LMBD1經由結合多巴胺轉運蛋白及自體受體D2調節多巴胺之攝取

LMBD1 protein regulates dopamine uptake through interacting with dopamine transporter and autoreceptor D2

張雅婷(Ya-Ting Chang)

指導教授 ： 張明富

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703065

摘要

神經傳遞物質的運輸對於神經系統正常的調控相當重要，若是在腦中神經傳遞物質的含量產生異常就會生成疾病，例如腦部發育以及行為上的異常，因此知曉調控神經傳遞物質的機制對於發展治療神經疾病的方法是相當重要的。LMBRD1基因可產生兩種主要的蛋白質，分別為LMBD1以及NESI。在過去的文獻指出LMBD1會協助維他命B12送出溶酶體，而實驗室更進一步發現LMBD1會調控由網格蛋白 (clathrin) 輔助的胰島素受體內化。另外實驗室亦發現Lmbrd1+/-基因剔除小鼠會表現出特殊行為，像是出現害怕緊張之情緒、對刺激的反應遲鈍以及學習記憶的不足，除此之外在正子放射斷層掃描圖譜對於Lmbrd1+/-基因剔除小鼠腦部的分析的結果中發現，與上述行為異常有關的紋狀體組織多巴胺含量有上升的現象。因此本研究的目的是想要探討LMBD1是藉由何種機制調控多巴胺運輸。在芳香族L-胺基酸脫羧酵素 (aromatic L-amino acid decarboxylase, AADC) 突變小鼠中找到了支持本研究對於LMBD1參與調控多巴胺運輸之假設的初步證據，結果顯示較年幼之AADC突變小鼠紋狀體中LMBD1表現量有下降的現象，推測AADC突變小鼠會藉由調控LMBD1表現量使得多巴胺含量上升來補償AADC突變小鼠無法生合成多巴胺的缺陷。在文獻中顯示多巴胺自體受體D2 (dopamine autoreceptor D2, D2autoR) 與多巴胺轉運蛋白 (dopamine transporter, DAT) 對於調控多巴胺的含量相當重要。多巴胺自體受體D2會藉由負回饋機制調控多巴胺的釋出，而多巴胺轉運蛋白則會直接運送多巴胺。除此之外，多巴胺自體受體D2會藉由PKA訊息傳遞路徑、PKCβ-ERK訊息傳遞路徑以及直接與多巴胺轉運蛋白交互作用以調控多巴胺轉運蛋白在細胞膜上的表現量。在本研究中發現將小鼠紋狀體細胞株STHdhQ7中Lmbrd1基因降低表現時多巴胺攝入量有提升的現象。在Lmbrd1+/-基因剔除小鼠紋狀體組織以及Lmbrd1基因低表現之紋狀體細胞STHdhQ7，PKA磷酸化有下降的現象，而ERK磷酸化則有上升的現象。在共軛焦顯微鏡觀察的結果中發現LMBD1與多巴胺自體受體D2的分佈有部分共位的現象。另外本研究利用免疫沉澱發現LMBD1與多巴胺轉運蛋白有結合的現象，並在共軛焦顯微鏡的觀察中發現有部分共位的現象，除此之外也利用GST pull-down assay發現了LMBD1主要以羧基端與多巴胺轉運蛋白結合。綜合以上研究結果，LMBD1可能藉由調節多巴胺自體受體D2以及多巴胺轉運蛋白的功能進而調節多巴胺運輸。

關鍵字

多巴胺轉運蛋白 ； 多巴胺自體受體D2 ； 多巴胺攝取 ； 多巴胺

並列摘要

The transport of neurotransmitters is essential for the regulation of nervous system. The abnormal level of neurotransmitters in brain often causes developmental problems and distinct behaviors. Therefore, understanding the mechanism of neurotransmitter transport is required for developing the treatment of neuron diseases. The limb region 1 domain containing 1 (LMBRD1) gene encodes two major proteins, named LMBD1 and NESI. The LMBD1 protein contains additional 73 amino acids residues at the N-terminus of NESI protein. Previous studies have shown that LMBD1 protein is a putative lysosomal vitamin B12 exporter. In addition, our studies further elucidated that LMBD1 protein serves as a specific adaptor protein and mediates the clathrin-mediated internalization of insulin receptor. Furthermore, our recent studies showed that Lmbrd1 heterozygous knockout (Lmbrd1+/-) mice exhibited distinct behaviors from normal mice such as fear, anxiety, pre-pulse inhibition, depression, sociability deficits, and insufficiency on learning memory. As the result of the abnormal behaviors of emotion and memory, our studies focus on striatum which involves in the regulation of emotion and memory reward. The data of positron emission tomography (PET) with 18F-DOPA showed an increase of dopamine level in Lmbrd1+/- mouse striatum. The specific aim of this study is to elucidate the mechanisms by which LMBD1 protein regulates dopamine transport. Results from the study of dopamine uptake in the aromatic L-amino acid decarboxylase (AADC) deficiency mouse model supported our hypothesis related to the function of LMBD1 protein in dopamine transport, indicating that the reduction of LMBD1 expression in the striatum of AADC deficiency mice with young age resulted in the dopamine accumulation to compensate the lack of dopamine biosynthesis. Dopamine autoreceptor D2 (D2autoR) and dopamine transporter (DAT) are responsible for dopamine transport. D2autoR regulates the dopamine exocytosis by negative feedback mechanism, whereas DAT directly transports extracellular dopamine into cells. In addition, D2autoR regulates the surface expression of DAT by PKA pathway, PKCβ-ERK pathway and by direct protein-protein interaction. In this study, dopamine uptake was increased when Lmbrd1 gene was knocked down in striatal STHdhQ7 cells. The phosphorylation of PKA was reduced in Lmbrd1+/- mouse striatum and Lmbrd1 knockdown STHdhQ7 cells, whereas the phosphorylation of ERK was increased. In addition, confocal image showed that LMBD1 partially colocalized with D2autoR. These data suggested that LMBD1 might play a role in regulating D2autoR and into downstream signal pathway. On the other hand, co-immunoprecipatation assay showed LMBD1 associated with DAT when co-expressed in HEK293T cells. GST pull down assay further demonstrated the interaction of the C terminus of LMBD1 with DAT present in STHdhQ7 cells. In addition, confocal image showed that DAT partially colocalized with LMBD1. These results suggested that LMBD1 might regulate D2autoR and DAT function involved in dopamine uptake.

並列關鍵字

LMBD1 ； dopamine ； dopamine transporter ； dopamine autoreceptor D2 ； dopamine uptake

參考文獻

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