Integrin Recycling Research Articles

PKD Controls αvβ3 Integrin Recycling and Tumor Cell Invasive Migration through Its Substrate Rabaptin-5

Integrin recycling is critical for cell migration. Protein kinase D (PKD) mediates signals from the platelet-derived growth factor receptor (PDGF-R) to control αvβ3 integrin recycling. We now show that Rabaptin-5, a Rab5 effector in endosomal membrane fusion, is a PKD substrate. PKD phosphorylates Rabaptin-5 at Ser407, and this is both necessary and sufficient for PDGF-dependent short-loop recycling of αvβ3, which in turn inhibits α5β1 integrin recycling. Rab4, but not Rab5, interacts with phosphorylated Rabaptin-5 toward the front of migrating cells to promote delivery of αvβ3 to the leading edge, thereby driving persistent cell motility and invasion that is dependent on this integrin. Consistently, disruption of Rabaptin-5 Ser407 phosphorylation reduces persistent cell migration in 2D and αvβ3-dependent invasion. Conversely, invasive migration that is dependent on α5β1 integrin is promoted by disrupting Rabaptin phosphorylation. These findings demonstrate that the PKD pathway couples receptor tyrosine kinase signaling to an integrin switch via Rabaptin-5 phosphorylation.

Cell Adhesion: A FERM Grasp of the Tail Sorts Out Integrins

As well as modulating integrin activation, a conserved NPxY motif in integrin cytoplasmic tails that binds the FERM-domain-containing proteins kindlin and sorting nexin 17 plays pivotal roles in integrin recycling and degradation.

Mechanistic Insights into Regulated Cargo Binding by ACAP1 Protein

Coat complexes sort protein cargoes into vesicular transport pathways. An emerging class of coat components has been the GTPase-activating proteins (GAPs) that act on the ADP-ribosylation factor (ARF) family of small GTPases. ACAP1 (ArfGAP with coiled-coil, ankyrin repeat, and PH domains protein 1) is an ARF6 GAP that also acts as a key component of a recently defined clathrin complex for endocytic recycling. Phosphorylation by Akt has been shown to enhance cargo binding by ACAP1 in explaining how integrin recycling is an example of regulated transport. We now shed further mechanistic insights into how this regulation is achieved at the level of cargo binding by ACAP1. We initially defined a critical sequence in the cytoplasmic domain of integrin β1 recognized by ACAP1 and showed that this sequence acts as a recycling sorting signal. We then pursued a combination of structural, modeling, and functional studies, which suggest that phosphorylation of ACAP1 relieves a localized mechanism of autoinhibition in regulating cargo binding. Thus, we have elucidated a key regulatory juncture that controls integrin recycling and also advanced the understanding of how regulated cargo binding can lead to regulated transport.

ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9β1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and β1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and β1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of β1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

Rab5c promotes AMAP1–PRKD2 complex formation to enhance β1 integrin recycling in EGF-induced cancer invasion

Epidermal growth factor receptor (EGFR) signaling is one of the crucial factors in breast cancer malignancy. Breast cancer cells often overexpress Arf6 and its effector, AMAP1/ASAP1/DDEF1; in these cells, EGFR signaling may activate the Arf6 pathway to induce invasion and metastasis. Active recycling of some integrins is crucial for invasion and metastasis. Here, we show that the Arf6-AMAP1 pathway links to the machinery that recycles β1 integrins, such as α3β1, to promote cell invasion upon EGFR stimulation. We found that AMAP1 had the ability to bind directly to PRKD2 and hence to make a complex with the cytoplasmic tail of the β1 subunit. Moreover, GTP-Rab5c also bound to AMAP1, and activation of Rab5c by EGFR signaling was necessary to promote the intracellular association of AMAP1 and PRKD2. Our results suggest a novel mechanism by which EGFR signaling promotes the invasiveness of some breast cancer cells via integrin recycling.

Sorting nexin 17 prevents lysosomal degradation of β1 integrins by binding to the β1-integrin tail

Integrin functions are controlled by regulating their affinity for ligand, and by the efficient recycling of intact integrins through endosomes. Here we demonstrate that the Kindlin-binding site in the β1-integrin cytoplasmic domain serves as a molecular switch enabling the sequential binding of two FERM-domain-containing proteins in different cellular compartments. When β1 integrins are at the plasma membrane, Kindlins control ligand-binding affinity. However, when they are internalized, Kindlins dissociate from integrins and sorting nexin 17 (SNX17) is recruited to free β1-integrin tails in early endosomes to prevent β1-integrin degradation, leading to their recycling back to the cell surface. Our results identify SNX17 as a β1-integrin-tail-binding protein that interacts with the free Kindlin-binding site in endosomes to stabilize β1 integrins, resulting in their recycling to the cell surface where they can be reused.

