Department of Orthopaedics, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325027 China.
School of Pathology and Laboratory Medicine, The University of Western Australia, Perth, WA 6009, Australia.
Sci Rep. 2016 Nov 29;6:37963. doi: 10.1038/srep37963.
Calmodulin is a highly versatile protein that regulates intracellular calcium homeostasis and is involved in a variety of cellular functions including cardiac excitability, synaptic plasticity and signaling transduction. During osteoclastic bone resorption, calmodulin has been reported to concentrate at the ruffled border membrane of osteoclasts where it is thought to modulate bone resorption activity in response to calcium. Here we report an interaction between calmodulin and Rab3D, a small exocytic GTPase and established regulator osteoclastic bone resorption. Using yeast two-hybrid screening together with a series of protein-protein interaction studies, we show that calmodulin interacts with Rab3D in a calcium dependent manner. Consistently, expression of a calcium insensitive form of calmodulin (i.e. CaM1234) perturbs calmodulin-Rab3D interaction as monitored by bioluminescence resonance energy transfer (BRET) assays. In osteoclasts, calmodulin and Rab3D are constitutively co-expressed during RANKL-induced osteoclast differentiation, co-occupy plasma membrane fractions by differential gradient sedimentation assay and colocalise in the ruffled border as revealed by confocal microscopy. Further, functional blockade of calmodulin-Rab3D interaction by calmidazolium chloride coincides with an attenuation of osteoclastic bone resorption. Our data imply that calmodulin- Rab3D interaction is required for efficient bone resorption by osteoclasts in vitro.
钙调蛋白是一种高度多功能的蛋白质，可调节细胞内钙稳态，并参与多种细胞功能，包括心脏兴奋性、突触可塑性和信号转导。在破骨细胞的骨吸收过程中，钙调蛋白已被报道集中在破骨细胞的皱襞边缘膜上，据认为它可以调节钙响应的骨吸收活性。在这里，我们报告了钙调蛋白与 Rab3D 之间的相互作用，Rab3D 是一种小的外排 GTPase，是破骨细胞骨吸收的既定调节剂。通过酵母双杂交筛选以及一系列蛋白质-蛋白质相互作用研究，我们表明钙调蛋白以钙离子依赖的方式与 Rab3D 相互作用。一致地，表达钙不敏感形式的钙调蛋白（即 CaM1234）通过生物发光共振能量转移（BRET）测定干扰钙调蛋白-Rab3D 相互作用。在破骨细胞中，钙调蛋白和 Rab3D 在 RANKL 诱导的破骨细胞分化过程中持续共表达，通过差速梯度沉降测定共占据质膜部分，并通过共聚焦显微镜显示在皱襞边缘共定位。此外，钙调蛋白-Rab3D 相互作用的功能阻断通过氯氮平盐酸盐与破骨细胞骨吸收的减弱一致。我们的数据表明，钙调蛋白-Rab3D 相互作用是破骨细胞体外有效骨吸收所必需的。
