scholarly journals Syndecan-1 Promotes Hepatocyte-Like Differentiation of Hepatoma Cells Targeting Ets-1 and AP-1

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1356
Author(s):  
Péter Hollósi ◽  
Lóránd Váncza ◽  
Katalin Karászi ◽  
Katalin Dobos ◽  
Bálint Péterfia ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Molecular Mechanisms ◽  
Signal Transduction Pathway ◽  
Normal Liver ◽  
Surface Expression ◽  
Hepatoma Cells ◽  
Full Length ◽  
Cell Surface Expression ◽  
Gene Assay

Syndecan-1 is a transmembrane heparan sulfate proteoglycan which is indispensable in the structural and functional integrity of epithelia. Normal hepatocytes display strong cell surface expression of syndecan-1; however, upon malignant transformation, they may lose it from their cell surfaces. In this study, we demonstrate that re-expression of full-length or ectodomain-deleted syndecan-1 in hepatocellular carcinoma cells downregulates phosphorylation of ERK1/2 and p38, with the truncated form exerting an even stronger effect than the full-length protein. Furthermore, overexpression of syndecan-1 in hepatoma cells is associated with a shift of heparan sulfate structure toward a highly sulfated type specific for normal liver. As a result, cell proliferation and proteolytic shedding of syndecan-1 from the cell surface are restrained, which facilitates redifferentiation of hepatoma cells to a more hepatocyte-like phenotype. Our results highlight the importance of syndecan-1 in the formation and maintenance of differentiated epithelial characteristics in hepatocytes partly via the HGF/ERK/Ets-1 signal transduction pathway. Downregulation of Ets-1 expression alone, however, was not sufficient to replicate the phenotype of syndecan-1 overexpressing cells, indicating the need for additional molecular mechanisms. Accordingly, a reporter gene assay revealed the inhibition of Ets-1 as well as AP-1 transcription factor-induced promoter activation, presumably an effect of the heparan sulfate switch.

scholarly journals Physical dissociation of the TCR-CD3 complex accompanies receptor ligation.

1995 ◽  
Vol 182 (6) ◽  
pp. 1997-2006 ◽  
Author(s):  
H Kishimoto ◽  
R T Kubo ◽  
H Yorifuji ◽  
T Nakayama ◽  
Y Asano ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Signal Transduction Pathway ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Levels ◽  
Before And After ◽  
Physical Dissociation ◽  
Receptor Ligation

Recent studies indicate that there may be functional uncoupling of the TCR-CD3 complex and suggest that the TCR-CD3 complex is composed of two parallel signal-transducing units, one made of gamma delta epsilon chains and the other of zeta chains. To elucidate the molecular mechanisms that may explain the functional uncoupling of TCR and CD3, we have analyzed their expression by using flow cytometry as well as immunochemical means both before and after stimulation with anti-TCR-beta, anti-CD3 epsilon, anti-CD2, staphylococcal enterotoxin B, and ionomycin. We present evidence that TCR physically dissociates from CD3 after stimulation of the TCR-CD3 complex. Stimulation with anti-CD3 resulted in down-modulation of TCR within 45 min whereas CD3 epsilon was still expressed on the cell surface as detected by flow cytometry. However, the cell surface expression of TCR and CD3 was not affected when cells were stimulated with anti-TCR-beta under the same conditions. In the case of anti-CD3 treatment of T cells, the TCR down-modulation appeared to be due to the internalization of TCR, as determined by immunoelectron microscopy. Immunochemical analysis of cells after stimulation with either anti-TCR or anti-CD3 mAbs revealed that the overall protein levels of TCR and CD3 were similar. More interestingly, the dissociation of the TCR-CD3 complex was observed with both treatments and occurred in a manner that the TCR and the associated TCR-zeta chain dissociated as a unit from CD3. These results provide the first report of physical dissociation of TCR and CD3 after stimulation through the TCR-CD3 complex. The results also suggest that the signal transduction pathway triggered by TCR may differ from that induced by CD3.

The NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform

2001 ◽  
Vol 114 (6) ◽  
pp. 1101-1113
Author(s):  
M. Gawaz ◽  
F. Besta ◽  
J. Ylanne ◽  
T. Knorr ◽  
H. Dierks ◽  
...  
Keyword(s):  
Steady State ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Chinese Hamster Ovary Cells ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Cell Surface Expression ◽  
Beta Chain ◽  
Single Chain ◽  
Beta3 Integrin

Beta3 integrin adhesion molecules play important roles in wound repair and the regulation of vascular development and three beta3 integrin isoforms (beta3-A, -B, -C) have been described so far. Surface expression of beta3 integrins is dynamically regulated through internalization of beta3 integrins, however, the molecular mechanisms are understood incompletely. To evaluate the role of the cytoplasmic domain of beta3 integrins for internalization, we have generated single chain chimeras with variant and mutated forms of beta3 cytoplasmic domains. Upon transient transfection into chinese hamster ovary cells, it was found that the beta3-A chimera had strongly reduced cell surface expression compared with the corresponding beta3-B, or beta3-C fusion proteins, or the tail-less constructs, whereas steady state levels of all chimeras were near identical. Studies employing cytoplasmic domain mutants showed that the NITY motif at beta3-A 756–759 is critical for plasma membrane expression of beta3-A. Furthermore, delivery of beta3-A to the cell surface was specifically modulated by the cytoplasmic protein beta3-endonexin, a previously described intracellular protein. Coexpression of the native, long form of beta3-endonexin, which does not interact with the beta3 tail, acted as a dominant negative inhibitor of beta3-A-internalization and enhanced steady-state surface expression of the beta3-A-chimera. Furthermore, anti-beta3 antibody-induced internalization of the native beta3 integrin (alpha(IIb)beta3 was dramatically reduced for the Tyr(759)-Ala substitution mutant (alpha(IIb)beta3) (Y759A) and expression of the long isoform of beta3-endonexin substantially decreased the internalization of wild-type alpha(IIb)beta3. Thus, the NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform and beta3-endonexin appears to couple the beta3-A isoform to a specific receptor-recycling pathway.

scholarly journals Differential Expression and Functional Characterization of Luteinizing Hormone Receptor Splice Variants in Human Luteal Cells: Implications for Luteolysis

Endocrinology ◽  
2009 ◽  
Vol 150 (6) ◽  
pp. 2873-2881 ◽  
Author(s):  
Rachel E. Dickinson ◽  
Alan J. Stewart ◽  
Michelle Myers ◽  
Robert P. Millar ◽  
W. Colin Duncan
Keyword(s):  
Cell Surface ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Splice Variants ◽  
Primary Cultures ◽  
Surface Expression ◽  
Full Length ◽  
Luteinizing Hormone Receptor ◽  
Cell Surface Expression ◽  
Truncated Form

The human LH receptor (LHR) plays a key role in luteal function and the establishment of pregnancy through its interaction with the gonadotropins LH and human chorionic gonadotropin. We previously identified four splice variants of the LHR in human luteinized granulosa cells (LGCs) and corpora lutea (CL). Real-time quantitative PCR revealed that expression of the full-length LHR (LHRa) and the most truncated form (LHRd) changed significantly in CL harvested at different stages of the ovarian cycle (P < 0.01, ANOVA). LHRa expression was reduced in the late luteal CL (P < 0.05). Conversely, an increase in LHRd expression was observed in the late luteal CL (P < 0.01). Chronic manipulation of human chorionic gonadotropin in LGC primary cultures supported the in vivo findings. LHRd encodes a protein lacking the transmembrane and carboxyl terminal domains. COS-7 cells expressing LHRd were unable to produce cAMP in response to LH stimulation. COS-7 cells coexpressing LHRd and LHRa also failed to generate cAMP in response to LH, suggesting that this truncated form has a negative effect on the signaling of LHRa. Immunofluorescence staining of LGC and COS-7 cells implied that there is a reduction in cell surface expression of LHRa when LHRd is present. Overall, these results imply expression of LHR splice variants is regulated in the human CL. Furthermore, during functional luteolysis a truncated variant could modulate the cell surface expression and activity of full-length LHR.

