RET ligand-induced internalization and its consequences for downstream signaling.
RET is a receptor tyrosine kinase (RTK) with roles in cell growth, differentiation and survival. Ligand-induced activation of RET results in stimulation of multiple signal transduction pathways, including the MAP kinase/Erk and PI3 kinase/Akt pathways. However, the mechanisms governing receptor internalization and signal down- regulation have not been explored. As other RTKs are internalized through the clathrin-coated pit pathway in a ligand-dependant manner, we have investigated whether RET is internalized through a similar process. Using a highly sensitive fluorescence resonance energy transfer (FRET)-based assay, we have shown that RET is internalized from the plasma membrane in a ligand-dependant manner that requires RET kinase activity as well as the GTPase activity of the clathrin-coated vesicle scission protein dynamin 2. Further, we have demonstrated that RET colocalizes with Rab5a, a marker of clathrin-coated vesicles and early endosomes, after internalization. Finally, we demonstrated that RET internalization is required for complete activation of Erk1/2, but not for activation of Akt signaling. Our data suggest that ligand-induced internalization of RET not only plays an overall role in downregulation and termination of signaling, but also functions to traffic RET to subcellular locations where it can fully activate certain downstream signaling pathways.
Richardson
D S
DS
Department of Pathology and Molecular Medicine, Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario, Canada.
Lai
A Z
AZ
Mulligan
L M
LM
D002462:Cell Membrane / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D008024:Ligands; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000378:metabolism*; D015398:Signal Transduction; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D008024:Ligands; D019869:Phosphatidylinositol 3-Kinases; D051096:Proto-Oncogene Proteins c-ret; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D020696:rab5 GTP-Binding Proteins