| Summary: | 碩士 === 國立成功大學 === 生理學研究所 === 93 === Discoidin domain receptor (DDR) is a receptor tyrosine kinase for collagen. We previously showed that over-expression of DDR1 in MDCK cells prevented cell spreading, whereas dominant negative DDR1 induced cell spreading on collagen gel-coated dish. Cell spreading is an important characteristic for cell migration, but the mechanism whereby DDR1-inhibited cell spreading is still unidentified. Cell spreading involves organization of actin cytoskeleton, which is mainly regulated by Rho family-GTPases. In order to examine whether Rho family-GTPases are involved in collagen gel-regulated cell spreading, we employed pull-down assay and transient transfection of constitutive active or dominant negative GTPases in MDCK cells and observed whether they could alter collagen-induced cell morphological changes. The results showed DDR1 decreased the activation of Rac1 and Cdc42, but had no effects on RhoA activity. Neither constitutive active nor dominant negative Rac1 could alter DDR1-inhibited cell spreading. However, constitutive active Cdc42 rescued the DDR1-inhibited cell spreading and dominant negative Cdc42 inhibited cell spreading in cells overexpressing dominant negative DDR1. These results indicate that DDR1-inhibited cell spreading is mediated by inactivation of Cdc42. With the use of 5E8, a potent a2b1 integrin blocking antibody, we found that collagen-induced activation of Cdc42 is mediated by a2b1 integrin. Furthermore, FRNK completely blocked collagen-induced decrease in Cdc42 activity, but DDR1 did not influence the phosphorylation levels of FAK. Taken together, DDR1 inhibits collagen-induced cell spreading by suppression of Cdc42 activation, which is mediated by a2b1 integrin through FAK.
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