Internalization-Dependent and -Independent Requirements for Transforming Growth Factor β Receptor Signaling via the Smad Pathway

Abstract

Members of the transforming growth factor β (TGF-β) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-β signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-β receptor (TGF-βR) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-βR internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-β-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-βR complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-βR endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.

We thank the laboratories of Richard Pagano and Mark McNiven for helpful comments during the course of these studies. We also thank John DiGuglielmo in the laboratory of Jeffrey Wrana for constructs and for help with the SARA association assays.

This work was supported by Public Health Service grants GM54200 and GM55816 from the National Institute of General Medical Science and the Mayo Foundation. S.G.P. was partially supported by National Cancer Institute postdoctoral training grant CA09441.