LMBD1蛋白質參與胰島素受體的訊息傳遞及心室肥大之機制

Abstract

LMBD1為Limb region 1 domain containing 1 gene (LMBRD1) 所轉譯的蛋白質，含有540個胺基酸的膜蛋白且具有九個穿膜區域 (transmembrane domain)，主要分佈於細胞膜、溶酶體膜及內質網上。先前LMBD1已被報導參與在維生素B12從溶酶體釋出至細胞質，本實驗室研究亦指出LMBD1蛋白質在胰島素受體 (insulin receptor; IR) 的內吞作用中扮演作為轉接器 (adaptor) 的角色，但不參與轉鐵蛋白質受體 (transferrin receptor; TrfR) 之內吞作用。另外，配合18F-FDG靜脈注射，在正子發射斷層掃描 (PET) 發現在Lmbrd1+/-小鼠心臟葡萄糖有高於wild type的現象。同時也觀察到Lmbrd1+/-小鼠其心臟有左心室肥大 (cardiac hypertrophy) 的症狀出現，並且在年齡較大時發生心臟收縮性功能障礙，顯示LMBD1蛋白質可能在心臟發育上扮演一個重要的角色。本研究主要探討在Lmbrd1+/-小鼠模式造成心室肥大的過程中，Lmbrd1基因是否透過參與胰島素受體訊息傳遞路徑調控心肌細胞的肥大。結果顯示，在Lmbrd1+/-基因剔除小鼠模式中觀察到其心臟組織IR、Akt及mTOR的磷酸化相較於wild type有上升的現象，並且心臟重量與脛骨長度比值也高於wild type，以rapamycin抑制mTOR的活化後Lmbrd1+/-小鼠的心臟大小恢復正常，心臟重量與脛骨長度比值也下降。同時，在大鼠的心肌母細胞H9C2中Lmbrd1基因表現下降時也觀察到IR及mTOR的磷酸化上升。在GST-pull down assay證實LMBD1會透過其羧基端 (胺基酸433-493) 與胰島素受體結合。利用dithiothreitol 和 2-mercaptoethanol破壞胰島素受體與β subunit間的雙硫鍵結，結果發現LMBD1會透過其羧基端與胰島素受體之β subunit作結合，但與 subunit 並沒有直接交互作用。GST-LMBD1 (胺基酸433-493) 競爭內生性之LMBD1蛋白質與胰島素受體結合，會使胰島素受體下游訊息傳遞路徑Akt磷酸化上升。綜合實驗結果可以推測Lmbrd1基因透過活化IR/Akt/mTOR的訊息傳遞路徑，導致Lmbrd1+/-小鼠出現心肌肥大症狀，而LMBD1羧基端可能在IR之內吞作用中扮演選擇性的角色。

LMBD1 protein is encoded by the limb region 1 domain containing 1 gene (LMBRD1) and consists of 540 amino acids residues with nine putative transmembrane domains. LMBD1 distributes over plasma membrane, ndoplasmic reticulum and lysosome. Previous study has shown that LMBD1 is involved in aiding the export of cobalamin from lysosome to the cytosol. In addition, our previous study demonstrated that LMBD1 is a specific adaptor for the insulin receptor (IR) endocytosis. The relactive 18F-FDG uptake in Lmbrd1+/- mouse herats was higher than that of the wildtype littermates. Lmbrd1 ubiquitous heterozygous knockout (Lmbrd1+/-) mice displayed cardiac hypertrophy and developed systolic cardiac dysfunction at an older age. These data suggest that LMBD1 is a critical component in the development of heart. This study aims to examine whether Lmbrd1 gene regulates cardiomyocyte hypertrophy through the insulin receptor signaling pathway and exhibits ventricular hypertrophy in Lmbrd1+/- mouse model. Results showed that IR kinase, along with its downstream signaling protein kinase Akt and mTOR, was hyperphosphorylated in Lmbrd1+/- mouse hearts. In addation, the ratio of heart weight to tibia length was increased in Lmbrd1+/- mice. When Lmbrd1+/- mice were treated with mTOR inhibitor rapamycin, the ratio of heart weight to tibia length decreased and heart size became normal. Furthrtmore, the phosophorylation of IR and mTOR were increasd in Lmbrd1 gene knocked down H9C2 cells. Moreover, the GST-pull down demonstrated an interacton of C-terminal domain of LMBD1 with the IR. Further studing using dithiothreitol and 2-mercaptoethanol to reduce disulfide bonds betweenIR subunits, results showed that GST-LMBD1(433-493) interacted with the subunit of IR. No direct interaction between LMBD1 and the  subunit of IR was observed. In addition, GST-LMBD1(433-493) competed with endogenous LMBD1 for interacting with IR and increased Akt phosphorylation, a downstream of IR signaling pathway. These data suggest that Lmbrd1+/- mice exhibit mTOR activation through IR-Akt axis, resulting in the progression of ventricular hypertrophy. In addation, LMBD1 interacts with IR through its subdomain from amino acid residues 433 to 493, with cargo selectivity in the IR endocytosis.