Differential Clathrin Binding and Subcellular Localization of OCRL1 Splice Isoforms
Differential Clathrin Binding and Subcellular Localization of OCRL1 Splice Isoforms
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DOI：
10.1074/jbc.m807442200
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发表时间：
2009-04-10
影响因子：
4.8
通讯作者：
Lowe, Martin
中科院分区：
文献类型：
--
作者：
Choudhury, Rawshan;Noakes, Christopher J.;Lowe, Martin
肌醇多磷酸5-磷酸酶OCRL 1的突变导致X-连锁疾病眼脑肾综合征，其特征是脑、肾和眼的缺陷。OCRL 1以两种剪接异构体存在，其差异在于编码8个氨基酸的单个外显子。较长的蛋白质，称为亚型a，是大脑中唯一的形式，而这两种亚型都存在于所有其他组织中。OCRL 1剪接的意义目前尚不清楚。鉴于其接近网格蛋白结合位点，我们假设剪接可能会改变OCRL 1的网格蛋白结合特性。在这里，我们表明，情况确实如此。OCRL 1亚型a与网格蛋白的结合亲和力高于亚型B，并且在网格蛋白包被的运输中间体中显著更富集。我们还确定了第二个网格蛋白结合位点OCRL 1，有助于网格蛋白结合的两种亚型。OCRL 1与网格蛋白包被的中间体的缔合需要通过与Rab GTP酶相互作用而非与网格蛋白衔接子AP 2结合的膜缔合。缺乏5-磷酸酶结构域的OCRL 1亚型a的表达损害转铁蛋白内吞作用，而亚型B的等价形式则不会。我们的研究结果表明，OCRL 1存在两个功能池，一个参与网格蛋白介导的贩运事件，如内吞作用，另一个是少得多或不参与这一过程。
Mutation of the inositol polyphosphate 5-phosphatase OCRL1 causes the X-linked disorder oculocerebrorenal syndrome of Lowe, characterized by defects in the brain, kidneys, and eyes. OCRL1 exists as two splice isoforms that differ by a single exon encoding 8 amino acids. The longer protein, termed isoform a, is the only form in brain, whereas both isoforms are present in all other tissues. The significance of OCRL1 splicing is currently unclear. Given its proximity to a clathrin-binding site, we hypothesized that splicing may alter the clathrin binding properties of OCRL1. Here we show that this is indeed the case. OCRL1 isoform a binds clathrin with higher affinity than isoform b and is significantly more enriched in clathrin-coated trafficking intermediates. We also identify a second clathrin-binding site in OCRL1 that contributes to clathrin binding of both isoforms. Association of OCRL1 with clathrin-coated intermediates requires membrane association through interaction with Rab GTPases but not binding to the clathrin adaptor AP2. Expression of OCRL1 isoform a lacking the 5-phosphatase domain impairs transferrin endocytosis, whereas an equivalent version of isoform b does not. Our results suggest that OCRL1 exists as two functional pools, one participating in clathrin-mediated trafficking events such as endocytosis and another that is much less or not involved in this process.