scholarly journals VEGF-A selectively inhibits FLT1 ectodomain shedding independent of receptor activation and receptor endocytosis

2018 ◽  
Vol 315 (2) ◽  
pp. C214-C224
Author(s):  
Nandita S. Raikwar ◽  
Masabumi Shibuya ◽  
Christie P. Thomas
Keyword(s):  
Cell Surface ◽  
Tyrosine Kinase ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
Surface Proteins ◽  
Hek293 Cells ◽  
Vegf Receptor ◽  
Ectodomain Shedding ◽  
Dependent Manner ◽  
Endosomal Acidification

Ectodomain shedding and regulated intracellular proteolysis can determine the fate or function of cell surface proteins. Fms-related tyrosine kinase (FLT) or VEGF receptor 1 is a high-affinity cell surface VEGF-A receptor tyrosine kinase that is constitutively cleaved to release an NH2-terminal VEGF-A binding ectodomain that, once shed, can antagonize the effects of VEGF-A in the extracellular milieu. We evaluated the effect of VEGF-A on FLT1 cleavage in native cells and in transient and stable expression systems. We demonstrate that VEGF-A inhibits FLT1 ectodomain cleavage in a time- and dose-dependent manner, whereas VEGF-A knockdown in HEK293 cells increases ectodomain shedding. Although kinase insert domain receptor (KDR) or VEGF receptor 2, analogous to FLT1, is also subject to extracellular and intracellular cleavage, VEGF-A does not inhibit KDR cleavage. VEGF-A inhibition of FLT1 cleavage is not dependent on FLT1 tyrosine kinase activity or the intracellular FLT1 residues. N-acetylleucylleucylnorleucinal (ALLN), a proteasomal inhibitor; bafilomycin A, an inhibitor of endosomal acidification; and dynasore, a dynamin inhibitor, all increase the abundance of FLT1 and the shed ectodomain, indicating that FLT1 is subject to dynamin-mediated endocytosis and susceptible to proteasomal and lysosomal degradation. VEGF-A inhibition of cleavage is not reversed by ALLN, bafilomycin A, or dynasore. However, a 30 AA deletion in the extracellular immunoglobulin 7 domain leads to enhanced cleavage of Flt1 with a significant reduction of the VEGF inhibitory effect. Our results indicate that the inhibition of FLT1 ectodomain cleavage by VEGF-A is dependent neither on receptor activation nor on internalization nor a consequence of receptor degradation and likely represents a direct inhibitory effect on receptor cleavage.

The K+-evoked release of [3H]acetylcholine from slices of rat globus pallidus: modulation by δ-opioid receptors

1991 ◽  
Vol 69 (3) ◽  
pp. 414-418 ◽  
Author(s):  
Bianca B. Ruzicka ◽  
Khem Jhamandas
Keyword(s):  
Globus Pallidus ◽  
Opioid Receptor ◽  
Opioid Receptors ◽  
Cholinergic Neurons ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Release Of Acetylcholine ◽  
Δ Opioid Receptor ◽  
Tritium Release

Previous investigations have shown that the activation of δ-opioid receptors depresses the release of acetylcholine (ACh) in the rat caudate putamen. This finding raised the possibility that the release of ACh is similarly modulated in the globus pallidus, a region containing a distinct population of cholinergic neurons and enriched in enkephalinergic nerve terminals. In the present study the pallidal release of ACh was characterized and the effects of δ-opioid receptor activation on this release were examined. The results show that this release is stimulated by high K+ in a concentration- and Ca2+-dependent manner. D-Pen2,L-Pen5-enkephalin (0.1 – 10 μM), a selective δ-opioid receptor agonist, produced a dose-related inhibition of the 25 mM K+-evoked tritium release. The maximal inhibitory effect, representing a 34% decrease in the K+-induced tritium release, was observed at a concentration of 1 μM. This opioid effect was attenuated by the selective δ-opioid receptor antagonist, ICI 174864 (1 μM). These findings support the role of a δ-opioid receptor in the modulation of ACh release in the rat globus pallidus.Key words: globus pallidus, acetylcholine, enkephalin, release.

