Transforming growth factor beta activates Smad2 in the absence of receptor endocytosis.
Like many other cell surface receptors, transforming growth factor beta (TGF-beta) receptors are internalized upon ligand stimulation. Given that the signaling-facilitating molecules Smad anchor for receptor activation (SARA) and Hrs are mainly localized in early endosomes, it was unclear whether receptor internalization is required for Smad2 activation. Using reversible biotin labeling, we directly monitored internalization of the TGF-beta type I receptor. Our data indicate that TGF-beta type I receptor is endocytosed via a clathrin-dependent mechanism and is effectively blocked by depletion of intracellular potassium or by expression of a mutant dynamin (K44A). However, blockage of receptor endocytosis by these two means has no effect on TGF-beta-mediated Smad2 activation. Furthermore, TGF-beta-induced Smad2 activation was unaffected by inhibition of hVPS34 activity with wortmannin or inhibitory anti-hVPS34 antibodies. Finally, we demonstrated that Smad2 interacted with cell surface receptors and that a SARA binding-deficient Smad2 mutant was phosphorylated by the receptors. Thus, our findings suggest that receptor endocytosis is dispersible for TGF-beta-mediated activation of Smad2 and that this activation can be mediated by both SARA-dependent and -independent mechanisms.
Lu
Zhongxian
Z
Division of Biomedical Sciences, University of California, Riverside, California 92521, USA.
Murray
James T
JT
Luo
Wenjie
W
Li
Hongling
H
Wu
Xiaoping
X
Xu
Huaxi
H
Backer
Jonathan M
JM
Chen
Ye-Guang
YG
D030201:Activin Receptors, Type I / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004705:Endocytosis; D006801:Humans; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D051899:Smad2 Protein; D015534:Trans-Activators / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000502:physiology*
D002966:Clathrin; D004268:DNA-Binding Proteins; D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I