The Art of “Cut and Run”: The Role of Rab14 GTPase in Regulating N-Cadherin Shedding and Cell Motility

Linford et al. define a Rab14-mediated endocytic recycling pathway that controls proteolytic N-cadherin cleavage by transporting ADAM10 protease to the plasma membrane. When this pathway is disrupted, diminished ADAM10-dependent N-cadherin shedding leads to increased cell-cell adhesion and inhibition of cell motility.

Abstract 4329: Modulation of metastatic behavior in breast cancers harboring p53 mutations

Abstract The mutation of p53 generally occurs during the later stages of cancer and leads to the increased metastatic potential of triple negative (ER_/PR_/Her2_) breast cancer. The low survival rates of advanced metastatic cancer make it critical not only to understand the mechanisms underlying the functional implications of p53 mutation, but also to identify ways to regulate its impact in cancer cell physiology. We have previously described the design, synthesis, and evaluation of a stabilized α-helix of p53 (SAH-p53) capable of inhibiting the p53-HDM2 and p53-HDMX interactions in order to restore the functionality of p53. Despite having low expression of both HDM2 and HDMX and mutations in p53, SAH-p53 treatment of the triple negative breast cancer cell lines MDA-MB-231 and HS578T results in a modest decrease in proliferative activity. While mutant p53 is known to promote metastatic behavior of breast cancer cells, the propensity of triple negative breast cancer cells to migrate and invade through Matrigel in the presence of EGF was decreased by treatment with SAH-p53. The anti-proliferative and anti-migratory effects of SAH-p53 treatment are independent of each other, yet both are accompanied by an alteration in cell morphology that includes the loss of cell membrane integrity and actin cytoskeletal organization. The surface level of β1 integrin, which is typically kept constant by mutant p53, is decreased in the presence of SAH-p53, pointing to possible alterations in integrin recycling. SAH-p53 also causes a change in the localization of the adhesion protein and stem cell marker CD44. Moreover, it has been suggested that mutant p53 enhances invasive properties by interacting with and inhibiting isoforms of p63, which have been implicated in the regulation of cell adhesion. Treatment of the triple negative breast cancer cells with a stabilized α-helix of p63 showed a significant decrease in cell viability as well as an enhanced inhibition of migration when treated simultaneously with SAH-p53. Targeted therapy for triple negative breast cancer is currently very limited and patients must rely on standard chemotherapeutic regimens. These data show that the use of SAH-p53 and SAH-p63 peptides provides a novel avenue to study the molecular interactions that drive metastatic behavior originated by p53 gain-of-function mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4329. doi:1538-7445.AM2012-4329

SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways

The FERM-like domain-containing sorting nexins of the SNX17/SNX27/SNX31 family have been proposed to mediate retrieval of transmembrane proteins from the lysosomal pathway. In this paper, we describe a stable isotope labeling with amino acids in culture-based quantitative proteomic approach that allows an unbiased, global identification of transmembrane cargoes that are rescued from lysosomal degradation by SNX17. This screen revealed that several integrins required SNX17 for their stability, as depletion of SNX17 led to a loss of β1 and β5 integrins and associated a subunits from HeLa cells as a result of increased lysosomal degradation. SNX17 bound to the membrane distal NPXY motif in β integrin cytoplasmic tails, thereby preventing lysosomal degradation of β integrins and their associated a subunits. Furthermore, SNX17-dependent retrieval of integrins did not depend on the retromer complex. Consistent with an effect on integrin recycling, depletion of SNX17 also caused alterations in cell migration. Our data provide mechanistic insight into the retrieval of internalized integrins from the lysosomal degradation pathway, a prerequisite for subsequent recycling of these matrix receptors.

