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.

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.

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 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 Trafficking of Kv1.4 potassium channels: interdependence of a pore region determinant and a cytoplasmic C-terminal VXXSL determinant in regulating cell-surface trafficking

2003 ◽  
Vol 375 (3) ◽  
pp. 761-768 ◽  
Author(s):  
Jing ZHU ◽  
Itaru WATANABE ◽  
Barbara GOMEZ ◽  
William B. THORNHILL
Keyword(s):  
Cell Surface ◽  
Cell Function ◽  
Cell Signalling ◽  
Surface Protein ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Cell Surface Expression ◽  
Pore Region ◽  
Expression Levels ◽  
Protein Levels

Kv1.4 and Kv1.1 potassium channel homomers have been shown to exhibit different intracellular trafficking programmes and cell-surface expression levels in cell lines: a determinant in the pore region of Kv1.4 and Kv1.1 [Zhu, Watanabe, Gomez and Thornhill (2001) J. Biol. Chem. 276, 39419–39427] and a cytoplasmic C-terminal VXXSL determinant on Kv1.4 [Li, Takimoto and Levitan (2000) J. Biol. Chem. 275, 11597–11602] have been described, which affected trafficking and cell-surface expression levels. In the present study, we examined whether trafficking pore determinants influenced any cytoplasmic C-terminal trafficking determinant. We found that removal of VXXSL from a Kv1.4 chimaera that contained the pore of Kv1.1 did not affect cell-surface trafficking. Therefore removal of the C-terminal VXXSL of Kv1.4 inhibited protein surface levels only in the presence of the Kv1.4 pore. In contrast, truncating the cytoplasmic C-terminus of Kv1.1 or truncating a Kv1.1 chimaera with the pore of Kv1.4, had little effect on surface protein levels. Furthermore, the subregion of the Kv1.4 pore trafficking determinant that was required for the inhibitory effect of VXXSL removal was mapped to a threonine residue in the deep pore region. Therefore the Kv1.4 pore determinant affected the trafficking and cell-surface levels directed by the C-terminal VXXSL determinant. Different Kv1 trafficking programmes would affect cell-surface expression levels either positively or negatively and also cell signalling. Cells may use differential trafficking programmes of membrane proteins as a post-translational mechanism to regulate surface protein levels and cell function.

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 CD33 SNP Genotype and Splice Variation Are Associated with CD33 Cell Surface Expression and SGN-CD33A Pharmacokinetics

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3942-3942
Author(s):  
Katherine Tarlock ◽  
Zixing Wang ◽  
Rory Rohm ◽  
Travis Biechele ◽  
Rhonda E. Ries ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Drug Exposure ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Set Domain ◽  
Employment Equity ◽  
Exon 2 ◽  
Splice Variation ◽  
Equity Ownership

