title: Prohibitin is involved in the activated internalization and degradation of protease-activated receptor 1.
abstract: The protease-activated receptor 1 (PAR1) is a G-protein-coupled receptor that is irreversibly activated by either thrombin or metalloprotease 1. Due this irrevocable activation, activated internalization and degradation are critical for PAR1 signaling termination. Prohibitin (PHB) is an evolutionarily conserved, ubiquitously expressed, pleiotropic protein and belongs to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain family. In a previous study, we found that PHB localized on the platelet membrane and participated in PAR1-mediated human platelet aggregation, suggesting that PHB likely regulates the signaling of PAR1. Unfortunately, PHB's exact function in PAR1 internalization and degradation is unclear. In the current study, flow cytometry revealed that PHB expressed on the surface of endothelial cells (HUVECs) but not cancer cells (MDA-MB-231). Further confocal microscopy revealed that PHB dynamically associates with PAR1 in a time-dependent manner following induction with PAR1-activated peptide (PAR1-AP), though differently between HUVECs and MDA-MB-231 cells. Depletion of PHB by RNA interference significantly inhibited PAR1 activated internalization and led to sustained Erk1/2 phosphorylation in the HUVECs; however, a similar effect was not observed in MDA-MB-231 cells. For both the endothelial and cancel cells, PHB repressed PAR1 degradation, while knockdown of PHB led to increased PAR1 degradation, and PHB overexpression inhibited PAR1 degradation. These results suggest that persistent PAR1 signaling due to the absence of membrane PHB and decreased PAR1 degradation caused by the upregulation of intracellular PHB in cancer cells (such as MDA-MB-231 cells) may render cells highly invasive. As such, PHB may be a novel target in future anti-cancer therapeutics, or in more refined cancer malignancy diagnostics.
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, Yunnan 650223, China.
authors.forename: Yan-Jie
authors.initials: YJ
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, Yunnan 650223, China; Kunming College of Life Science, University of Chinese Academy of Sciences Kunming, Yunnan 650204, China.
authors.forename: Xiao-Long
authors.initials: XL
authors.lastname: Guo
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, Yunnan 650223, China.
authors.forename: Sheng-An
authors.initials: SA
authors.lastname: Li
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, Yunnan 650223, China.
authors.forename: Yu-Qi
authors.initials: YQ
authors.lastname: Zhao
authors.affiliation: Department of Life Science and Technology, Kunming University, Kunming, Yunnan 650214, China.
authors.forename: Zi-Chao
authors.initials: ZC
authors.lastname: Liu
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, Yunnan 650223, China.
authors.forename: Wen-Hui
authors.initials: WH
authors.lastname: Lee
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, Yunnan 650223, China. Electronic address: yang_xiang000@hotmail.com.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xiang
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, Yunnan 650223, China. Electronic address: zhangy@mail.kiz.ac.cn.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics* / Q000378:metabolism; D009928:Organ Specificity; D010455:Peptides / Q000494:pharmacology; D000091402:Prohibitins; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D010975:Platelet Aggregation Inhibitors
chemical_list: C000717328:PHB protein, human; D010455:Peptides; D000091402:Prohibitins; D034741:RNA, Small Interfering; D044463:Receptor, PAR-1; D012097:Repressor Proteins; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010975:Platelet Aggregation Inhibitors
keywords: Activated internalization; Erk1/2 signaling degradation; PAR1; Prohibitin