Dynamic turnover of integrin cell adhesion molecules to and from the cell surface is central to cell migration. We report for the first time an association between integrins and Rab proteins, which are small GTPases involved in the traffic of endocytotic vesicles. Rab21 (and Rab5) associate with the cytoplasmic domains of α-integrin chains, and their expression influences the endo/exocytic traffic of integrins. This function of Rab21 is dependent on its GTP/GDP cycle and proper membrane targeting. Knock down of Rab21 impairs integrin-mediated cell adhesion and motility, whereas its overexpression stimulates cell migration and cancer cell adhesion to collagen and human bone. Finally, overexpression of Rab21 fails to induce cell adhesion via an integrin point mutant deficient in Rab21 association. These data provide mechanistic insight into how integrins are targeted to intracellular compartments and how their traffic regulates cell adhesion.
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.
Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PIP3 and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.
Integrin-mediated cell adhesion regulates a multitude of cellular responses, including proliferation, survival and cross-talk between different cellular signalling pathways. So far, integrins have been mainly shown to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signalling. Here we show that a collagen-binding integrin alpha(1)beta(1) functions as a negative regulator of epidermal growth factor receptor (EGFR) signalling through the activation of a protein tyrosine phosphatase. The cytoplasmic tail of alpha(1) integrin selectively interacts with a ubiquitously expressed protein tyrosine phosphatase TCPTP (T-cell protein tyrosine phosphatase) and activates it after cell adhesion to collagen. The activation results in reduced EGFR phosphorylation after EGF stimulation. Introduction of the alpha(1) cytoplasmic domain peptide into cells induces phosphatase activation and inhibits EGF-induced cell proliferation and anchorage-independent growth of malignant cells. These data are the first demonstration of the regulation of TCPTP activity in vivo and represent a new molecular paradigm of integrin-mediated negative regulation of receptor tyrosine kinase signalling.
Objective-Coagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine, and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a noncoagulant state and to generating prothrombotic TF remain incompletely understood. Approach and Results-Here, we study the activation of TF in primary macrophages by a combination of pharmacological, genetic, and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. After cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF + microvesicles. TF cell surface availability for release onto microvesicles is regulated by the GTPase arf6 associated with integrin α4β1. Furthermore, microvesicles proteome analysis identifies activation of Gα i2 as a participating factor in the release of microvesicles with prothrombotic activity in flowing blood. ATP not only prevents TF and phosphatidylserine internalization but also induces TF conversion to a conformation with high affinity for its ligand, coagulation factor VII. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant phosphatidylserine, the resulting TF + microvesicles distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for microvesicles generated by thrombo-inflammatory P2rx7 activation. Conclusions-These data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic microvesicles from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional microvesicles-based diagnostic approaches. Visual Overview-An online visual overview is available for this article. The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/ Highlights • Tissue factor (TF) cell surface availability is controlled by integrin α4β1-and arf6-regulated trafficking. • Microvesicles generated by pharmacological interruption of TF-integrin internalization differ in protein composition and function from mi-crovesicles released by P2rx7 cell injury signaling. • Maturation of TF to a high affinity state is a key determinant for the prothrombotic activity of TF + microvesicles in blood.
Mutations of the tumor suppressor TP53 are present in many forms of human cancer and are associated with increased tumor cell invasion and metastasis. Several mechanisms have been identified for promoting dissemination of cancer cells with TP53 mutations, including increased targeting of integrins to the plasma membrane. Here, we demonstrate a role for the filopodia-inducing motor protein Myosin-X (Myo10) in mutant p53-driven cancer invasion. Analysis of gene expression profiles from 2 breast cancer data sets revealed that MYO10 was highly expressed in aggressive cancer subtypes. Myo10 was required for breast cancer cell invasion and dissemination in multiple cancer cell lines and murine models of cancer metastasis. Evaluation of a Myo10 mutant without the integrin-binding domain revealed that the ability of Myo10 to transport β 1 integrins to the filopodia tip is required for invasion. Introduction of mutant p53 promoted Myo10 expression in cancer cells and pancreatic ductal adenocarcinoma in mice, whereas suppression of endogenous mutant p53 attenuated Myo10 levels and cell invasion. In clinical breast carcinomas, Myo10 was predominantly expressed at the invasive edges and correlated with the presence of TP53 mutations and poor prognosis. These data indicate that Myo10 upregulation in mutant p53-driven cancers is necessary for invasion and that plasma-membrane protrusions, such as filopodia, may serve as specialized metastatic engines.
SummaryPhosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P 3 ] is a key regulator of cell signaling that acts by recruiting proteins to the cell membrane, such as at the leading edge during cell migration. Here, we show that PtdIns (3,4,5)P 3 plays a central role in filopodia formation via the binding of myosin-X (Myo10), a potent promoter of filopodia. We found that the second pleckstrin homology domain (Myo10-PH2) of Myo10 specifically binds to PtdIns(3,4,5)P 3 , and that disruption of this binding led to impairment of filopodia and partial re-localization of Myo10 to microtubule-associated Rab7-positive endosomal vesicles. Given that the localization of Myo10 was dynamically restored to filopodia upon reinstatement of PtdIns(3,4,5)P 3 -binding, our results indicate that PtdIns(3,4,5)P 3 binding to the Myo10-PH2 domain is involved in Myo10 trafficking and regulation of filopodia dynamics.
Regulated turnover of integrin receptors is essential for cell adhesion and migration. Pathways selectively regulating β1-integrin recycling are implicated in cancer invasion and metastasis, yet proteins required for the internalization of this pro-invasive integrin remain to be identified. Here, we uncover formin-like 2 (FMNL2) as a critical regulator of β1-integrin internalization downstream of protein kinase C (PKC). PKCα associates with and phosphorylates FMNL2 at S1072 within its Diaphanous autoregulatory region, leading to the release of formin autoinhibition. Phosphorylation of FMNL2 triggers its rapid relocation and promotes its interaction with the cytoplasmic tails of the α-integrin subunits for β1-integrin endocytosis. FMNL2 drives β1-integrin internalization and invasive motility in a phosphorylation-dependent manner, while a FMNL2 mutant defective in actin assembly interferes with β1-integrin endocytosis and cancer cell invasion. Our data establish a role for FMNL2 in the regulation of β1-integrin and provide a mechanistic understanding of the function of FMNL2 in cancer invasiveness.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.