Dynamic regulation of mammalian numb by G protein-coupled receptors and protein kinase C activation: Structural determinants of numb association with the cortical membrane
Dynamic regulation of mammalian numb by G protein-coupled receptors and protein kinase C activation: Structural determinants of numb association with the cortical membrane
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DOI：
10.1091/mbc.e06-02-0097
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发表时间：
2006-09-01
影响因子：
3.3
通讯作者：
McGlade, C. Jane
中科院分区：
文献类型：
--
作者：
Dho, Sascha E.;Trejo, JoAnn;McGlade, C. Jane
细胞命运决定因子 Numb 是一种膜相关衔接蛋白，参与发育和细胞内囊泡运输。它具有磷酸酪氨酸结合 (PTB) 结构域和含有 a-adaptin 和 Eps15 同源结构域的蛋白质的 COOH 末端内吞结合基序。 Numb 的四种亚型在脊椎动物中表达，其中两种亚型选择性地与皮质膜结合。在这项研究中，我们表征了 Numb 的皮质池，它与 AP2 和 Eps15 共定位于基质质膜点和皮质膜相关囊泡处。 Numb 的绿色荧光蛋白 (GFP) 标记突变体用于鉴定定位所需的结构决定因素。除了之前描述的 PTB 结构域与质膜的关联之外，我们还发现 AP2 结合基序促进 Numb 与皮质膜点和囊泡的关联。我们还发现，与磷脂酶 Co 和蛋白激酶 C (PKC) 激活相关的 G 蛋白偶联受体 (GPCR) 的激动剂刺激会导致 Numb 从皮质膜重新分布到细胞质。这种效应与 Numb 磷酸化及其 Triton X-100 溶解度的增加相关。对突变体的实时成像分析确定了 Numb 内独立响应 GPCR 介导的脂质水解和 PKC 激活的两个区域：PTB 结构域和包含至少三个推定 PKC 磷酸化位点的区域。我们的数据表明 Numb 的膜定位受到 GPCR 激活的磷脂水解和 PKC 依赖性磷酸化事件的动态调节。
The cell fate determinant Numb is a membrane-associated adaptor protein involved in both development and intracellular vesicular trafficking. It has a phosphotyrosine-binding (PTB) domain and COOH-terminal endocytic-binding motifs for a-adaptin and Eps15 homology domain-containing proteins. Four isoforms of Numb are expressed in vertebrates, two of which selectively associate with the cortical membrane. In this study, we have characterized a cortical pool of Numb that colocalizes with AP2 and Eps15 at substratum plasma membrane punctae and cortical membrane-associated vesicles. Green fluorescent protein (GFP)-tagged mutants of Numb were used to identify the structural determinants required for localization. In addition to the previously described association of the PTB domain with the plasma membrane, we show that the AP2-binding motifs facilitate the association of Numb with cortical membrane punctae and vesicles. We also show that agonist stimulation of G protein-coupled receptors (GPCRs) that are linked to phospholipase Co and protein kinase C (PKC) activation causes redistribution of Numb from the cortical membrane to the cytosol. This effect is correlated with Numb phosphorylation and an increase in its Triton X-100 solubility. Live-imaging analysis of mutants identified two regions within Numb that are independently responsive to GPCR-mediated lipid hydrolysis and PKC activation: the PTB domain and a region encompassing at least three putative PKC phosphorylation sites. Our data indicate that membrane localization of Numb is dynamically regulated by GPCR-activated phospholipid hydrolysis and PKC-dependent phosphorylation events.