Distinct Recycling of Active and Inactive β1 Integrins

Integrin trafficking plays an important role in cellular motility and cytokinesis. Integrins undergo constant endo/exocytic shuttling to facilitate the dynamic regulation of cell adhesion. Integrin activity toward the components of the extracellular matrix is regulated by the ability of these receptors to switch between active and inactive conformations. Several cellular signalling pathways have been described in the regulation of integrin traffic under different conditions. However, the interrelationship between integrin activity conformations and their endocytic fate have remained incompletely understood. Here, we have investigated the endocytic trafficking of active and inactive β1 integrins in cancer cells. Both conformers are endocytosed in a clathrin- and dynamin-dependent manner. The net endocytosis rate of the active β1 integrins is higher, whereas endocytosis of the inactive β1 integrin is counteracted by rapid recycling back to the plasma membrane via an ARF 6- and early endosome antigen 1-positive compartment in an Rab 4a- and actin-dependent manner. Owing to these distinct trafficking routes, the two receptor pools display divergent subcellular localization. At steady state, the inactive β1 integrin is mainly on the plasma membrane, whereas the active receptor is predominantly intracellular. These data provide new insights into the endocytic traffic of integrins and imply the possibility of a previously unappreciated crosstalk between pathways regulating integrin activity and traffic.

Diacylglycerol kinase α controls RCP-dependent integrin trafficking to promote invasive migration

Inhibition of αvβ3 integrin or expression of oncogenic mutants of p53 promote invasive cell migration by enhancing endosomal recycling of α5β1 integrin under control of the Rab11 effector Rab-coupling protein (RCP). In this paper, we show that diacylglycerol kinase α (DGK-α), which phosphorylates diacylglycerol to phosphatidic acid (PA), was required for RCP to be mobilized to and tethered at the tips of invasive pseudopods and to allow RCP-dependent α5β1 recycling and the resulting invasiveness of tumor cells. Expression of a constitutive-active mutant of DGK-α drove RCP-dependent invasion in the absence of mutant p53 expression or αvβ3 inhibition, and conversely, an RCP mutant lacking the PA-binding C2 domain was not capable of being tethered at pseudopod tips. These data demonstrate that generation of PA downstream of DGK-α is essential to connect expression of mutant p53s or inhibition of αvβ3 to RCP and for this Rab11 effector to drive the trafficking of α5β1 that is required for tumor cell invasion through three-dimensional matrices.

Phosphoinositide specificity determines which cytohesins regulate β1 integrin recycling

Recycling of internalized integrins is a crucial step in adhesion remodeling and cell movement. Recently, we determined that the ADP-ribosylation factor-guanine nucleotide exchange factors (ARF-GEFs) cytohesin 2/ARNO and cytohesin 3/GRP1 have opposing effects on adhesion and stimulated β1 integrin recycling even though they are very closely related proteins (80% sequence identity). We have now determined the sequence differences underlying the differential actions of cytohesin 2/ARNO and cytohesin 3/GRP1. We found that the ability of cytohesins to promote β1 integrin recycling and adhesion depends upon the presence or absence of a key glycine residue in their pleckstrin homology (PH) domains. This glycine residue determines the phosphoinositide specificity and affinity of cytohesin PH domains. Switching the number of glycines in the PH domains of cytohesin 2 and cytohesin 3 is sufficient to reverse their effects on adhesion and spreading and to reverse their subcellular locations. Importantly, we also find that a mutant form of cytohesin 3/GRP1 that has three rather than two glycines in its PH domain rescues β1 integrin recycling in cytohesin 2/ARNO knockdown cells. Conversely, a mutant form of cytohesin 2/ARNO with two glycines in its PH domain fails to rescue β1 integrin recycling. Therefore, we conclude that phosphoinositide specificity is the sole functional difference that determines which cytohesin can promote integrin recycling.

The Bro1 domain-containing Myopic/HDPTP coordinates with Rab4 to regulate cell adhesion and migration

Protein tyrosine phosphatases (PTPs) are a group of tightly regulated enzymes that coordinate with protein tyrosine kinases to control protein phosphorylation during various cellular processes. Using genetic analysis in Drosophila non-transmembrane PTPs, we identified one role that Myopic (Mop), the Drosophila homolog of the human His domain phosphotyrosine phosphatase (HDPTP), plays in cell adhesion. Depletion of Mop results in aberrant integrin distribution and border cell dissociation during Drosophila oogenesis. Interestingly, Mop phosphatase activity is not required for its role in maintaining border cell cluster integrity. We further identified Rab4 GTPase as a Mop interactor in a yeast two-hybrid screen. Expression of the Rab4 dominant-negative mutant leads to border cell dissociation and suppression of Mop-induced wing-blade adhesion defects, suggesting a critical role of Rab4 in Mop-mediated signaling. In mammals, it has been shown that Rab4-dependent recycling of integrins is necessary for cell adhesion and migration. We found that human HDPTP regulates the spatial distribution of Rab4 and integrin trafficking. Depletion of HDPTP resulted in actin reorganization and increased cell motility. Together, our findings suggest an evolutionarily conserved function of HDPTP-Rab4 in the regulation of endocytic trafficking, cell adhesion and migration.

