Mechanisms of Transforming Growth Factor-β Receptor Endocytosis and Intracellular Sorting Differ between Fibroblasts and Epithelial Cells

Abstract

Transforming growth factor-␤s (TGF-␤) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-␤ type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-␤ receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling. INTRODUCTIONTransforming growth factor-␤s (TGF-␤) control a variety of cellular processes as diverse as mitotic inhibition or stimulation (Massagué, 1996;Moses and Serra, 1996). It is unclear how the same receptor complex can mediate such different cellular phenotypes. The most commonly accepted receptor model for TGF-␤ action consists of a heteromeric complex composed of type I and type II receptors (Wrana et al., 1992(Wrana et al., , 1994. Once associated, the type I receptor becomes phosphorylated primarily within the juxtamembrane GS domain (amino acids 185-192) by the constitutive serine/threonine kinase activity of the type II receptor (Franzén et al., 1995;Wieser et al., 1995). Phosphorylation of the GS domain is proposed to activate the type I receptor, resulting in signal propagation to downstream effector molecules (Massagué, 1998). In addition, specific residues in nearby regions have also been suggested to have both positive and negative regulatory functions (Wieser et al., 1995;Charng et al., 1996;Souchelnytskyi et al., 1996;Doré et al., 1998). For instance, threonine 200 has been shown to have a fundamental role in mediating all aspects of TGF-␤ signaling (Wieser et al., 1995), whereas replacement of threonine 204 with an acidic residue, such as aspartate, can generate a type I receptor capable of signaling (albeit to a lesser extent) independent of ligand or an associated type II receptor (Wieser et al., 1995;Charng et al., 1996;Luo and Lodish, 1996). What makes these data most i...

Endocytic regulation of TGF-β signaling

“…Contrary to what is observed in fibroblasts [46], the kinase activity of TbRII is not essential for downregulation of heteromeric TGF-b receptor complexes in epithelial cells [56]. In addition to this, a signaling-incompetent mutation of TbRI -T200V -abolishes endocytosis/ receptor downregulation in fibroblasts, whereas it has no such effect in epithelial cells [56].…”

“…There are two putative tyrosine-based YXX∅ motifs in the intracellular domain of TbRI: Y182DMT and Y249QTV. However, mutation of either tyrosine residue had no effect on TbRI endocytosis [56]. Instead, deletion and substitution mutation studies have identified a motif (R482LTALRIKKTL492) close to the C-terminal tail that is important for TbRI downregulation [47].…”

“…In addition to this, a signaling-incompetent mutation of TbRI -T200V -abolishes endocytosis/ receptor downregulation in fibroblasts, whereas it has no such effect in epithelial cells [56].…”

“…Both type I and type II TGF-b receptors seem to constantly undergo internalization [49,52,55]. Once internalized into the cytoplasmic compartments, TGF-b receptor molecules seem to be recycled back to the cell surface in the absence of ligand [56]. The recycling of the internalized TGF-b receptors is impaired by dominant-negative Rab11 [57], suggesting that the recycling takes place in the perinuclear recycling endosomes [58] (Figure 2).…”

When cell biology meets development: endocytic regulation of signaling pathways

“…Both heteromeric and homomeric TGF-␤ receptor complexes undergo ligand-dependent internalization. Once internalized, if the ligand dissociates from the complex, TGF-␤ receptors are constitutively recycled to the cell surface similar to the constitutive recycling observed for EGFR (Dore et al , 2001). However, if the ligand is associated with the complex, the TGF-␤ receptors can be targeted for either recycling or lysosomal degradation depending on the complex composition.…”