Characterization of the Structural Domains in the Type V TGF-beta Receptor Required for Mediating the Growth Inhibitory Response to IGFBP-3 and TGF-beta

• Main Authors: Wen-fang Tseng, 曾文芳
• Other Authors: Jung San Huang
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/73362015054714052901

博士 === 國防醫學院 === 生命科學研究所 === 93 === The type V Transforming growth factor beta- receptor (T-beta-R-V) mediates the IGF-independent growth inhibition induced by IGFBP-3. It also mediates the growth inhibitory response to TGF-beta-1 in concert with other TGF-beta- receptor types, and its loss may contribute to the malignant phenotype of human carcinoma cells. Recently, by MALDI-TOF analysis of tryptic peptides of T-beta-R-V purified from bovine liver, T-beta-R-V and Low density lipoprotein related protein (LRP-1) were found to share the same amino sequence. The roles that LRP-1 plays in cell physical mechanisms are extremely diverse, it involve in lipoprotein metabolism, removing of Alpha 2 marcoglobulin (Alpha2M) -protease complexes, platelet-derived growth factor (PDGF) signaling, inflammation, embryonic development, cellular entry of toxin and virus, activation of lysosomal enzymes, neurotransmission, Alzheimer disease, coagulation, blood-brain-barrier (BBB) transportation, and cell migration. In this study, we charactize T-beta-R-V by knocking, rescueing, and dominant negative expression of LRP-1 gene. To create a LRP-1 deficient Mv1Lu cell line, we treated cell with mutagene and selection with Pseudomonas exotoxin (PEA), which is endocytosised by LRP-1. We obtained a PEA resistant cell line, PEA-C11 cells, and found its 125I-TGF-b1 affinity labeling of TbR-V was diminished, and its growth inhibitory responses to IGFBP-3 and TGF-beta-1 were reduced, too. However, the TGF-beta- type I and type II receptor (Tbeta-R-I, Tbeta-R-II) mediated Smad pathway is not altered. Furthermore, by sequence the mutated LRP-1 cDNA, a 65-nucleotide-deletion was detected which results in early termination of LRP-1 at the the cell surface part of the light chain. Additionally, we generated the same deletion in human LRP-1 cDNA construct and then stably expressed this mutated construct in Mv1Lu cells, we aquired a stable clone named as #4-23. By analyzing #4-23 cells, we proved this deletion contributes to the dominant negative effect. On the other hand, we determined the effects of IGFBP-3 and TGF-beta-1 induced growth suppression in other LRP-1 deficient cells including PEA-13, H1299, and CHO-LRP-1- cells. All of the three cells were found lacking the growth inhibitory response to TGF-beta-1. Additionally, in H1299 cell and CHO-LRP-1- cells, we demonstrated that this response can be restored by over expressing LRP-1 cDNA. The LRP-1/T-beta-R-V is a high molecular (~600 kDa) member of LDL receptor gene family, which is an ancient family of endocytic receptor. It is synthesized as a single chain molecule, but in mature LRP-1, it is processed into a 515-kDa heavy chain and an 85-kDa light chain that contains the transmembrane domain. The heavy chain contains four ligand binding domains and associate with light chain on the cell surface. To dissect the function of each ligand binding domains, we constructed four membrane- bounding-minireceptor cDNAs (mLRPI, mLRPII, mLRPIII, mLRPIV) each contains one of the four ligand binding domains and one light chain, and then stably express these cDNAs in CHO-LRP-1- and CHO-K1 cells respectively. After analyzed these clones, we obtained the following discovery: (1) expression of mLRPs in CHO-LRP-1- cells can not mediate cell growth inhibition response to TGF-beta- and IGFBP-3. However, overexpression of mLRPs attenuates the growth inhibitory response to TGF-beta-1 in CHO-K1 cells. (2)125I-IGFBP-3 affinity-labeled mini LRP II and mini LRPIV were detected and this result indicated that IGFBP-3 bind to ligand domain II and IV of LRP-1/T-beta-R-V/IGFBP-3 receptor. (3) Overexpression of mLRPs does not significantly affect the endocytosis rate of endogenous LRP-1 and TGF-beta-1-induced Plasminogen activator inhibitor-I (PAI-1) expression, which is an indicator of Smads signaling. In summary, through knock out, rescue, and dominant negative experiments, we prove that the well known multiple functional receptor, LRP-1, is identical to T-beta-R-V. In addition, we prove that T-beta-R-V receptor is required for the growth inhibitory response to IGFBP-3 and TGF-beta-1 and its signaling pathway is different form Smad signaling pathway mediated by Tbeta-R-I and Tbeta-R-II. Furthermore, by analyzing the stable clones that express mLRPs in CHO-K1 cells, we identified that IGFBP-3 bind to ligand binding domain II and IV of LRP-1. In addition, expressing any of the four mini receptors in CHO-K1 can cause a dominant negative effect of endogenous LRP-1 and this effect does not cause by alteration of endocytosis rate or Smads pathway.