Characterization of Recombinant ELMOD ( Cell Engulfment and Motility Domain) Proteins as GTPase-activating Proteins ( GAPs) for ARF Family GTPases
Characterization of Recombinant ELMOD ( Cell Engulfment and Motility Domain) Proteins as GTPase-activating Proteins ( GAPs) for ARF Family GTPases
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DOI：
10.1074/jbc.m114.548529
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发表时间：
2014-04-18
影响因子：
4.8
通讯作者：
Kahn, Richard A.
中科院分区：
文献类型：
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作者：
Ivanova, Anna A.;East, Michael P.;Kahn, Richard A.
背景：ELMOD 蛋白是 ARF 家族的非典型 GAP，与耳聋有关。结果：ELMOD 蛋白对 ARF 蛋白具有独特的特异性活性，并结合非阿片类药物 sigma-1 受体。结论：预计 ELMOD1-3 的不同特定活性是不同细胞功能的基础。意义：ELMOD 的特异性和调节有助于了解它们在细胞信号传导和内耳中的作用。调节性 GTP 酶的 ARF 家族属于 RAS 超家族，在哺乳动物中由大约 30 个成员组成，包括多达 6 个 ARF 和至少 18 个 ARF 样 (ARL) 蛋白。它们表现出显着的结构和生化保守性，并调节各种重要的细胞过程，包括膜运输、细胞分裂和能量代谢；每一个都与人类疾病有关。我们之前将 ELMOD 家族的成员鉴定为 ARL2 的 GTP 酶激活蛋白 (GAP)，也对 ARF 表现出交叉活性。为了进一步表征三种人类 ELMOD 作为 GAP 的 GAP 活性，我们在人胚胎肾 (HEK293T) 细胞中过表达后开发了每种 ELMOD 的新制剂。这使得纯化的蛋白质具有更高的比活性并增强稳定性和溶解度。确定了 ELMOD1-3 作为 ARF 家族六个不同成员的 GAP 的特异性，并发现其表现出广泛的变化，我们相信这将揭示家族成员细胞功能的差异。非阿片类药物 sigma-1 受体 (S1R) 被确定为 ELMOD1-3 蛋白 GAP 活性的新型效应器，因为它直接结合 ELMOD1 或 ELMOD2 导致 GAP 活性丧失。这些发现对于理解 ELMOD 蛋白在一般细胞信号传导和内耳细胞信号传导中的作用至关重要，并为探索通过 S1R 激动剂或拮抗剂调节其 GAP 活性打开了大门。
Background: ELMOD proteins are atypical GAPs for the ARF family with links to deafness. Results: ELMOD proteins have distinct specific activities for ARF proteins and bind the non-opioid sigma-1 receptor. Conclusion: Different specific activities of ELMOD1-3 are predicted to underlie different cellular functions. Significance: Specificities and regulation of ELMODs contribute to understanding their roles in cell signaling and the inner ear.The ARF family of regulatory GTPases, within the RAS superfamily, is composed of approximate to 30 members in mammals, including up to six ARF and at least 18 ARF-like (ARL) proteins. They exhibit significant structural and biochemical conservation and regulate a variety of essential cellular processes, including membrane traffic, cell division, and energy metabolism; each with links to human diseases. We previously identified members of the ELMOD family as GTPase-activating proteins (GAPs) for ARL2 that displayed crossover activity for ARFs as well. To further characterize the GAP activities of the three human ELMODs as GAPs we developed new preparations of each after overexpression in human embryonic kidney (HEK293T) cells. This allowed much higher specific activities and enhanced stability and solubility of the purified proteins. The specificities of ELMOD1-3 as GAPs for six different members of the ARF family were determined and found to display wide variations, which we believe will reveal differences in cellular functions of family members. The non-opioid sigma-1 receptor (S1R) was identified as a novel effector of GAP activity of ELMOD1-3 proteins as its direct binding to either ELMOD1 or ELMOD2 resulted in loss of GAP activity. These findings are critical to understand the roles of ELMOD proteins in cell signaling in general and in the inner ear specifically, and open the door to exploration of the regulation of their GAP activities via agonists or antagonists of the S1R.