Role of cations in the flocculation of Saccharomyces cerevisiae and discrimination of the corresponding proteins

1991 ◽  
Vol 37 (5) ◽  
pp. 397-403 ◽  
Author(s):  
Hiroshi Kuriyama ◽  
Itaru Umeda ◽  
Harumi Kobayashi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Inhibitory Effect ◽  
Surface Proteins ◽  
Promoting Effect ◽  
Yeast Strains ◽  
Yeast Flocculation ◽  
Different Types ◽  
Anionic Groups

Asexual yeast flocculation was studied using strong flocculents of Saccharomyces cerevisiae. The inhibitory effect of cations on flocculation is considered to be caused by competition between those cations and Ca2+ at the binding site of the Ca2+-requiring protein that is involved in flocculation. Inhibition of flocculation by various cations occurred in the following order: La3+, Sr2+, Ba2+, Mn2+, Al3+, and Na+. Cations such as Mg2+, Co2+, and K+ promoted flocculation. This promoting effect may be based on the reduction of electrostatic repulsive force between cells caused by binding of these cations anionic groups present on the cell surface. In flocculation induced by these cations, trace amounts of Ca2+ excreted on the cell surface may activate the corresponding protein. The ratio of Sr2+/Ca2+ below which cells flocculated varied among strains: for strains having the FLO5 gene, it was 400 to 500; for strains having the FLO1 gene, about 150; and for two alcohol yeast strains, 40 to 50. This suggests that there are several different types of cell surface proteins involved in flocculation in different yeast strains. Key words: yeast, flocculation, protein, cation, calcium.

Tyrosine Phosphorylation Enhances ITIM-Dependent Internalization of CD33, the Target for the Anti-AML Immunoconjugate, Gemtuzumab Ozogamicin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2581-2581
Author(s):  
Roland B. Walter ◽  
Brian W. Raden ◽  
Irwin D. Bernstein ◽  
Jonathan A. Cooper
Keyword(s):  
Cell Surface ◽  
Tyrosine Kinase ◽  
Gene Silencing ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Myeloid Cell ◽  
Gemtuzumab Ozogamicin ◽  
Dependent Manner ◽  
Wild Type ◽  
Phosphorylation State

Abstract Background: CD33, the target for the anti-AML immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), contains two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). We have previously shown that these motifs control uptake of antibody-bound CD33 and GO-induced cytotoxicity. In this study, we determined which phosphorylation state favors uptake of antibody-bound CD33, identified proteins that bind to CD33 in an ITIM-dependent manner, and assessed their importance for CD33 internalization by siRNA-based gene silencing. Methods: Internalization of anti-CD33 antibodies was measured by flow cytometry in the presence or absence of the tyrosine phosphatase inhibitor, pervanadate, in human CD33+ AML cell lines (ML-1, HL-60, NB4, U937, TF-1) and CD33− Jurkat T cells infected with wild-type and mutant CD33. Pull-down experiments were performed with glutathione S-transferase (GST) proteins fused to phosphorylated cytoplasmic tails of CD33, using human myeloid cell lysates. Co-immunoprecipitations were performed with myeloid cell lines expressing HA-tagged wild-type CD33. Lentivirus-based siRNA constructs were generated for gene silencing, and expressed in human CD33+ AML cell lines. Results: Pervanadate significantly increased uptake of anti-CD33 antibodies in human AML cell lines; this effect was dependent upon the integrity of the ITIMs and was prevented by co-treatment with the Src tyrosine kinase inhibitor PP2, suggesting that Src family kinase-dependent phosphorylation of the ITIMs critically controls uptake of antibody-bound CD33, possibly by altering which proteins binds to CD33 or by facilitating binding of adaptor-proteins required for endocytosis. We identified several proteins, including the tyrosine phophatases, SHP-1 and SHP-2, and the non-receptor tyrosine kinase, Syk, which bound to phosphorylated wild-type and mutant CD33 in a manner that paralleled the endocytic properties of the corresponding CD33 protein. Since these three proteins have been implicated in endocytic processes of other cell surface proteins, we assessed their role in uptake of antibody-bound CD33 by siRNA-mediated gene silencing. Simultaneous depletion of SHP-1 and SHP-2, but not SHP-1 or SHP-2 alone, significantly increased internalization of antibody-bound CD33 in the two AML cell lines with the highest cell surface expression of CD33, whereas no effect was seen in two other cell lines with lower CD33 expression levels. In contrast, depletion of Syk, whose expression has previously been correlated to the inhibitory effect of anti-CD33 antibodies on AML cell growth, failed to affect antibody internalization in the cell lines assessed. Conclusion: These studies indicate that the phosphorylation status of the ITIMs controls uptake of antibody-bound CD33. In line with this model, SHP-1 and SHP-2, which have been shown to dephosphorylate CD33 in vitro, can affect this endocytic process. Thus, our data imply manipulation of the phosphorylation state of CD33, e.g. by activating Src family kinases or interfering with phosphatases as a novel means to increase uptake of anti-CD33 antibody-based therapeutics such as GO. Finally, the variable effect of SHP-1 and SHP-2 depletion suggests that there are significant cell-type specific differences in the response to anti-CD33 antibody ligation, for example differences in tyrosine phosphorylation levels and/or activation/recruitment or redundancies of tyrosine phosphatases.

