LMBD1羧基端區域對胰島素受體訊息傳遞之重要性

Abstract

細胞內能量平衡對於維持細胞正常生理功能扮演重要角色。LMBRD1基因所表現之LMBD1蛋白質先前被報導參與在溶酶體之維生素B12輸出。LMBD1被預測有九個穿膜功能區，主要分布於細胞膜、溶酶體及內質網上。先前實驗室研究指出LMBD1蛋白質選擇性調控心臟胰島素受體(IR)之內吞作用，而不參與轉鐵蛋白質受體(TrfR)之內吞作用。進一步結果顯示LMBD1蛋白質作為胰島素受體內吞作用之adaptor之角色。胰島素受體調控細胞內葡萄糖之恆定、細胞分裂、發育及代謝，以雙硫鍵形成雙體結構後始具有生理功能。細胞膜上胰島素受體之含量由依賴網格蛋白的胞吞作用(clathrin-mediated endocytosis)所調控。依賴網格蛋白的胞吞作用調控真核細胞之代謝及恆定，胞吞作用對於cargo之選擇性是由adaptor所提供。胰島素受體及轉鐵蛋白質受體皆由AP-2所調控，顯示LMBD1蛋白質可能提供胰島素受體於依賴網格蛋白胞吞作用之專一性adaptor。先前結果顯示LMBD1蛋白質與AP-2之間會透過YXXφ與WXXF功能區進行交互作用幫忙IR的內吞作用，TrfR的內吞作用則不經由LMBD1的協助，更進一步顯示LMBD1蛋白質在心臟胰島素受體內吞作用之重要性。 小鼠Lmbrd1異體蛋白質在心臟組織主要以LMBD1之形式存在。胰島素受體下游調控細胞內葡萄糖含量之GLUT4，在H9C2心肌細胞中Lmbrd1 knockdown後，發現細胞膜上GLUT4表現增加。本研究利用細胞培養及蛋白質化學方式欲釐清LMBD1蛋白質如何選擇性調控胰島素受體之內吞作用。結果顯示LMBD1蛋白質會利用其羧基端(433-493 a.a.)，而非(433-540 a.a.)，與胰島素受體進行交互作用。利用GST-LMBD1(433-493)競爭內生性之LMBD1蛋白質與IR的結合，確實會使IR下游訊息傳遞路徑之Akt磷酸化上升。綜合實驗結果可以推測LMBD1羧基端可能在IR之內吞作用中扮演具有選擇性之角色。

Energy homeostasis plays a critical role in maintaining cell normal functions. The LMBD1 protein which is encoded by the limb region 1 (LMBR1) domain containing 1 gene ( LMBRD1 ) has been suggested to be involved in the export of cobalamin in the lysosome. The LMBD1 protein possesses 9 putative transmembrane domains and distributes over plasma membrane, lysosome and endoplasmic reticulum. Our previous study showed that LMBD1 protein serves as specific adaptor for insulin receptor (IR) endocytosis. The IR encoded by a single gene INSR is a member of tyrosine kinase family and plays critical regulatory roles in glucose homeostasis, development, cell division, and metabolism. The recycle of IR on plasma membrane is controlled by clathrin-mediated endocytosis which is a common way to internalize cargos in eukaryotic cells such as, receptors and nutrients. In the process of endocytosis, the cargo specificity is determined by cargo-specific adaptors. LMBD1 use YXXφ and WXXF motifs to interact with adaptor protein-2 (AP-2) and is involved in the unique endocytosis of IR. Although TrfR and IR use the same adaptor AP-2, LMBD1 specially regulates the insulin receptor internalization. It seems that LMBD1 protein may be through interacting with IR to provide the selectivity to recognize. In this study, how LMBD1 protein provides the selectivity in the endocytosis of IR was examined. To dissect the functional motifs of LMBD1 protein, GST-pull down assay was performed. Results showed that LMBD1(433-499), but not LMBD1(433-540) interacted with IR. GST-LMBD1(433-493) competed the interaction of endogenous LMBD1 with IR, and increased the phosphorylation of Akt in the IR downstream signaling pathway. These results provide further evidence in understanding how LMBD1, through the domain from amino acid residues 433 to 493, interacts with IR with cargo selectivity in the IR endocytosis.