scholarly journals p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

2009 ◽  
Vol 20 (7) ◽  
pp. 1970-1980 ◽  
Author(s):  
Christine M. Chiasson ◽  
Kristin B. Wittich ◽  
Peter A. Vincent ◽  
Victor Faundez ◽  
Andrew P. Kowalczyk
Keyword(s):  
Plasma Membrane ◽  
Rho Gtpase ◽  
Retention Mechanism ◽  
Endocytic Pathway ◽  
Membrane Domains ◽  
P120 Catenin ◽  
Rhoa Activity ◽  
Endocytic Machinery ◽  
Endocytic Compartments ◽  
The Relationship

p120-catenin is a cytoplasmic binding partner of cadherins and functions as a set point for cadherin expression by preventing cadherin endocytosis, and degradation. p120 is known to regulate cell motility and invasiveness by inhibiting RhoA activity. However, the relationship between these functions of p120 is not understood. Here, we provide evidence that p120 functions as part of a plasma membrane retention mechanism for VE-cadherin by preventing the recruitment of VE-cadherin into membrane domains enriched in components of the endocytic machinery, including clathrin and the adaptor complex AP-2. The mechanism by which p120 regulates VE-cadherin entry into endocytic compartments is dependent on p120's interaction with the cadherin juxtamembrane domain, but occurs independently of p120's prevention of Rho GTPase activity. These findings clarify the mechanism for p120's function in stabilizing VE-cadherin at the plasma membrane and demonstrate a novel role for p120 in modulating the availability of cadherins for entry into a clathrin-dependent endocytic pathway.

scholarly journals The VE-cadherin cytoplasmic domain undergoes proteolytic processing during endocytosis

2017 ◽  
Vol 28 (1) ◽  
pp. 76-84 ◽  
Author(s):  
Wenji Su ◽  
Andrew P. Kowalczyk
Keyword(s):  
Plasma Membrane ◽  
Adhesion Strength ◽  
Turnover Rate ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Proteolytic Processing ◽  
Endocytic Pathway ◽  
Cytoplasmic Domains ◽  
P120 Catenin ◽  
Binding Domains

VE-cadherin trafficking to and from the plasma membrane has emerged as a critical mechanism for regulating cadherin surface levels and adhesion strength. In addition, proteolytic processing of cadherin extracellular and cytoplasmic domains has been reported to regulate cadherin adhesion and signaling. Here we provide evidence that VE-cadherin is cleaved by calpain upon entry into clathrin-enriched domains. This cleavage event occurs between the β-catenin and p120-binding domains within the cadherin cytoplasmic tail. Of interest, VE-cadherin mutants that are resistant to endocytosis are similarly resistant to cleavage. Furthermore, p120-catenin overexpression blocks cadherin internalization and cleavage, coupling entry into the endocytic pathway with proteolytic processing. Of importance, the cleavage of the VE-cadherin tail alters the postendocytic trafficking itinerary of the cadherin, resulting in a higher turnover rate due to decreased recycling and increased degradation. In conclusion, this study identifies a novel proteolytic event that regulates the trafficking of VE-cadherin after endocytosis.

scholarly journals Endocytosis of GPI-linked membrane folate receptor-alpha.

1996 ◽  
Vol 132 (1) ◽  
pp. 35-47 ◽  
Author(s):  
S Rijnboutt ◽  
G Jansen ◽  
G Posthuma ◽  
J B Hynes ◽  
J H Schornagel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Folate Receptor ◽  
Large Fraction ◽  
Integral Membrane Protein ◽  
Endocytic Pathway ◽  
Reduced Folate Carrier ◽  
Membrane Domains ◽  
Chemotherapeutic Drugs ◽  
Folate Receptors ◽  
Triton X

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.

scholarly journals The kinetic aspects of intracellular fluorescence labeling with TMA-DPH support the maturation model for endocytosis in L929 cells.

1994 ◽  
Vol 125 (4) ◽  
pp. 783-794 ◽  
Author(s):  
D Illinger ◽  
J G Kuhry
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Intensity ◽  
Release Kinetics ◽  
Aqueous Media ◽  
Endocytic Pathway ◽  
Fluorescence Labeling ◽  
Cell Periphery ◽  
Membrane Composition ◽  
Endocytic Compartments ◽  
Intracellular Fluorescence

TMA-DPH (1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene), a hydrophobic fluorescent membrane probe, interacts with living cells by instantaneous incorporation into the plasma membrane, where it becomes fluorescent. It then follows the intracellular constitutive membrane traffic and acts as a bulk membrane marker of the endocytic pathway (Illinger, D., P. Poindron, P. Fonteneau, M. Modolell, and J. G. Kuhry. 1990. Biochim. Biophys. Acta. 1030:73-81; Illinger, D., P. Poindron, and J. G. Kuhry. 1991. Biol. Cell. 73:131-138). As such, TMA-DPH displays particular properties mainly due to partition between membranes and aqueous media. From these properties, original arguments can be inferred in favor of the maturation model for the endocytic pathway, against that of pre-existing compartments, in L929 cultured mouse fibroblasts. (a) TMA-DPH labeling is seen to progress from the cell periphery to perinuclear regions during endocytosis without any noticeable loss in fluorescence intensity; with a vesicle shuttle model this evolution would be accompanied by probe dilution with a decrease in the overall intracellular fluorescence intensity, and the labeling of the inner (late) compartments could in no way become more intense than that of the peripheral (early) ones. (b) From TMA-DPH fluorescence anisotropy assays, it is concluded that membrane fluidity is the same in the successive endocytic compartments as in the plasma membrane, which probably denotes a similar phospholipidic membrane composition, as might be expected in the maturation model. (c) TMA-DPH internalization and release kinetics are more easily described with the maturation model.