scholarly journals Brefeldin A inhibits the endocytosis of plasma-membrane-associated heparan sulphate proteoglycans of cultured rat ovarian granulosa cells

1995 ◽  
Vol 310 (1) ◽  
pp. 271-278 ◽  
Author(s):  
L Uhlin-Hansen ◽  
M Yanagishita
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Granulosa Cells ◽  
Protein Transport ◽  
Heparan Sulphate ◽  
Brefeldin A ◽  
Inhibitory Effect ◽  
Surface Proteins ◽  
Gel Chromatography ◽  
Ovarian Granulosa Cells

Rat ovarian granulosa cells were labelled with [35S]sulphate for 0.5-20 h and chased in the presence or absence of 1-2 micrograms/ml of brefeldin A (BFA) for up to 21 h. Heparan [35S]sulphate (HS) proteoglycans from the culture medium, plasma membrane and intracellular fractions were then analysed by gel chromatography. In the absence of BFA, about 85% of the plasma membrane-associated HS proteoglycans were endocytosed and subsequently degraded intracellularly. Recirculation of the HS proteoglycans between the intracellular pool and the cell surface was not observed. Exposing the cells to BFA for less than 1 h did not influence the turnover of the HS proteoglycans, whereas the effect of the drug on the Golgi functions reached a maximum in approx. 10 min. When the cells were treated with BFA for more than 1-2 h, the rate of endocytosis of HS proteoglycans was reduced to about 50% of the control. The delivery of endocytosed HS proteoglycans to lysosomes were not affected by the drug. Cycloheximide also reduced the endocytosis of HS proteoglycans, but not as much as BFA, indicating that the inhibitory effect of BFA can be only partly accounted for by a block of protein transport from the endoplasmic reticulum to the plasma membrane. In contrast with the endocytosis of HS proteoglycans, neither that of 125I-transferrin, known to be mediated by clathrin-coated vesicles, nor that of 125I-ricin, a marker molecule for bulk endocytosis, was affected by BFA. The half-life of 125I-transferrin and 125I-ricin in the plasma membrane was about 10 and 25 min respectively compared with about 5 h for the HS proteoglycans. Altogether, these results indicate that the endocytosis of plasma-membrane-associated HS proteoglycans is mediated by different mechanisms than the endocytosis of most other cell-surface proteins. Further, the mechanisms involved in the endocytosis of HS proteoglycans are sensitive to BFA.

scholarly journals Molecular mechanism of Fast Endophilin-Mediated Endocytosis

2020 ◽  
Vol 477 (12) ◽  
pp. 2327-2345 ◽  
Author(s):  
Alessandra Casamento ◽  
Emmanuel Boucrot
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Molecular Mechanism ◽  
Receptor Activation ◽  
Membrane Curvature ◽  
Surface Proteins ◽  
Resting Cells ◽  
Cellular Functions ◽  
Surface Receptors ◽  
Key Steps

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Clathrin-mediated endocytosis (CME) is the housekeeping pathway in resting cells but additional Clathrin-independent endocytic (CIE) routes, including Fast Endophilin-Mediated Endocytosis (FEME), internalize specific cargoes and support diverse cellular functions. FEME is part of the Dynamin-dependent subgroup of CIE pathways. Here, we review our current understanding of the molecular mechanism of FEME. Key steps are: (i) priming, (ii) cargo selection, (iii) membrane curvature and carrier formation, (iv) membrane scission and (v) cytosolic transport. All steps are controlled by regulatory mechanisms mediated by phosphoinositides and by kinases such as Src, LRRK2, Cdk5 and GSK3β. A key feature of FEME is that it is not constitutively active but triggered upon the stimulation of selected cell surface receptors by their ligands. In resting cells, there is a priming cycle that concentrates Endophilin into clusters on discrete locations of the plasma membrane. In the absence of receptor activation, the patches quickly abort and new cycles are initiated nearby, constantly priming the plasma membrane for FEME. Upon activation, receptors are swiftly sorted into pre-existing Endophilin clusters, which then bud to form FEME carriers within 10 s. We summarize the hallmarks of FEME and the techniques and assays required to identify it. Next, we review similarities and differences with other CIE pathways and proposed cargoes that may use FEME to enter cells. Finally, we submit pending questions and future milestones and discuss the exciting perspectives that targeting FEME may boost treatments against cancer and neurodegenerative diseases.