Abstract The cell surface antigen CD33 is expressed on the majority of AML blasts and is appropriate for immunotherapeutic targeting with antibody drug conjugates (ADCs). Expression of CD33 is in part mediated by splicing of the CD33 transcript, and has been demonstrated to be one of the factors that may mediate response to the ADC gemtuzumab ozogamicin, which results in significant benefit in some patients but lacks responses in others. Splicing of the CD33 transcript is in part regulated by a single nucleotide polymorphism (SNP) in exon 2 (e2) that causes a C>T substitution and the resultant skipping of e2. CD33 thus exists in 2 main isoforms, as either a full length (FL) transcript or a truncated version missing e2 (Δe2), which includes the IgV binding domain that is the epitope for diagnostic and therapeutic antibodies (Ab). The CC genotype encodes the FL isoform at a higher rate compared to the CT or TT, and the TT genotype encodes the short isoform at a higher rate compared to CT or CC. SGN-CD33A is a CD33-directed ADC, utilizing a pyrolobenzodiazepine (PBD) dimer. SGN-CD33A has been evaluated in multiple clinical trials as either monotherapy or in combination. We hypothesized that the patient's CD33 genotype would impact CD33 expression as well as response characteristics following treatment with SGN-CD33A. We analyzed CD33 genotype variation in bone marrow (BM) or peripheral blood (PB) samples from patients treated with SGN-33A as either monotherapy (NCT01902329; n=133) or in combination with hypomethylating agents (HMAs; NCT02785900; n=83). CD33 SNP genotyping was determined on gDNA using RFLP PCR with 2 restriction enzymes recognizing cut sites generated by the C and T polymorphisms and genotype confirmed using fragment length analysis (CC=108, CT=86, TT=22). CD33 surface expression on the AML blasts was determined by flow cytometric analysis using the human anti-CD33 monoclonal Abs HIM3-4 and H212, which bind to the membrane-proximal C2-set and V-set domain, respectively. HIM-34 measured CD33 levels independent of SNP-driven splice variation. The h2H12 epitope is within e2, thus its binding may be susceptible to splice variation. We subsequently evaluated the association of CD33 genotype with pharmacokinetic (PK), clinical and other variables using a generalized regression model. Patients classified as CC genotype had significantly higher surface CD33 expression as determined by flow cytometry in both BM and PB. In accordance with observed differences in CD33 expression, we also found drug exposure demonstrated an inverse relationship according to CC genotype in both mono and combination therapy trials. For monotherapy, compared to patients with CC and CT genotypes, patients with TT genotypes had significantly higher drug exposure following SGN-CD33A. Patients with TT had higher AUCs following the first and last doses of SGN-33A (p < 10-4 -; Fig 1). Cmax following SGN-CD33A exposure was higher in TT genotype patients compared to the CT and CC (p< 10-1.5 for Cmax following the first dose and p<10-1.6 for Cmax over all treatments;Fig 1). In combination with HMAs, the TT genotype was also associated with significantly higher SGN-CD33A AUC and Cmax (p-values ranging from 10-3.3 - 10-9.7). We next examined expression and subcellular localization of CD33 to elucidate the mechanism by CD33 variation contributes to cell surface presentation. Transfection of cDNA encoding the FL CD33 transcript resulted in increased cell surface expression, as indicated by flow cytometry with both HIM3-4 and h2H12. In contrast, both Abs failed to detect cell surface CD33 following transfection with cDNA encoding the Δe2 variant. Examination of the intracellular compartment revealed that HIM3-4, but not 2H12, binds to the Δe2 variant in a pattern localized proximal to the nucleus. Taken together, our findings suggest that the Δe2 splice CD33 variant lacks the portion of the V-set domain required for h2H12/SGN-CD33A binding and does not efficiently traffic to the cell surface. We show that CD33 SNP genotype is associated with CD33 expression, with CC patients demonstrating higher CD33 as detected by flow cytometry; and that CD33 SNP genotype affects the PK profile of SGN-CD33A, with TT patients having higher levels of drug exposure. Our findings suggest that the CD33 genotype can impact CD33 expression, PK profile, and trafficking of bound agents and thus may impact therapeutic targeting of CD33-directed agents. Disclosures Wang: Seattle Genetics: Employment, Equity Ownership. Rohm:Seattle Genetics: Employment, Equity Ownership. Biechele:Seattle Genetics: Employment, Equity Ownership. Means:Seattle Genetics: Employment, Equity Ownership. Thurman:Seattle Genetics: Employment, Equity Ownership. Arthur:Seattle Genetics: Employment, Equity Ownership.

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 Cell-surface expression of PrPC and the presence of scrapie prions in the blood of goats

2012 ◽  
Vol 93 (5) ◽  
pp. 1127-1131 ◽  
Author(s):  
Rohana P. Dassanayake ◽  
David A. Schneider ◽  
Lynn M. Herrmann-Hoesing ◽  
Thomas C. Truscott ◽  
William C. Davis ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Whole Blood ◽  
Surface Expression ◽  
Infected Sheep ◽  
Cell Surface Expression ◽  
Prion Infectivity ◽  
Infected Goat ◽  
Scrapie Infection ◽  
Relative Differences

Although host-encoded prion protein (PrPC) expression in ovine PBMCs and prion infectivity in scrapie-infected sheep blood have been demonstrated, such studies have not been reported in goats. Therefore, this study characterized cell-surface expression of PrPC on PBMC subsets derived from normal goats and sheep, by flow cytometry, and determined prion infectivity in blood from a scrapie-infected goat using a transfusion bioassay in goat kids. Cell-surface PrPC expression was detected on all subsets of goat PBMCs. The highest PrPC cell-surface expression was found in CD2+ T lymphocytes in goats. Transmission of infection was detected in all three recipients who received whole blood from a goat with classical scrapie. It was concluded that caprine PBMCs express PrPC similarly to sheep but with relative differences among PBMCs subsets, and that blood-borne infectious prions can be detected in scrapie-infected goats. Thus, similar to sheep, goat blood may be a suitable diagnostic target for the detection of scrapie infection.

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