Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.
Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.
J Mol Biol. 2015 May 22;427(10):1934-48. doi: 10.1016/j.jmb.2015.03.001. Epub 2015 Mar 9.
We have expressed and purified three soluble fragments of the human LRIG1-ECD (extracellular domain): the LRIG1-LRR (leucine-rich repeat) domain, the LRIG1-3Ig (immunoglobulin-like) domain, and the LRIG1-LRR-1Ig fragment using baculovirus vectors in insect cells. The two LRIG1 domains crystallised so that we have been able to determine the three-dimensional structures at 2.3Å resolution. We developed a three-dimensional structure for the LRIG1-ECD using homology modelling based on the LINGO-1 structure. The LRIG1-LRR domain and the LRIG1-LRR-1Ig fragment are monomers in solution, whereas the LRIG1-3Ig domain appears to be dimeric. We could not detect any binding of the LRIG1 domains or the LRIG1-LRR-1Ig fragment to the EGF receptor (EGFR), either in solution using biosensor analysis or when the EGFR was expressed on the cell surface. The FLAG-tagged LRIG1-LRR-1Ig fragment binds weakly to colon cancer cells regardless of the presence of EGFRs. Similarly, neither the soluble LRIG1-LRR nor the LRIG1-3Ig domains nor the full-length LRIG1 co-expressed in HEK293 cells inhibited ligand-stimulated activation of cell-surface EGFR.
我们使用杆状病毒载体在昆虫细胞中表达和纯化了人 LRIG1-ECD（细胞外结构域）的三个可溶性片段：LRIG1-LRR（亮氨酸丰富重复）结构域、LRIG1-3Ig（免疫球蛋白样）结构域和 LRIG1-LRR-1Ig 片段。这两个 LRIG1 结构域结晶，使我们能够以 2.3Å 的分辨率确定其三维结构。我们使用基于 LINGO-1 结构的同源建模方法为 LRIG1-ECD 开发了一个三维结构。LRIG1-LRR 结构域和 LRIG1-LRR-1Ig 片段在溶液中为单体，而 LRIG1-3Ig 结构域似乎为二聚体。我们无法在溶液中使用生物传感器分析或在 EGFR 表达于细胞表面时，检测到 LRIG1 结构域或 LRIG1-LRR-1Ig 片段与 EGF 受体（EGFR）的任何结合。FLAG 标记的 LRIG1-LRR-1Ig 片段与结肠癌细胞结合较弱，无论 EGFR 是否存在。同样，在 HEK293 细胞中表达的可溶性 LRIG1-LRR 或 LRIG1-3Ig 结构域或全长 LRIG1 均不能抑制配体刺激的细胞表面 EGFR 激活。
