 mol of Pi were maximally incorporated per mol of R2D5 antigen by CaM kinase II and A kinase, respectively. Detailed immunohistochemical study showed that R2D5 antigen is also expressed in a variety of ependymal cells in the rabbit central nervous system. Aside from ubiquitous calmodulin, R2D5 antigen is the first identified calcium-binding protein in olfactory receptor neurons that may modulate olfactory signal transduction. Furthermore our results indicate that olfactory receptor neurons and ependymal cells have certain signal transduction components in common, suggesting a novel physiological process in ependymal cells.
R2D5是一种标记兔嗅觉受体神经元的小鼠单克隆抗体。免疫印迹分析表明，单克隆抗体R2D5识别一种表观pI为4.8的22-kD蛋白，该蛋白大量存在于嗅上皮和嗅球中。我们分离了R2D5抗原的cDNA，并通过Northern分析和神经元耗竭技术证实，R2D5抗原主要但并非仅在嗅觉受体神经元中表达。对推导的一级结构的分析表明，R2D5抗原由189个氨基酸组成，计算的M(r)为20,864，pI为4.74，有三个钙结合EF手结构，并且有钙调蛋白依赖性蛋白激酶II（CaM激酶II）和环磷酸腺苷依赖性蛋白激酶（A激酶）的可能磷酸化位点。使用细菌表达的蛋白，我们直接检测了R2D5抗原的生化特性。R2D5抗原结合Ca2+并以类似于钙调蛋白的方式发生构象变化。R2D5抗原在体外被CaM激酶II和A激酶在不同位点磷酸化，CaM激酶II和A激酶分别使每摩尔R2D5抗原最大掺入1.81和0.80摩尔的无机磷酸（Pi）。详细的免疫组织化学研究表明，R2D5抗原也在兔中枢神经系统的多种室管膜细胞中表达。除了普遍存在的钙调蛋白外，R2D5抗原是嗅觉受体神经元中第一个被鉴定的可能调节嗅觉信号转导的钙结合蛋白。此外，我们的结果表明，嗅觉受体神经元和室管膜细胞具有某些共同的信号转导成分，提示室管膜细胞中存在一种新的生理过程。