Rab25 and CLIC3 Collaborate to Promote Integrin Recycling from Late Endosomes/Lysosomes and Drive Cancer Progression

SummaryHere we show that Rab25 permits the sorting of ligand-occupied, active-conformation α5β1 integrin to late endosomes/lysosomes. Photoactivation and biochemical approaches show that lysosomally targeted integrins are not degraded but are retrogradely transported and recycled to the plasma membrane at the back of invading cells. This requires CLIC3, a protein upregulated in Rab25-expressing cells and tumors, which colocalizes with active α5β1 in late endosomes/lysosomes. CLIC3 is necessary for release of the cell rear during migration on 3D matrices and is required for invasion and maintenance of active Src signaling in organotypic microenvironments. CLIC3 expression predicts lymph node metastasis and poor prognosis in operable cases of pancreatic ductal adenocarcinoma (PDAC). The identification of CLIC3 as a regulator of a recycling pathway and as an independent prognostic indicator in PDAC highlights the importance of active integrin trafficking as a potential drive to cancer progression in vivo.

The Arp2/3 activator WASH regulates α5β1-integrin-mediated invasive migration.

The actin cytoskeleton provides scaffolding and physical force to effect fundamental processes such as motility, cytokinesis and vesicle trafficking. The Arp2/3 complex nucleates actin structures and contributes to endocytic vesicle invagination and trafficking away from the plasma membrane. Internalisation and directed recycling of integrins are major driving forces for invasive cell motility and potentially for cancer metastasis. Here, we describe a direct requirement for WASH and Arp2/3-mediated actin polymerisation on the endosomal membrane system for α5β1 integrin recycling. WASH regulates the trafficking of endosomal α5β1 integrin to the plasma membrane and is fundamental for integrin-driven cell morphology changes and integrin-mediated cancer cell invasion. Thus, we implicate WASH and Arp2/3-driven actin nucleation in receptor recycling leading to invasive motility.

Rab25 Mediates Integrin Recycling for Tumor Cell Migration in 3D

Prior to the publication of this report, the importance of endocytotic and exocytotic recycling of integrins was just beginning to be recognized. However, the findings in this report provided significant new insights into the mechanisms mediating integrin recycling and the critical importance of spatially regulated recycling in controlling specific aspects of cell migration and invasion. The Rab family GTPase RAB25, which has been implicated in tumor progression, was found to bind directly to β1 integrin and, through this interaction, specifically deliver α5β1-containing vesicles to the tips of pseudopodial extensions. This RAB25-integrin interaction was found to be critical for directional invasion of tumor cells in 3D extracellular matrix environments. I liked this report not only for the elegance of the experiments and fundamental insights into matrix receptor recycling, but also because it provided mechanistic insights into how aberrant vesicle trafficking and receptor recycling could contribute to tumorigenesis. This PaperPick refers to Rab25 associates with α5β1 integrin to promote invasive migration in 3D microenvironments, by P.T. Caswell, H.J. Spence, M. Parsons, D.P. White, K. Clark, K.W. Cheng, G.B. Mills, M.J. Humphries, A.J. Messent, K.I. Anderson, M.W. McCaffrey, B.W. Ozanne, and J.C. Norman, published in October 2007. Video Abstract Jim Norman and Pat Caswell discuss their 2007 paper, in which they found that an interaction between a Rab GTPase and an integrin is important for cell migration and tumor metastasis.

Integrin Trafficking and Tumor Progression

Integrins are major mediators of cancer cell adhesion to extracellular matrix. Through this interaction, integrins play critical roles in cell migration, invasion, metastasis, and resistance to apoptosis during tumor progression. Recent studies highlight the importance of integrin trafficking, endocytosis and recycling, for the functions of integrins in cancer cells. Understanding the molecular mechanisms of integrin trafficking is pivotal for understanding tumor progression and for the development of anticancer drugs.

Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated α5β1 Integrin Recycling

The α5β1 integrin heterodimer regulates many processes that contribute to embryonic development and angiogenesis, in both physiological and pathological contexts. As one of the major adhesion complexes on endothelial cells, it plays a vital role in adhesion and migration along the extracellular matrix. We recently showed that angiogenesis is modulated by syntaxin 6, a Golgi- and endosome-localized t-SNARE, and that it does so by regulating the post-Golgi trafficking of VEGFR2. Here we show that syntaxin 6 is also required for α5β1 integrin-mediated adhesion of endothelial cells to, and migration along, fibronectin. We demonstrate that syntaxin 6 and α5β1 integrin colocalize in EEA1-containing early endosomes, and that functional inhibition of syntaxin 6 leads to misrouting of β1 integrin to the degradation pathway (late endosomes and lysosomes) rather transport along recycling pathway from early endosomes; an increase in the pool of ubiquitinylated α5 integrin and its lysosome-dependent degradation; reduced cell spreading on fibronectin; decreased Rac1 activation; and altered Rac1 localization. Collectively, our data show that functional syntaxin 6 is required for the regulation of α5β1-mediated endothelial cell movement on fibronectin. These syntaxin 6-regulated membrane trafficking events control outside-in signaling via haptotactic and chemotactic mechanisms.

Competitive binding of Rab21 and p120RasGAP to integrins regulates receptor traffic and migration

Integrin trafficking from and to the plasma membrane controls many aspects of cell behavior including cell motility, invasion, and cytokinesis. Recruitment of integrin cargo to the endocytic machinery is regulated by the small GTPase Rab21, but the detailed molecular mechanisms underlying integrin cargo recruitment are yet unknown. Here we identify an important role for p120RasGAP (RASA1) in the recycling of endocytosed α/β1-integrin heterodimers to the plasma membrane. Silencing of p120RasGAP attenuated integrin recycling and augmented cell motility. Mechanistically, p120RasGAP interacted with the cytoplasmic domain of integrin α-subunits via its GAP domain and competed with Rab21 for binding to endocytosed integrins. This in turn facilitated exit of the integrin from Rab21- and EEA1-positive endosomes to drive recycling. Our results assign an unexpected role for p120RasGAP in the regulation of integrin traffic in cancer cells and reveal a new concept of competitive binding of Rab GTPases and GAP proteins to receptors as a regulatory mechanism in trafficking.

Proteome-wide Dysregulation by PRA1 Depletion Delineates a Role of PRA1 in Lipid Transport and Cell Migration

We have previously identified prenylated Rab acceptor 1 (PRA1) as a novel cellular interacting partner for Epstein-Barr virus-encoded oncoprotein, latent membrane protein 1 (LMP1). The intracellular trafficking and full signaling of LMP1 requires its interaction with PRA1. To further explore the role of PRA1 in Epstein-Barr virus-associated nasopharyngeal carcinoma (NPC) cells, we generated several PRA1-knockdown cell clones, which exhibited altered cell morphology and increased cell motility. We identified proteins differentially expressed in the knockdown clones by means of isobaric mass tags labeling coupled with multidimensional liquid chromatography-mass spectrometry. We validated a panel of proteins, which showed consistent up-regulation in PRA1-knockdown clones and participated in regulating lipid homeostasis and cell migration. Immunofluorescence staining further revealed altered localization of these proteins and accumulation of intracellular cholesterol in PRA1-knockdown clones. These effects were phenocopied by treatment with a cholesterol transport inhibitor, U18666A. Moreover, overexpressed PRA1 was able to alleviate the dysregulation of these affected proteins either from PRA1 knockdown or U18666A treatment, implying a role for PRA1 in regulating the levels of these affected proteins in response to altered cholesterol homeostasis. We further demonstrated that LMP1 expression caused PRA1 sequestration in NPC cells, leading to a consequence reminiscent of PRA1 knockdown. Finally, the immunohistochemistry showed a physiological relevance of the PRA1-associated proteome-wide changes in NPC biopsy tissues. In sum, our findings delineated novel roles of PRA1 in lipid transport and cell migration, and provided additional insights into the molecular basis of NPC morphogenesis, namely a consequence of LMP1-PRA1 interaction.