酪氨酸激酶接受器(discoidin domain receptor 1-DDR1)為一具酪胺激酶活性之膠原蛋白受體，主要表現於上皮細胞中。本實驗室近來的研究證實活化的酪氨酸激酶接受器藉由抑制Cdc42藉導的內吞作用(endocytosis)，促進上皮細胞鈣黏附因子(E-cadherin)之穩定性。然而，受酪氨酸激酶接受器抑制之上皮細胞鈣黏附因子的內吞作用，其訊息傳遞機制目前仍不清楚。以往的研究表明上皮細胞鈣黏附因子，α-catenin及肌動蛋白(actin)所形成的複合物能增加上皮細胞鈣黏附因子在細胞膜上之穩定性並減少其流失率(turnover rate)。在肝細胞生長因子(hepatocyte growth factor)刺激下，活化的Cdc42透過Src誘導上皮細胞鈣黏附因子之酪氨酸磷酸化，然後造成上皮細胞鈣黏附因子，α-catenin及肌動蛋白所形成的複合物之分離，隨後導致上皮細胞鈣黏附因子透過網格蛋白(clathrin)依賴的途徑進行內吞作用。因此，我們假設酪氨酸激酶接受器透過抑制Src的活性及網格蛋白依賴的內吞途徑，穩定上皮細胞鈣黏附因子，α-catenin及肌動蛋白所形成的複合物。首先我們想了解是否knockdown 酪氨酸激酶接受器會促進上皮細胞鈣黏附因子藉由網格蛋白進行內吞作用。免疫沉澱的結果顯示knockdown DDR1 (Sh-DDR1) 促進了上皮細胞鈣黏附因子和網格蛋白間的交互作用，並且網格蛋白依賴之內吞作用抑制劑dynasore可抑制上皮細胞鈣黏附因子的內吞作用,其呈現時間與劑量依賴的趨勢，顯示受酪氨酸激酶接受器抑制之上皮細胞鈣黏附因子內吞作用是透過網格蛋白依賴之途徑。為了證明Src的活化是關鍵的中繼者，我們對Src酪氨酸418在Sh - DDR1細胞中的磷酸化水平進行評估。其結果顯示，knockdown酪氨酸激酶接受器增加細胞與細胞交界處的Src酪氨酸418磷酸化。此外，Src抑制劑PP2可抑制knockdown 酪氨酸激酶接受器所引起的上皮細胞鈣黏附因子內吞作用。為了更進一步研究knockdown酪氨酸激酶接受器如何增加Src之活性，我們利用阻斷抗體4B4來阻斷整合素β1之信號。4B4降低Src活性並且使Sh-DDR1細胞內的上皮細胞鈣黏附因子再度位在細胞交界處，顯示knockdown酪氨酸激酶接受器藉由抑制整合素β1之信號增加Src之活性。光轉化及溶解度實驗顯示抑制Src的活性可增加上皮細胞鈣黏附因子及上皮細胞鈣黏附因子，α-catenin及肌動蛋白所形成的複合物之穩定度。免疫螢光染色的結果也顯示抑制整合素β1-Src信號可減少Sh-DDR1細胞中的應力纖維。整合這些結果，我們發現，knockdown酪氨酸激酶接受器透過整合素β1增強的Src磷酸化，及對肌動蛋白骨架的修改，減少位於細胞交界處的上皮細胞鈣黏附因子。

Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase of collagen, is mainly expressed in epithelial cells. Our recent works have shown that activation of DDR1 results in the stabilization of E-cadherin via inhibition of Cdc42-mediated endocytosis. However, the signaling mechanism in DDR1-inhibited E-cadherin endocytosis remains unclear. Previous studies have shown that E-cadherin-catenin-actin complexes increases E-cadherin membrane stability and reduces its turnover rate. Upon HGF treatment, active Cdc42 induces E-cadherin phosphorylation via Src-dependent pathway, which in turn causes the disruption of E-cadherin-catenin-actin complex and endocytosis of E-cadherin via clathrin-dependent pathway. We then proposed that DDR1 stabilized E-cadherin-catenin-actin complex through inactivation of Src and subsequent clathrin-dependent endocytosis pathway. To test this hypothesis, we first aim to investigate whether knockdown of DDR1 (Sh-DDR1) would result in the increase of E-cadherin endocytosis through clathrin-dependent pathway. We found that Sh-DDR1 decreased junctional localization and increased endocytosis of E-cadherin without affecting protein abundance of E-cadherin. The immunoprecipitation result showed that Sh-DDR1 increased the physical interactions between E-cadherin and clathrin and the treatment of clathrin-dependent endocytosis inhibitor, dynasore, suppressed Sh-DDR1-induced E-cadherin endocytosis in a time- and dose-dependent manner, indicating that DDR1-inhibited E-cadherin endocytosis was mediated through clathrin-dependent pathway. To confirm that Src activation is the key mediator, the phosphorylation level of Src tyrosine 418 in Sh-DDR1 cells was assessed. The result showed that knockdown of DDR1 increased the phosphorylation level of Src tyrosine 418 in cell-cell junctions. Moreover, Src inhibitor, PP2, inhibited Sh-DDR1-induced E-cadherin endocytosis. To further investigate how knockdown of DDR1 increases Src activity, we blocked integrin β1 signal by blocking antibody, 4B4. 4B4 treatment decreased Src activity and rescued E-cadherin junctional localization in Sh-DDR1 cells, which indicates knockdown of DDR1 increases Src activity via integrin β1 signaling. The results of photoconversion and triton solubility showed inhibition of Src activity rescued E-cadherin membrane stability and the stability of E-cadherin-catenin-actin complex. The immunofluorescence study also showed that inhibition of integrin β1-Src signaling decreased stress fibers in Sh-DDR1 cells. Taken together, we showed that knockdown of DDR1 decreased the membrane localization of E-cadherin via clathrin-dependent endocytosis pathway, which is mediated by integrin β1-augmented Src phosphorylation and modification of actin cytoskeleton.

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