title: Determining TGF-β Receptor Levels in the Cell Membrane.
abstract: Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that signals via transmembrane TGF-β type I and type II serine/threonine kinases receptors, i.e., TβRI and TβRII. Upon TGF-β-induced receptor complex formation, the TβRII kinase phosphorylates TβRI. Subsequently, the activated TβRI induces the phosphorylation of receptor regulated SMAD2 and SMAD3, which can form heteromeric complexes with Smad4. These heteromeric SMAD complexes accumulate in the nucleus, where they regulate target gene expression. The stability and membrane localization of TβRI is an important determinant to control the intensity and duration of TGF-β signaling. TβRI is targeted for poly-ubiquitylation-mediated proteasomal degradation by the SMAD7-SMURF E3 ligase complex. We recently identified another important regulatory factor that controls TβRI levels in the cell membrane. As a strong inducer of TGF-β signaling, ubiquitin-specific protease (USP) 4 was found to directly interact with TβRI and act as a deubiquitylating enzyme, thereby stabilizing TβRI levels at the plasma membrane. This chapter introduces methods for examining cell membrane receptor (TβRI) levels.
authors.affiliation: Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Postbus 9600 2300 RC, Leiden, The Netherlands.|Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, Zhejiang, 310058, China.
authors.forename: Long
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, Zhejiang, 310058, China.|Institutes of Biology and Medical Sciences, Soochow University, Suzhou Industrial Park, Suzhou, Jiangsu, China.
authors.forename: Fangfang
authors.initials: F
authors.lastname: Zhou
authors.affiliation: Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Postbus 9600 2300 RC, Leiden, The Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: van Dinther
authors.affiliation: Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Postbus 9600 2300 RC, Leiden, The Netherlands. p.ten_dijke@lumc.nl.|Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, Zhejiang, 310058, China. p.ten_dijke@lumc.nl.|Ludwig Institute for Cancer Research, Uppsala University, Box 595, Uppsala, 75124, Sweden. p.ten_dijke@lumc.nl.
authors.forename: Peter
authors.initials: P
authors.lastname: Ten Dijke
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D008024:Ligands; D008565:Membrane Proteins; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta
keywords: Deubiquitylating enzyme; E3 ubiquitin ligase; TGF-β; TβRI; Ubiquitination