Abdel-Ghany Mossaad, Cheng Hung-Chi, Elble Randolph C, Lin Haiqun, DiBiasio John, Pauli Bendicht U
Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA.
J Biol Chem. 2003 Dec 5;278(49):49406-16. doi: 10.1074/jbc.M309086200. Epub 2003 Sep 25.
CLCA (chloride channel, calcium-activated) proteins are novel pulmonary vascular addresses for blood-borne, lung-metastatic cancer cells. They facilitate vascular arrest of cancer cells via adhesion to beta4 integrin and promote early, intravascular, metastatic growth. Here we identify the interacting binding domains of endothelial CLCA proteins (e.g. hCLCA2, mCLCA5, mCLCA1, and bCLCA2) and beta4 integrin. Endothelial CLCAs share a common beta4-binding motif (beta4BM) in their 90- and 35-kDa subunits of the sequence F(S/N)R(I/L/V)(S/T)S, which is located in the second extracellular domain of the 90-kDa CLCA and near the N terminus of the 35-kDa CLCA, respectively. Using enzyme-linked immunosorbent, pull-down, and adhesion assays, we showed that glutathione S-transferase fusion proteins of beta4BMs from the 90- and 35-kDa CLCA subunits bind to the beta4 integrin in a metal ion-dependent manner. Fusion proteins from fibronectin and the integrins beta1 and beta3 served as negative controls. beta4BM fusion proteins competitively blocked the beta4/CLCA adhesion and prevented lung colonization of MDA-MB-231 breast cancer cells. A disrupted beta4BM in hCLCA1, which is not expressed in endothelia, failed to interact with beta4 integrin. The corresponding CLCA-binding domain of the beta4 integrin is localized to the specific determining loop (SDL). Again enzyme-linked immunosorbent, pull-down, and adhesion assays were used to confirm the interaction with CLCA proteins using a glutathione S-transferase fusion protein representing the C-terminal two-thirds of beta4 SDL (amino acids 184-203). A chimeric beta4 integrin in which the indicated SDL sequence had been replaced with the corresponding sequence from the beta1 integrin failed to bind hCLCA2. The dominance of the CLCA ligand in beta4 activation and outside-in signaling is discussed in reference to our previous report that beta4/CLCA ligation elicits selective signaling via focal adhesion kinase to promote metastatic growth.
CLCA（氯离子通道，钙激活）蛋白是血源性肺转移癌细胞新的肺血管靶向分子。它们通过与β4整合素黏附促进癌细胞的血管停滞，并促进早期血管内转移生长。在此，我们鉴定了内皮CLCA蛋白（如hCLCA2、mCLCA5、mCLCA1和bCLCA2）与β4整合素的相互作用结合结构域。内皮CLCA在其90 kDa和35 kDa亚基中共享一个共同的β4结合基序（β4BM），序列为F(S/N)R(I/L/V)(S/T)S，分别位于90 kDa CLCA的第二个细胞外结构域和35 kDa CLCA的N端附近。使用酶联免疫吸附、下拉和黏附试验，我们表明来自90 kDa和35 kDa CLCA亚基的β4BM的谷胱甘肽S-转移酶融合蛋白以金属离子依赖的方式与β4整合素结合。来自纤连蛋白以及整合素β1和β3的融合蛋白作为阴性对照。β4BM融合蛋白竞争性阻断β4/CLCA黏附，并阻止MDA-MB-231乳腺癌细胞在肺部定植。hCLCA1中一个破坏的β4BM（在内皮细胞中不表达）未能与β4整合素相互作用。β4整合素相应的CLCA结合结构域定位于特定决定环（SDL）。再次使用酶联免疫吸附、下拉和黏附试验，使用代表β4 SDL C端三分之二（氨基酸184 - 203）的谷胱甘肽S-转移酶融合蛋白来确认与CLCA蛋白的相互作用。一个嵌合β4整合素，其中指定的SDL序列已被β1整合素的相应序列取代，未能结合hCLCA2。参考我们之前的报告，即β4/CLCA连接通过黏着斑激酶引发选择性信号传导以促进转移生长，讨论了CLCA配体在β4激活和外向内信号传导中的主导作用。
