Cell Migration Is Regulated by Platelet-Derived Growth Factor Receptor Endocytosis

Abstract

Cell migration requires spatial and temporal processes that detect and transfer extracellular stimuli into intracellular signals. The platelet-derived growth factor (PDGF) receptor is a cell surface receptor on fibroblasts that regulates proliferation and chemotaxis in response to PDGF. How the PDGF signal is transmitted accurately through the receptor into cells is an unresolved question. Here, we report a new intracellular signaling pathway by which DOCK4, a Rac1 guanine exchange factor, and Dynamin regulate cell migration by PDGF receptor endocytosis. We showed by a series of biochemical and microscopy techniques that Grb2 serves as an adaptor protein in the formation of a ternary complex between the PDGF receptor, DOCK4, and Dynamin, which is formed at the leading edge of cells. We found that this ternary complex regulates PDGFdependent cell migration by promoting PDGF receptor endocytosis and Rac1 activation at the cell membrane. This study revealed a new mechanism by which cell migration is regulated by PDGF receptor endocytosis.Chemoattractants bind to cell surface receptors, resulting in the cytoskeletal reorganization that permits the migration of cells toward a stimulus. In fibroblasts, the platelet-derived growth factor receptor ␤ (PDGFR␤) is a cell surface receptor tyrosine kinase (RTK) that regulates cell proliferation and chemotaxis in response to PDGF. PDGF binding activates PDGF receptor autophosphorylation, which in turn mediates a series of intracellular signaling cascades initiated by the association of SH2 domain-containing adaptor proteins (25). The adaptor protein Grb2 at the plasma membrane binds to Ras exchange factor Sos1, activating mitogen-activated protein kinase (MAPK) and cell proliferation signals (19). Grb2 also plays a critical role in receptor internalization via its interaction with dynamin, an exchange factor that facilitates receptor entry into endocytic vesicles (32). Grb2 regulates ubiquitination and the degradation of the receptor via its interaction with Cbl, an E3 ubiquitin ligase (33). While the role of Grb2 in modulating receptor levels and facilitating growth factor-dependent mitogenic signals is defined, its role in coordinating receptor-dependent chemotaxis has not been elucidated.The small GTPase Rac1 plays a crucial role in PDGF-mediated chemotaxis by regulating cortical actin at the leading edge of cells. PDGF receptor activation promotes GTP loading and the translocation of Rac1 to the cell membrane via guanine exchange factors (GEFs). The DOCK family of Rac1 GEFs, also called CDM proteins (for Caenorhabditis elegans ced-5, vertebrate DOCK180, and Drosophila myoblast city), are regulators of cell migration and have been implicated in various biological processes, such as lymphocyte migration, phagocytosis, and cancer progression (6,10,30,35). In migrating fibroblasts, DOCK proteins localize to the cell's leading edge via their interaction with the phospholipid PIP3, but a direct molecular link to PDGF has not been established (5). Biochemical studies show that R...

On the move: endocytic trafficking in cell migration

“…Like in the case of border cells [28], blocking RTK endocytosis in NIH3T3 fibroblasts causes impaired migration [33]. Of note, internalized PDGFR recycled via Rab11 or Rab4 positive endosomes to the surface of NHI3T3 cells [33], whereas in human fibroblasts the same receptor is predominantly degraded in lysosomes [34]. Consistent with the importance of balanced PDGFR trafficking for cell migration and other processes, it is subject to regulation by kinases and phosphatases.…”

“…Upon ligand binding, PDGFRb becomes tyrosine phosphorylated enabling its interaction with the adaptor protein Grb2 which recruits DOCK4 and dynamin2 resulting in receptor endocytosis and Rac activation. Like in the case of border cells [28], blocking RTK endocytosis in NIH3T3 fibroblasts causes impaired migration [33]. Of note, internalized PDGFR recycled via Rab11 or Rab4 positive endosomes to the surface of NHI3T3 cells [33], whereas in human fibroblasts the same receptor is predominantly degraded in lysosomes [34].…”

“…For instance, in case of the PDGFRb, an intracellular signaling pathway was unraveled by which the Rac1 GEF DOCK4 and the GTPase dynamin2 control NIH3T3 cell migration in response to PDGF [33]. Upon ligand binding, PDGFRb becomes tyrosine phosphorylated enabling its interaction with the adaptor protein Grb2 which recruits DOCK4 and dynamin2 resulting in receptor endocytosis and Rac activation.…”

Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration

“…Rab11 effectors are also involved in mammalian cell migration (46)(47)(48). More specifically, PDGF receptor-dependent cell migration has been shown to be regulated by endocytosis in a mammalian cell culture assay (49) and the recycling endosome has been indirectly implicated in the regulation of migration guided by the EGFR (47). Given the involvement of the recycling endosome in so many processes, targeting its function to reduce metastasis is unlikely to be efficient.…”

The Atypical Guanine Nucleotide Exchange Factor Dock4 Regulates Neurite Differentiation through Modulation of Rac1 GTPase and Actin Dynamics

“…Dock4 was originally identified as a gene disrupted during tumorigenesis (20). Multiple studies in fibroblasts then confirmed that Dock4 is capable of controlling cell migration by transducing several upstream signals, such as Wnt, platelet-derived growth factor, and RhoG, toward activation of Rac1 (21)(22)(23)(24). However, the functional roles of Dock4 in the central nervous system are only beginning to be understood (25)(26)(27).…”