scholarly journals GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

2016 ◽  
Vol 36 (7) ◽  
pp. 1152-1163 ◽  
Author(s):  
Maoxiang Zhang ◽  
Jason E. Davis ◽  
Chunman Li ◽  
Jie Gao ◽  
Wei Huang ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Loop ◽  
Interaction Sites ◽  
G Protein Coupled

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at thetrans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.

scholarly journals High throughput Characterization of KCNB1 variants Associated with Developmental and Epileptic Encephalopathy

2019 ◽  
Author(s):  
Seok Kyu Kang ◽  
Carlos G. Vanoye ◽  
Sunita N. Misra ◽  
Dennis M. Echevarria ◽  
Jeffrey D. Calhoun ◽  
...  
Keyword(s):  
Cell Surface ◽  
High Throughput ◽  
Molecular Mechanisms ◽  
Ion Selectivity ◽  
Surface Expression ◽  
Rapid Screening ◽  
Cell Surface Expression ◽  
Voltage Sensitivity ◽  
Pathogenic Variants ◽  
Functional Defects

ABSTRACTPathogenic variants in KCNB1, encoding the voltage-gated potassium channel Kv2.1, are associated with developmental and epileptic encephalopathies (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. We evaluated a series of 17 KCNB1 variants associated with DEE or neurodevelopmental disorder (NDD) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant Kv2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage-dependence of activation and/or inactivation, as homotetramers or when co-expressed with wild-type Kv2.1. Quantification of protein expression also identified variants with reduced total Kv2.1 expression or deficient cell-surface expression.Our study establishes a platform for rapid screening of functional defects of KCNB1 variants associated with DEE and other NDDs, which will aid in establishing KCNB1 variant pathogenicity and may enable discovery of targeted strategies for therapeutic intervention based on molecular phenotype.

scholarly journals The Majority of Adrenocorticotropin Receptor (Melanocortin 2 Receptor) Mutations Found in Familial Glucocorticoid Deficiency Type 1 Lead to Defective Trafficking of the Receptor to the Cell Surface

2008 ◽  
Vol 93 (12) ◽  
pp. 4948-4954 ◽  
Author(s):  
T. T. Chung ◽  
T. R. Webb ◽  
L. F. Chan ◽  
S. N. Cooray ◽  
L. A. Metherell ◽  
...  
Keyword(s):  
Cell Surface ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
A Cell ◽  
Glucocorticoid Deficiency ◽  
Deficiency Type

Context: There are at least 24 missense, nonconservative mutations found in the ACTH receptor [melanocortin 2 receptor (MC2R)] that have been associated with the autosomal recessive disease familial glucocorticoid deficiency (FGD) type 1. The characterization of these mutations has been hindered by difficulties in establishing a functional heterologous cell transfection system for MC2R. Recently, the melanocortin 2 receptor accessory protein (MRAP) was identified as essential for the trafficking of MC2R to the cell surface; therefore, a functional characterization of MC2R mutations is now possible. Objective: Our objective was to elucidate the molecular mechanisms responsible for defective MC2R function in FGD. Methods: Stable cell lines expressing human MRAPα were established and transiently transfected with wild-type or mutant MC2R. Functional characterization of mutant MC2R was performed using a cell surface expression assay, a cAMP reporter assay, confocal microscopy, and coimmunoprecipitation of MRAPα. Results: Two thirds of all MC2R mutations had a significant reduction in cell surface trafficking, even though MRAPα interacted with all mutants. Analysis of those mutant receptors that reached the cell surface indicated that four of six failed to signal, after stimulation with ACTH. Conclusion: The majority of MC2R mutations found in FGD fail to function because they fail to traffic to the cell surface.

