Neuronal Secretion Group, Department of Neuroscience, University of Copenhagen, København, Denmark.
Department of Clinical Genetics, Center for Neurogenomics and Cognitive Research, VU Medical Center, Amsterdam, Netherlands.
Elife. 2017 Dec 23;6:e27000. doi: 10.7554/eLife.27000.
Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B - inhibition of release during sustained calcium elevations - depends on an overlapping protein domain (the MID-domain), but is separate from its Ca-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.
Doc2B 是一种胞质蛋白，具有与 Munc13 和 Tctex-1（动力蛋白轻链）结合的位点，以及两个结合磷脂、Ca 和 SNARE 的 C2 结构域。Doc2B 是否像突触融合蛋白一样充当钙传感器，或者以其他非钙依赖或钙依赖的方式发挥作用，这是有争议的。我们通过突变和过表达显示，Doc2B 在小鼠肾上腺嗜铬细胞中的两个连续的引发步骤中发挥不同的作用。突变 C2A 结构域中协调 Ca 的天冬氨酸可使 Doc2B 永久定位于质膜，使上游引发步骤不依赖 Ca，而下游引发步骤中的一个单独功能则依赖 SNARE 结合、Doc2B 的 C2B 结构域结合 Ca、与 ubMunc13-2 的相互作用和突触融合蛋白-1 的存在。Doc2B 的另一个功能——在持续的钙升高期间抑制释放——取决于重叠的蛋白结构域（MID 结构域），但与其 Ca 依赖性引发功能是分开的。我们得出结论，Doc2B 是一种囊泡引发蛋白。
