Complex formation among transforming growth factor-β (TGF-β) receptors and its modulation by coreceptors represent an important level of regulation for TGF-β signaling. Oligomerization of ALK5 and the type II TGF-β receptor (TβRII) has been thoroughly investigated, both in vitro and in intact cells. However, such studies, especially in live cells, are missing for the endothelial cell coreceptor endoglin and for the ALK1 type I receptor, which enables endothelial cells to respond to TGF-β by activation of both Smad2/3 and Smad1/5/8. Here we combined immunoglobulin G-mediated immobilization of one cell-surface receptor with lateral mobility studies of a coexpressed receptor by fluorescence recovery after photobleaching (FRAP) to demonstrate that endoglin forms stable homodimers that function as a scaffold for binding TβRII, ALK5, and ALK1. ALK1 and ALK5 bind to endoglin with differential dependence on TβRII, which plays a major role in recruiting ALK5 to the complex. Signaling data indicate a role for the quaternary receptor complex in regulating the balance between TGF-β signaling to Smad1/5/8 and to Smad2/3.

The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and -SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3' terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane.

Transforming growth factor-β (TGF-β) receptor oligomerization has important roles in signaling. Complex formation among type I and type II (TβRI and TβRII) TGF-β receptors is well characterized and is essential for signal transduction. However, studies on their interactions with the type III TGF-β coreceptor (TβRIII) in live cells and their effects on TGF-β signaling are lacking. Here we investigated the homomeric and heteromeric interactions of TβRIII with TβRI and TβRII in live cells by combining IgG-mediated patching/immobilization of a given TGF-β receptor with fluorescence recovery after photobleaching studies on the lateral diffusion of a coexpressed receptor. Our studies demonstrate that TβRIII homo-oligomerization is indirect and depends on its cytoplasmic domain interactions with scaffold proteins (mainly GIPC). We show that TβRII and TβRI bind independently to TβRIII, whereas TβRIII augments TβRI/TβRII association, suggesting that TβRI and TβRII bind to TβRIII simultaneously but not as a complex. TβRIII expression inhibited TGF-β-mediated Smad2/3 signaling in MDA-MB-231 cell lines, an effect that depended on the TβRIII cytoplasmic domain and did not require TβRIII ectodomain shedding. We propose that independent binding of TβRI and TβRII to TβRIII competes with TβRI/TβRII signaling complex formation, thus inhibiting TGF-β-mediated Smad signaling.

Fibronectin (FN) is a critical regulator of extracellular matrix (ECM) remodeling through its availability and stepwise polymerization for fibrillogenesis. Availability of FN is regulated by its synthesis and turnover, and fibrillogenesis is a multistep, integrin-dependent process essential for cell migration, proliferation, and tissue function. Transforming growth factor β (TGF-β) is an established regulator of ECM remodeling via transcriptional control of ECM proteins. Here we show that TGF-β, through increased FN trafficking in a transcription- and SMAD-independent manner, is a direct and rapid inducer of the fibrillogenesis required for TGF-β-induced cell migration. Whereas TGF-β signaling is dispensable for rapid fibrillogenesis, stable interactions between the cytoplasmic domain of the type II TGF-β receptor (TβRII) and the FN receptor (α5β1 integrin) are required. We find that, in response to TGF-β, cell surface-internalized FN is not degraded by the lysosome but instead undergoes recycling and incorporation into fibrils, a process dependent on TβRII. These findings are the first to show direct use of trafficked and recycled FN for fibrillogenesis, with a striking role for TGF-β in this process. Given the significant physiological consequences associated with FN availability and polymerization, our findings provide new insights into the regulation of fibrillogenesis for cellular homeostasis.