A specific dileucine motif is required for the GGA-dependent entry of newly synthesized insulin-responsive aminopeptidase into the insulin-responsive compartment
A specific dileucine motif is required for the GGA-dependent entry of newly synthesized insulin-responsive aminopeptidase into the insulin-responsive compartment
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DOI：
10.1074/jbc.m601583200
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发表时间：
2006-11-03
影响因子：
4.8
通讯作者：
Watson, Robert T.
中科院分区：
文献类型：
--
作者：
Hou, June Chunqiu;Suzuki, Naoko;Watson, Robert T.
在肌肉和脂肪细胞中，胰岛素应答性氨肽酶（IRAP）定位于细胞内储存位点，并经历胰岛素依赖性再分布至细胞表面。表达后，新合成的IRAP蛋白运输到核周胰岛素敏感区室，并在生物合成后6-9小时获得胰岛素敏感性。通过RNA干扰敲低GGA 1阻止IRAP进入但不退出胰岛素应答区室。在位置76和77处的双亮氨酸基序（EGFP-IRAP/AA（76，77））的突变，而不是在位置53和54处的双亮氨酸基序的突变，导致在生物合成后3小时开始，报道分子快速默认到细胞表面。丙氨酸取代9个残基的氨基或羧基末端LL 76，77没有扰乱基础的细胞内螯合或废除胰岛素刺激的IRAP易位。此外，显性干扰GGA突变体（VHS-GAT）有效地抑制EGFP-IRAP/WT的胰岛素刺激的易位，但不阻断EGFP-IRAP/AA的组成性胞吐运输（76，77）。此外，EGFP-IRAP/WT和EGFP-IRAP/AA（76，77）构建体在核周区域占据形态上不同的管泡隔室。总之，这些数据表明，LL 76，77在新制备的IRAP的GGA依赖性分选进入胰岛素响应性储存隔室期间起作用。
In muscle and adipose cells, the insulin-responsive aminopeptidase ( IRAP) is localized to intracellular storage sites and undergoes insulin-dependent redistribution to the cell surface. Following expression, the newly synthesized IRAP protein traffics to the perinuclear insulin-sensitive compartment and acquires insulin sensitivity 6-9 h following biosynthesis. Knockdown of GGA1 by RNA interference prevented IRAP from entering, but not exiting, the insulin-responsive compartment. Mutation of the dileucine motif at positions 76 and 77 (EGFP-IRAP/AA(76,77)),but not the dileucine motif at positions 53 and 54, resulted in the rapid default of the reporter to the cell surface beginning at 3 h following biosynthesis. Alanine substitution of 9 residues amino- or carboxyl-terminal to LL76,77 did not perturb basal intracellular sequestration or abrogate insulin-stimulated IRAP translocation. Moreover, a dominant interfering GGA mutant (VHS-GAT) potently inhibited insulin-stimulated translocation of EGFP-IRAP/WT but did not block the constitutive exocytotic trafficking of EGFP-IRAP/AA(76,77). In addition, the EGFP-IRAP/WT and EGFP-IRAP/AA(76,77) constructs occupied morphologically distinct tubulovesicular compartments in the perinuclear region. Taken together, these data indicate that LL76,77 functions during the GGA-dependent sorting of newly made IRAP into the insulin-responsive storage compartment.