title: Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.
abstract: The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.
authors: Division of Cancer Biology and Genetics, Cancer Research Institute and Department of Pathology & Molecular Medicine, Queen's University, Kingston, Ontario, K7L 3N6, Canada.
authors: Mathieu J F
authors: http://orcid.org/0000-0002-4452-2383
authors: MJ
authors: Crupi
authors: Division of Cancer Biology and Genetics, Cancer Research Institute and Department of Pathology & Molecular Medicine, Queen's University, Kingston, Ontario, K7L 3N6, Canada.
authors: Piriya
authors: P
authors: Yoganathan
authors: Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, M5B 2K3, Canada.
authors: Leslie N
authors: LN
authors: Bone
authors: Division of Cancer Biology and Genetics, Cancer Research Institute and Department of Pathology & Molecular Medicine, Queen's University, Kingston, Ontario, K7L 3N6, Canada.
authors: Eric
authors: E
authors: Lian
authors: Division of Cancer Biology and Genetics, Cancer Research Institute and Department of Pathology & Molecular Medicine, Queen's University, Kingston, Ontario, K7L 3N6, Canada.
authors: Andrew
authors: A
authors: Fetz
authors: Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, M5B 2K3, Canada.
authors: Costin N
authors: CN
authors: Antonescu
authors: Division of Cancer Biology and Genetics, Cancer Research Institute and Department of Pathology & Molecular Medicine, Queen's University, Kingston, Ontario, K7L 3N6, Canada.
authors: Lois M
authors: LM
authors: Mulligan
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
chemical_list: D033962:Adaptor Protein Complex 2; D020033:Protein Isoforms
keywords: RET receptor; adaptor protein complex 2; clathrin; internalization; total internal reflection fluorescence microscopy