scholarly journals Mechanisms of Melanocortin-2 Receptor (MC2R) Internalization and Recycling in Human Embryonic Kidney (HEK) Cells: Identification of Key Ser/Thr (S/T) Amino Acids

2011 ◽  
Vol 25 (11) ◽  
pp. 1961-1977 ◽  
Author(s):  
Simon Roy ◽  
Sébastien Jean Roy ◽  
Sandra Pinard ◽  
Louis-Daniel Taillefer ◽  
Mohamed Rached ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Surface Expression ◽  
Receptor Internalization ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Camp Accumulation ◽  
Human Embryonic Kidney ◽  
And Function

Abstract ACTH is the most important stimulus of the adrenal cortex. The precise molecular mechanisms underlying the ACTH response are not yet clarified. The functional ACTH receptor includes melanocortin-2 receptor (MC2R) and MC2R accessory proteins (MRAP). In human embryonic kidney 293/Flp recombinase target cells expressing MC2R, MRAP1 isoforms, and MRAP2, we found that ACTH induced a concentration-dependent and arrestin-, clathrin-, and dynamin-dependent MC2R/MRAP1 internalization, followed by intracellular colocalization with Rab (Ras-like small guanosine triphosphate enzyme)4-, Rab5-, and Rab11-positive recycling endosomes. Preincubation of cells with monensin and brefeldin A revealed that 28% of the internalized receptors were recycled back to the plasma membrane and participated in total accumulation of cAMP. Moreover, certain intracellular Ser and Thr (S/T) residues of MC2R were found to play important roles not only in plasma membrane targeting and function but also in promoting receptor internalization. The S/T residues T131, S140, T204, and S280 were involved in MRAP1-independent cell-surface MC2R expression. Other mutants (S140A, S208A, and S202D) had lower cell-surface expressions in absence of MRAPβ. In addition, T143A and T147D drastically impaired cell-surface expression and function, whereas T131A, T131D, and S280D abrogated MC2R internalization. Thus, the modification of MC2R intracellular S/T residues may positively or negatively regulate its plasma membrane expression and the capacity of ACTH to induce cAMP accumulation. Mutations of T131, T143, T147, and S280 into either A or D had major repercussions on cell-surface expression, cAMP accumulation, and/or internalization parameters, pointing mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation.

scholarly journals Subunit Interactions Influence TSHR Multimerization

2010 ◽  
Vol 24 (10) ◽  
pp. 2009-2018 ◽  
Author(s):  
Rauf Latif ◽  
Krzysztof Michalek ◽  
Terry F. Davies
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Surface ◽  
Hormone Receptors ◽  
Fluorescent Protein ◽  
Surface Expression ◽  
Full Length ◽  
Cell Surface Expression ◽  
Receptor Interaction ◽  
Glycoprotein Hormone ◽  
Green Fluorescent