scholarly journals p120-Catenin Regulates Clathrin-dependent Endocytosis of VE-Cadherin

2005 ◽  
Vol 16 (11) ◽  
pp. 5141-5151 ◽  
Author(s):  
Kanyan Xiao ◽  
Jennifer Garner ◽  
Kathleen M. Buckley ◽  
Peter A. Vincent ◽  
Christine M. Chiasson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Lysosomal Degradation ◽  
P120 Catenin ◽  
Binding Partner ◽  
Endocytic Machinery ◽  
Green Fluorescent ◽  
Retention Signal

VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin–p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.

scholarly journals Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine

2015 ◽  
Vol 112 (51) ◽  
pp. E7138-E7147 ◽  
Author(s):  
David S. Wheeler ◽  
Suzanne M. Underhill ◽  
Donna B. Stolz ◽  
Geoffrey H. Murdoch ◽  
Edda Thiels ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Dopamine Transporter ◽  
Rho Gtpase ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dopamine Neurons ◽  
Rhoa Activity ◽  
Dependent Inactivation ◽  
Independent Process ◽  
Dopamine Efflux

Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH’s effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action.

scholarly journals Association of Rho-associated protein kinase 1 with E-cadherin complexes is mediated by p120-catenin

2012 ◽  
Vol 23 (1) ◽  
pp. 99-110 ◽  
Author(s):  
Andrew L. Smith ◽  
Michael R. Dohn ◽  
Meredith V. Brown ◽  
Albert B. Reynolds
Keyword(s):  
Protein Kinase ◽  
Rho Gtpase ◽  
Actin Dynamics ◽  
Physical Interaction ◽  
P120 Catenin ◽  
Proteomic Approach ◽  
Downstream Effectors ◽  
Blocking Antibodies ◽  
The Relationship ◽  
E Cadherin

The dynamic functional linkage of cadherins with the underlying actin cytoskeleton is tightly regulated to achieve proper cell–cell adhesion. p120-catenin (p120) regulates both cadherin stability and actin dynamics, but the relationship between these two functions remains unclear. Using a novel proteomic approach called reversible cross-link immunoprecipitation, or ReCLIP, we previously identified a physical interaction between p120 and Rho-associated protein kinase 1 (ROCK1), a major effector of RhoA. In this paper, we show that a discrete fraction of cellular ROCK1 coimmunoprecipitates with p120 and precisely colocalizes to adherens junctions (AJs). Manipulation of AJs using a calcium-switch assay and cadherin-blocking antibodies indicates direct recruitment of ROCK1 to newly forming junctions. Importantly, we find that p120 links ROCK1 to the cadherin complex, as ROCK1 coimmunoprecipitates with wild-type but not p120-uncoupled E-cadherin. Moreover, depletion of ROCK1 using short-hairpin RNA results in dramatic mislocalization of the cadherin complex and junctional actin. These data are consistent with a model in which p120 dynamically regulates Rho-GTPase activity at the cadherin complex through transient interaction with several of its up- and downstream effectors, including ROCK1.

scholarly journals Interplay between actomyosin and E-cadherin dynamics regulates cell shape in the Drosophila embryonic epidermis

2020 ◽  
Vol 133 (15) ◽  
pp. jcs242321
Author(s):  
Joshua Greig ◽  
Natalia A. Bulgakova
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Cell Elongation ◽  
Cell Shape ◽  
Intercellular Adhesion ◽  
Epithelial Tissue ◽  
P120 Catenin ◽  
Endocytic Machinery ◽  
E Cadherin

ABSTRACTPrecise regulation of cell shape is vital for building functional tissues. Here, we study the mechanisms that lead to the formation of highly elongated anisotropic epithelial cells in the Drosophila epidermis. We demonstrate that this cell shape is the result of two counteracting mechanisms at the cell surface that regulate the degree of elongation: actomyosin, which inhibits cell elongation downstream of RhoA (Rho1 in Drosophila) and intercellular adhesion, modulated via clathrin-mediated endocytosis of E-cadherin (encoded by shotgun in flies), which promotes cell elongation downstream of the GTPase Arf1 (Arf79F in Drosophila). We show that these two mechanisms do not act independently but are interconnected, with RhoA signalling reducing Arf1 recruitment to the plasma membrane. Additionally, cell adhesion itself regulates both mechanisms – p120-catenin, a regulator of intercellular adhesion, promotes the activity of both Arf1 and RhoA. Altogether, we uncover a complex network of interactions between cell–cell adhesion, the endocytic machinery and the actomyosin cortex, and demonstrate how this network regulates cell shape in an epithelial tissue in vivo.

scholarly journals Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes

2011 ◽  
Vol 22 (12) ◽  
pp. 2068-2082 ◽  
Author(s):  
Karine Laulagnier ◽  
Nicole L. Schieber ◽  
Tanja Maritzen ◽  
Volker Haucke ◽  
Robert G. Parton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Cytosolic Calcium ◽  
Endocytic Pathway ◽  
Secretory Process ◽  
Microtubule Depolymerization ◽  
Lysosome Related Organelles ◽  
Endocytic Compartments

Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

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