Alternative promoters regulate transcription of the gene that encodes stem cell surface protein AC133

Blood ◽  
2004 ◽  
Vol 103 (6) ◽  
pp. 2055-2061 ◽  
Author(s):  
Sergey V. Shmelkov ◽  
Lin Jun ◽  
Ryan St Clair ◽  
Deirdre McGarrigle ◽  
Christopher A. Derderian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Surface ◽  
Progenitor Cells ◽  
Cell Biology ◽  
Surface Proteins ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Alternative Promoters ◽  
Tissue Specific

Abstract AC133 is a member of a novel family of cell surface proteins with 5 transmembrane domains. The function of AC133 is unknown. Although AC133 mRNA is detected in different tissues, its expression in the hematopoietic system is restricted to CD34+ stem cells. AC133 is also expressed on stem cells of other tissues, including endothelial progenitor cells. However, despite the potential importance of AC133 to the field of stem cell biology, nothing is known about the transcriptional regulation of AC133 expression. In this report we showed that the human AC133 gene has at least 9 distinctive 5′–untranslated region (UTR) exons, resulting in the formation of at least 7 alternatively spliced 5′-UTR isoforms of AC133 mRNA, which are expressed in a tissue-dependent manner. We found that transcription of these AC133 isoforms is controlled by 5 alternative promoters, and we demonstrated their activity on AC133-expressing cell lines using a luciferase reporter system. We also showed that in vitro methylation of 2 of these AC133 promoters completely suppresses their activity, suggesting that methylation plays a role in their regulation. Identification of tissue-specific AC133 promoters may provide a novel method to isolate tissue-specific stem and progenitor cells.

scholarly journals Bovine CD18 Is Necessary and Sufficient To Mediate Mannheimia (Pasteurella) haemolytica Leukotoxin-Induced Cytolysis

2002 ◽  
Vol 70 (9) ◽  
pp. 5058-5064 ◽  
Author(s):  
M. S. Deshpande ◽  
T. C. Ambagala ◽  
A. P. N. Ambagala ◽  
M. E. Kehrli ◽  
S. Srikumaran
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Surface Proteins ◽  
Polymorphonuclear Neutrophils ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Dependent Manner ◽  
Β Subunit ◽  
Concentration Dependent Manner ◽  
Necessary And Sufficient

ABSTRACT Leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica is an RTX toxin which is specific for ruminant leukocytes. Lkt binds to β2 integrins on the surface of bovine leukocytes. β2 integrins have a common β subunit, CD18, that associates with three distinct α chains, CD11a, CD11b, and CD11c, to give rise to three different β2 integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (CR4), respectively. Our earlier studies revealed that Lkt binds to all three β2 integrins, suggesting that the common β subunit, CD18, may be the receptor for Lkt. In order to unequivocally elucidate the role of bovine CD18 as a receptor for Lkt, a murine cell line nonsusceptible to Lkt (P815) was transfected with cDNA for bovine CD18. One of the transfectants, 2B2, stably expressed bovine CD18 on the cell surface. The 2B2 transfectant was effectively lysed by Lkt in a concentration-dependent manner, whereas the P815 parent cells were not. Immunoprecipitation of cell surface proteins of 2B2 with monoclonal antibodies specific for bovine CD18 or murine CD11a suggested that bovine CD18 was expressed on the cell surface of 2B2 as a heterodimer with murine CD11a. Expression of bovine CD18 and the Lkt-induced cytotoxicity of 2B2 cells were compared with those of bovine polymorphonuclear neutrophils and lymphocytes. There was a strong correlation between cell surface expression of bovine CD18 and percent cytotoxicity induced by Lkt. These results indicate that bovine CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of target cells.

scholarly journals Cell-specific cargo delivery using synthetic bacterial spores

2020 ◽  
Author(s):  
Minsuk Kong ◽  
Taylor B. Updegrove ◽  
Maira Alves Constantino ◽  
Devorah L. Gallardo ◽  
I-Lin Wu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cancer Cells ◽  
Xenograft Model ◽  
Surface Proteins ◽  
Tumor Xenograft ◽  
Ovarian Cancer Cells ◽  
Target Cells ◽  
Mouse Tumor ◽  
Dependent Manner ◽  
Zebrafish Model