Abstract The TSH receptor (TSHR) is the key molecule influencing thyroid growth and development and is an antigenic target in autoimmune thyroid disease. The TSHR exists in monomeric and multimeric forms, and it has been shown previously that multimeric complexes of the TSHR preferentially localize in lipid rafts. However, unlike other glycoprotein hormone receptors, the TSHR exists in several forms on the cell membrane due to intramolecular cleavage of its ectodomain, which causes the production of α- and β-subunits of various lengths. After cleavage and reduction of disulfide bonds, α-subunits consisting of the receptor ectodomain may be lost from the cell surface by receptor shedding, leading to accumulation of excess β-subunits within the membrane. Because cell surface expression of these various forms of the TSHR is critical to receptor signaling and autoimmune responses, we set out to model the influence of β-subunits on full-length TSHRs. To study this interaction, we generated three truncated ectodomain β-subunits linked to green fluorescent protein (named β-316, -366, and -409) as examples of native cleaved forms of the TSHR. These constructs were transfected into human embryonic kidney 293 cells in the presence and absence of the full-length receptor. Whereas the β-316 and β-366 forms showed cell surface expression, the expression of β-409 was primarily intracellular. Cotransfection of the β-subunits with a full-length hemagglutinin-tagged wild-type (WT) receptor (HT-WT-TSHR) in both transient and stable systems caused a significant decrease in surface expression of the full-length WT receptors. This decrease was not seen with control plasmid consisting of a plasma membrane-targeted protein tagged to red fluorescent protein. To ascertain if this response was due to homointeraction of the truncated β-constructs with the WT-TSHRs, we immunoprecipitated membranes prepared from the cotransfected cells using antihemagglutinin and then probed with anti-green fluorescent protein. These studies confirmed dimerization of the β-subunits with the WT full-length receptor, and this interaction was further observed in vivo by fluorescence resonance energy transfer. We then studied the functional consequences of this interaction on TSHR signaling by examining Gαs-mediated signals. The well-expressed truncated constructs, when coexpressed with full-length TSHR, did not alter constitutive cAMP levels, but there was a significant decrease in TSH-induced cAMP generation. Furthermore, we observed that truncated β-316 and β-366 had faster internalization rate, which may lead to a significant decrease in the expression of the full-length receptor on the cell surface, thus contributing to the decreased signaling response. However, the decrease in surface receptors may also be due to inhibition of newly formed receptors reaching the surface as result of receptor-receptor interaction. It is well known that under normal physiological conditions both cleaved and uncleaved TSHR forms coexist on the cell surface of normal thyrocytes. Our studies allow us to conclude, therefore, that multimerization of cleaved/ truncated forms of the β-subunits with the full-length TSHR has a profound influence on TSHR internalization and signaling. Hence, the degree of intramolecular cleavage must also modulate TSHR signaling.

Mechanisms That Regulate the Function of the Selectins and Their Ligands

1999 ◽  
Vol 79 (1) ◽  
pp. 181-213 ◽  
Author(s):  
DIETMAR VESTWEBER ◽  
JAMES E. BLANKS
Keyword(s):  
Cell Surface ◽  
Molecular Mechanisms ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cell Contact ◽  
Endothelial Cell Surface ◽  
And Function ◽  
Blocking Antibodies ◽  
Selectin Binding

Vestweber, Dietmar, and James E. Blanks. Mechanisms That Regulate the Function of the Selectins and Their Ligands. Physiol. Rev. 79: 181–213, 1999. — Selectins are a family of three cell adhesion molecules (L-, E-, and P-selectin) specialized in capturing leukocytes from the bloodstream to the blood vessel wall. This initial cell contact is followed by the selectin-mediated rolling of leukocytes on the endothelial cell surface. This represents the first step in a cascade of molecular interactions that lead to leukocyte extravasation, enabling the processes of lymphocyte recirculation and leukocyte migration into inflamed tissue. The central importance of the selectins in these processes has been well documented in vivo by the use of adhesion-blocking antibodies as well as by studies on selectin gene-deficient mice. This review focuses on the molecular mechanisms that regulate expression and function(s) of the selectins and their ligands. Cell-surface expression of the selectins is regulated by a variety of different mechanisms. The selectins bind to carbohydrate structures on glycoproteins, glycolipids, and proteoglycans. Glycoproteins are the most likely candidates for physiologically relevant ligands. Only a few glycoproteins are appropriately glycosylated to allow strong binding to the selectins. Recently, more knowledge about the structure and the regulated expression of some of the carbohydrates on these ligands necessary for selectin binding has been accumulated. For at least one of these ligands, the physiological function is now well established. A novel and exciting aspect is the signaling function of the selectins and their ligands. Especially in the last two years, convincing data have been published supporting the idea that selectins and glycoprotein ligands of the selectins participate in the activation of leukocyte integrins.

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