2016-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/670333摘要：Clathrin 所媒介之內吞作用(endocytosis)是受體內在化(internalization)的主要路徑，對於在細胞膜上蛋白質和受體之再循環及訊息傳遞是必需的。Clathrin-coated pits 的組裝，包括接合蛋白質和 clathrin 次單元之徵募是以準確性和時空調節性的方式緊密合作。同時， cargo的加入，clathrin網格及 clathrin相關之分類蛋白質聚集形成一個架構，可以促進穩定、內吞、細胞膜重新塑型，最終將 coated vesicles切割分離而進入細胞內。胰島素受體是 tyrosine kinase受體，可以調節哺乳類細胞生長和維持脂質和葡萄糖之恆定。當胰島素結合至其受體，可以透過對其結構改變，放大胰島素訊息，活化 tyrosinekinase及其下游 PI3K/Akt路徑，使葡萄糖運送蛋白質 GLUT4從細胞質移至細胞膜以吸收葡萄糖並進行代謝。胰島素受體訊息傳遞是由受體所參與內吞作用後啟動，在內質區經 tyrosine phosphatase PIP1B的作用而終止。雖然胰島素受體靠近膜區域之 dileucine對於 AP-2-clathrin所參與之內吞作用是必需的。但是，接合蛋白質如何專一性調控胰島素受體之內吞作用，之前仍然未被鑑定出。我們最近的研究證明細胞膜上 LMBD1蛋白質其功能是經由與 AP-2結合，為 clathrin所媒介之胰島素受體內吞作用之結合蛋白質。由於 cargo內吞作用為非競爭性及非飽和性，使 cargo的選擇性被觀察到。LMBD1蛋白質不參與輸鐵蛋白質(transferrin)之內吞作用。而對於 LMBD1參與胰島素受體內吞作用之分子機轉及專一性並不完全明瞭。為了回答這些問題，下列為本研究計畫之主要目標:1. 分析與胰島素受體結合之 LMBD1蛋白質功能區2. 檢視心肌細胞中，是否胰島素受體之自體磷酸化為與 LMBD1蛋白質結合之先決條件3. 鑑定與胰島素受體內吞作用所需之細胞表面 LMBD1結合蛋白質4. 分析這些細胞表面 LMBD1結合蛋白質參與胰島素受體內吞作用之功能角色5. 檢視 LMBD1蛋白質是否透過 PIP1B的結合，參與胰島素受體之再循環6. 探討 LMBD1蛋白質參與心臟胰島素受體之內吞與葡萄糖吸收，是否亦發生於對胰島素敏感之脂肪組織及骨骼肌7. 測試 PI3K/Akt抑制劑是否可調節心臟葡萄糖吸收及在 lmbrd1基因敲除鼠所導致之心室肥大的救援8. 鑑定由 LMBD1蛋白質所媒介內吞作用之其他受體/輸送體<br> Abstract: Clathrin-mediated endocytosis is a major pathway for receptor internalization, which isessential for both the recycling of proteins and receptors on the plasma membrane (PM) and theintracellular signaling. The assembling of clathrin-coated pits, including the recruitment ofadaptor proteins and clathrin triskelion, is tightly coordinated in a precise, spatiotemporal manner.Concurrently with the cargo incorporation, clathrin lattice and clathrin-associated sortingproteins are clustered forming a scaffold that facilitates the stabilization, invagination, membranereshaping, and eventually scission of the coated vesicles. The insulin receptor (IR) is a receptortyrosine kinase that controls mammalian growth and maintains the homeostasis of lipid andglucose metabolism. When insulin binds to the IR, it amplifies insulin signals by stimulating astructural change in the IR, allowing the activation of its tyrosine kinase activity and thedownstream PI3K/Akt pathway, resulting in the translocation of glucose transporter 4 from thecytoplasm to the PM for glucose uptake and metabolism. IR signaling is terminated throughreceptor-mediated endocytosis followed by the action of tyrosine phosphatase PTP1B in theendosomal compartments. Although the juxtamembrane dileucine motif of the IR is essential forAP2-clathrin-mediated endocytosis, the adaptor protein that specifically regulates theinternalization of the IR remains unidentified. Our recent studies demonstrated that PM-localizedLMBD1 protein functions as a specific adaptor for the clathrin-mediated endocytosis of the IRthrough its interaction with AP-2. Because cargoes are internalized in a non-competitive,non-saturable manner, cargo selectivity is observed. LMBD1 is not involved in the transferrinreceptor endocytosis. Whereas, molecular mechanisms and the specificity of LMBD1 involved inthe IR endocytsis are not fully understood. To address these questions, specific aims of this studyare described as follows.1. To dissect the functional motifs of LMBD1 protein required for the interaction with insulinreceptor2. To examine whether autophosphorylation of insulin receptor is prerequisite for the interactionwith LMBD1 protein in cardiomyocytes3. To identify surface LMBD1-interacting proteins required for the clathrin-mediatedinternalization of insulin receptor4. To analyze functional roles of the surface LMBD1-associated proteins involved in the IRendocytosis5. To examine whether LMBD1 protein involves in the recycling of insulin receptor through theinteraction with PTP1B6. To investigate whether LMBD1 protein involves in the insulin receptor endocytosis andglucose uptake in insulin-sensitive adipose tissue and skeletal muscle7. To test whether PI3K/Akt inhibitors can modulate the cardiac glucose uptake and rescue thephenotype of cardiac hypertrophy in the lmbrd1+/- knockout mice8. To identify additional putative receptors/transporters that undergo endocytosis through theLMBD1-mediated mechanismclathrin所媒介之內吞作用胰島素受體接合蛋白質clathrin-mediated endocytosisinsulin receptoradaptor protein