ABSTRACTSSHELs are synthetic bacterial spore-like particles wherein the spore’s cell surface is partially reconstituted around 1 µm-diameter silica beads coated with a lipid bilayer. Via a unique cysteine engineered in one of the surface proteins, the surface of SSHELs may be covalently decorated with molecules of interest. Here, we modified SSHELs with an affibody directed against HER2, a cell surface protein overexpressed in some breast and ovarian cancer cells, and loaded them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduced tumor growth with lower toxicity in a mouse tumor xenograft model compared to free drug by specifically binding to HER2-positive cancer cells. We show that SSHELs bound to target cells are taken up and trafficked to acidic compartments, whereupon the cargo is released in a pH-dependent manner. Finally, we demonstrate that SHELLs can clear small tumor lesions in a complex tumor microenvironment in a zebrafish model of brain metastasis. We propose that SSHELs represent a versatile strategy for targeted drug delivery.

scholarly journals Heterogeneity in VEGF Receptor-2 mobility and organization on the endothelial cell surface leads to diverse activation models by VEGF

2019 ◽  
Author(s):  
Bruno da Rocha-Azevedo ◽  
Sungsoo Lee ◽  
Aparajita Dasgupta ◽  
Anthony R. Vega ◽  
Luciana R. de Oliveira ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Molecule ◽  
Receptor Activation ◽  
Cell Surface Receptor ◽  
Vegf Receptor ◽  
Surface Receptors ◽  
Endothelial Cell Surface ◽  
Heterogeneous Nature ◽  
Surface Receptor ◽  
Complex Relationships

SummaryThe nanoscale organization of cell surface receptors plays an important role in signaling. We determined this organization and its relation to receptor activation for VEGF Receptor-2 (VEGFR-2), a critical receptor tyrosine kinase in endothelial cells (ECs), by combining live-cell single-molecule imaging of endogenous VEGFR-2 with rigorous computational analysis. We found that surface VEGFR-2 can be mobile or immobile/confined, and monomeric or non-monomeric, with a complex interplay between the two. The mobility and interaction heterogeneity of VEGFR-2 in the basal state led to heterogeneity in the sequence of steps leading to VEGFR-2 activation by VEGF. Specifically, we found that VEGF can bind to both monomeric and non-monomeric VEGFR-2, and, when binding to monomeric VEGFR-2, promotes dimer formation but only for immobile/confined receptors. Overall, our study highlights the dynamic and heterogeneous nature of cell surface receptor organization and its complex relationships with receptor activation and signaling.

scholarly journals The c-Met receptor: Implication for targeted therapies in colorectal cancer

Tumor Biology ◽  
2017 ◽  
Vol 39 (5) ◽  
pp. 101042831769911 ◽  
Author(s):  
Elmira Safaie Qamsari ◽  
Sepideh Safaei Ghaderi ◽  
Bahareh Zarei ◽  
Ruhollah Dorostkar ◽  
Salman Bagheri ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Surface ◽  
Tyrosine Kinase ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Tumor Formation ◽  
Surface Proteins ◽  
Tyrosine Kinase Receptor ◽  
Met Receptor ◽  
Surface Receptors

c-Met (mesenchymal–epithelial transition factor) is a tyrosine kinase receptor activated by hepatocyte growth factor and regulates multiple biological processes, such as cell scattering, survival, and proliferation. Aberrant c-Met signaling has been implicated in a variety of cancer types, including colorectal cancer. c-Met is genetically altered through various mechanisms that is associated with colorectal cancer progression and metastasis. Especially, in colorectal cancer, preclinical evidence for the aberrant activation of the c-Met signaling exists. Accordingly, molecular targeting of c-Met receptor could be a promising strategy, in the treatment of colorectal cancer patients. Recently, it was also shown that crosstalk between c-Met and other cell surface receptors attributes to tumorigenesis and development of therapeutic resistance. Characterization of the molecular mechanisms through which c-Met crosstalks with other receptors in favor of tumor formation and progression remains to explore. This review will describe the mechanisms of aberrant c-Met signaling in colorectal cancer and discuss on additional roles for c-Met receptor through crosstalk with other tyrosine kinase receptors and cell surface proteins in colorectal cancer. Novel therapeutic approaches for c-Met pathway targeting will also be discussed.

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