Members of the transforming growth factor ␤ (TGF-␤) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-␤ 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-␤ receptor (TGF-␤R) 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-␤R internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-␤-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-␤R complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-␤R endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.

Transforming growth factor ␤ (TGF-␤) causes growth arrest in epithelial cells and proliferation and morphological transformation in fibroblasts. Despite the ability of TGF-␤ to induce various cellular phenotypes, few discernible differences in TGF-␤ signaling between cell types have been reported, with the only well-characterized pathway (the Smad cascade) seemingly under identical control. We determined that TGF-␤ receptor signaling activates the STE20 homolog PAK2 in mammalian cells. PAK2 activation occurs in fibroblast but not epithelial cell cultures and is independent of Smad2 and/or Smad3. Furthermore, we show that TGF-␤-stimulated PAK2 activity is regulated by Rac1 and Cdc42 and dominant negative PAK2 or morpholino antisense oligonucleotides to PAK2 prevent the morphological alteration observed following TGF-␤ addition. Thus, PAK2 represents a novel Smad-independent pathway that differentiates TGF-␤ signaling in fibroblast (growth-stimulated) and epithelial cell (growth-inhibited) cultures.

Transforming growth factor ␤ (TGF␤) superfamily polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGF␤ type I and type II receptors. Signal propagation is dependent upon heteromeric (type I-type II) complex formation and transphosphorylation of the type I receptor by the type II receptor. While many of the phosphorylation events necessary for receptor signaling have recently been characterized, the role of TGF␤ receptor kinase activity in modulating receptor endocytosis has not been addressed. To that end, we have used chimeric receptors consisting of the extracellular domain of the granulocyte/macrophage colony-stimulating factor ␣ and ␤ receptors spliced to the TGF␤ type I and type II transmembrane and cytoplasmic domains to address the specific role of type I and/or type II receptor kinase activity in TGF␤ receptor internalization, downregulation, and signaling. To inactivate chimeric receptor kinase activity, point mutations in the ATP binding site were made at amino acids 232 and 277 in the type I and type II receptor, respectively. Either of these mutations abolished plasminogen activator inhibitor 1 protein expression stimulated by granulocyte/macrophage colony-stimulating factor activation of chimeric heteromeric type I-type II TGF␤ receptors. They did not, however, modulate TGF␤ signaling stimulated through the endogenous TGF␤ receptor. Although TGF␤ receptor signaling was dependent upon the kinase activity of both chimeric receptors, the initial endocytic response was distinctly regulated by type I and/or type II receptor kinase activity. For instance, while heteromeric receptor complexes containing a kinase-inactive type I receptor were endocytosed similarly to wild type complexes, the kinase activity of the type II TGF␤ receptor was necessary for optimal internalization and receptor down-regulation. Furthermore, these responses were shown to occur independently of type II receptor autophosphorylation but require a type II receptor capable of transphosphorylation.The transforming growth factor ␤ (TGF␤) 1 superfamily of proteins regulate a number of diverse biologic processes (1-3). While the cellular response can be as distinct as growth stimulation or growth inhibition, it appears as though a similar receptor system is utilized for both pathways. Understanding how the receptors are regulated for one family of proteins will ultimately extend the knowledge for the entire superfamily. The model most commonly accepted for receptor activation requires oligomerization of a type I and type II TGF␤ receptor (4 -7). This occurs through ligand binding to a type II receptor and recruitment of a type I receptor into a dimeric and/or tetrameric complex (7-11). The serine/threonine kinase activity of the type I receptor is then activated by specific type II receptor phosphorylations in the juxtamembrane region of the type I receptor (12-16). This cascade of receptor interactions and phosphorylations ultimately results in the propagation of the T...

Matrix remodeling, degradation, inflammation and invasion liberate peptide fragments that can subsequently interact with cells in an attachment-independent manner. Such 'soluble' matrix components, including collagens, fibronectin and laminin, induced Smad activation (termed crosstalk signaling), which follows a similar chronological sequence and R-Smad specificity as induced by transforming growth factor (TGF)-b1. Smad4 nuclear translocation occurred in response to collagen binding, indicating downstream signal propagation. TGF-b scavenging antibody affected only TGF-b1, but not crosstalk-induced responses. TGF-b type II receptor mutation (DR26D25), which is deficient in TGF-b type I receptor recruitment to the ligand, induced a heterotetramer signaling complex, and propagated Smad2 activation only through collagen induction and not TGF-b signaling. Consequentially, TGF-b ligand participation is not required for crosstalk signaling. This signaling requires a functional integrin b1 receptor as showed by RNA interference. Co-immunoprecipitation (co-IP) and fluorescent microscopy indicate the involvement of focal adhesion kinase (FAK) and Src activity in collagen-induced signal propagation, and suggest a membrane signaling complex formation that includes both TGF-b receptors and integrins. The related gene expressional responses are distinct from that evoked by TGF-b1, supporting its separate function. This signaling mechanism expands and partially explains TGFb receptor dynamics and consequential signaling diversityrelated gene expressional plasticity.