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Mechanisms of transforming growth factor-beta receptor endocytosis and intracellular sorting differ between
1Thoracic Diseases Research Unit and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Abstract:
Transforming growth factor-betas (TGF-beta) 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-beta 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-beta 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.
Insights
Transforming growth factor-betas (TGF-beta) signaling pathways involve specific receptor residues that regulate endocytosis and cell response. Key mutations reveal differences in how mesenchymal and epithelial cells handle TGF-beta receptor trafficking.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Transforming growth factor-betas (TGF-beta) are crucial regulators of cell proliferation and differentiation.
- TGF-beta exerts its effects through a receptor complex comprising type I and type II serine/threonine kinase receptors.
- Cellular responses to TGF-beta are diverse, varying by cell type, and mediated by a single receptor complex.
Purpose of the Study:
- To investigate the roles of specific amino acid residues in TGF-beta type I and II receptors in regulating ligand-mediated endocytosis and signaling.
- To elucidate the mechanisms of TGF-beta receptor trafficking and signaling in fibroblasts and epithelial cells.
- To identify key residues critical for TGF-beta receptor internalization, recycling, and down-regulation.
Main Methods:
- Construction of chimeric receptors by fusing ligand-binding domains of the granulocyte/macrophage colony-stimulating factor receptor to TGF-beta receptor signaling domains.
- Introduction of point mutations at specific residues (Y182, T200, Y249 in type I; K277, P525 in type II) within the TGF-beta receptor complex.
- Analysis of endocytosis, signaling, and receptor down-regulation in response to TGF-beta stimulation in fibroblasts and epithelial cells.
Main Results:
- Mutations at Y182 and Y249, within putative internalization motifs, did not affect TGF-beta receptor endocytosis or signaling.
- Mutation of T200 to valine abolished TGF-beta signaling in both cell types and receptor down-regulation in fibroblasts.
- Fibroblasts and epithelial cells constitutively internalize and recycle TGF-beta receptors in the absence of ligand.
Conclusions:
- Specific amino acid residues, particularly T200 in the type I receptor, are critical for TGF-beta-mediated signaling and receptor regulation.
- Ligand binding diverts heteromeric TGF-beta receptors from a default recycling pathway to one involving down-regulation and signaling.
- Mesenchymal and epithelial cells exhibit fundamental differences in the endocytic sorting of TGF-beta receptors.