scholarly journals Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis

2011 ◽  
Vol 438 (2) ◽  
pp. 359-367 ◽  
Author(s):  
Andrew Botham ◽  
Xiaodan Guo ◽  
Yu Pei Xiao ◽  
Alyn H. Morice ◽  
Steven J. Compton ◽  
...  
Keyword(s):  
Cell Line ◽  
Mutant Cell ◽  
Calcium Signalling ◽  
Surface Expression ◽  
Cysteine Residue ◽  
Receptor Internalization ◽  
Cell Surface Expression ◽  
Extracellular Signal ◽  
Proteinase Activated Receptors ◽  
Kinase Phosphorylation

hPAR2 (human proteinase-activated receptor-2) is a member of the novel family of proteolytically activated GPCRs (G-protein-coupled receptors) termed PARs (proteinase-activated receptors). Previous pharmacological studies have found that activation of hPAR2 by mast cell tryptase can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR2 that can regulate function, we have explored the functional role of the intracellular cysteine residue Cys361. We have demonstrated, using autoradiography, that Cys361 is the primary palmitoylation site of hPAR2. The hPAR2C361A mutant cell line displayed greater cell-surface expression compared with the wt (wild-type)-hPAR2-expressing cell line. hPAR2C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV. In stark contrast, hPAR2C361A triggered greater and more prolonged ERK (extracellular-signal-regulated kinase) phosphorylation compared with that of wt-hPAR2 possibly through Gi, since pertussis toxin inhibited the ability of this receptor to activate ERK. Finally, flow cytometry was utilized to assess the rate and extent of receptor internalization following agonist challenge. hPAR2C361A displayed faster internalization kinetics following trypsin activation compared with wt-hPAR2, whereas SLIGKV had a negligible effect on internalization for either receptor. In conclusion, palmitoylation plays an important role in the regulation of PAR2 expression, agonist sensitivity, desensitization and internalization.

scholarly journals Glycosylation of human proteinase-activated receptor-2 (hPAR2): role in cell surface expression and signalling

2002 ◽  
Vol 368 (2) ◽  
pp. 495-505 ◽  
Author(s):  
Steven J. COMPTON ◽  
Sabrina SANDHU ◽  
Suranga J. WIJESURIYA ◽  
Morley D. HOLLENBERG
Keyword(s):  
Cell Surface ◽  
Western Blot ◽  
Molecular Mass ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mutant Cell ◽  
Surface Expression ◽  
Receptor Expression ◽  
Cell Surface Expression

We have analysed the role of N-linked glycosylation in regulating human proteinase-activated receptor-2 (hPAR2) expression and function. Epitope-tagged wild-type hPAR2 (wt-hPAR2) or hPAR2 that lacked glycosylation sequons (following site-directed mutagenesis) in either the N-terminus [hPAR2N30A (Asn30→Ala)], extracellular loop 2 [ECL2; hPAR2N222Q (Asn222→Gln) or hPAR2N222A (Asn222→Ala)] or both (hPAR2N30A,N222A or hPAR2N30A,N222Q) were expressed in the Chinese-hamster ovary (CHO) fibroblast cell line, Pro5. Western blot analysis of wt-hPAR2 showed mature wt-hPAR2 to have a molecular mass of 55—100kDa, and 33—48kDa following N-glycosidase F deglycosylation. FACS analysis and immunocytochemistry of the wt-hPAR2 and PAR2 mutant cell lines revealed that removal of both glycosylation sequons decreases (50% of wt-hPAR2) cell surface expression. Western blot analysis indicated that both N-linked sites are glycosylated. In functional studies, hPAR2N30A displayed a selective and significant increase in sensitivity towards tryptase. Interestingly, hPAR2N222A displayed a loss in sensitivity towards all PAR2 agonists tested. However, further analysis revealed receptor sensitivity to alanine mutations in this domain, as the more conservative substitution hPAR2N222Q displayed no change in response to PAR2 agonists. hPAR2N30A,N222Q displayed increased sensitivity towards tryptase, but a loss in sensitivity towards trypsin and the synthetic peptide SLIGRL-NH2, although this loss in sensitivity towards trypsin and SLIGRL-NH2 was secondary to changes in cell-surface expression. Finally, expression of sialic-acid-deficient wt-hPAR2 in the CHO Lec2 glycosylation-deficient mutant cell line, showed a 40kDa loss in molecular mass, in addition to a marked and selective increase in sensitivity towards tryptase. We conclude that hPAR2 N-linked glycosylation and sialylation regulates receptor expression and/or signalling.

scholarly journals Cell surface expression of murine, rat, and human Fc receptors by Xenopus oocytes.

1984 ◽  
Vol 160 (2) ◽  
pp. 606-611 ◽  
Author(s):  
E Pure ◽  
A D Luster ◽  
J C Unkeless
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Surface Expression ◽  
Gamma Interferon ◽  
Membrane Receptors ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
High Avidity ◽  
U937 Cells ◽  
Mouse Igg2a

We report that Xenopus laevis oocytes can efficiently translate and insert heterologous membrane receptors into the oocyte plasma membrane, where they can be detected by the binding of either monoclonal antibodies or ligands. Thus, oocytes injected with mRNA from the mouse J774 macrophage-like cell line, the rat RBL-1 basophilic leukemia, and the U937 promonocyte cell line, bound 2.4G2 Fab, rat IgE, and mouse IgG2a, respectively. The increase in the high avidity Fc gamma R observed after gamma-interferon induction of U937 cells was also observed after injection of mRNA from gamma-interferon-induced U937 cells into oocytes. This suggests either much greater message stability or a greater rate of transcription of Fc gamma Rhi mRNA in the gamma-interferon-induced cells. The assay affords a sensitive method for the detection of rare mRNA species that code for plasma membrane proteins.

Assessing factors for predictive response to ADC targets in clinical tissue for companion diagnostic development.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22187-e22187
Author(s):  
Steven James Potts ◽  
Joseph Krueger ◽  
Holger Lange ◽  
David Eberhard ◽  
George David Young
Keyword(s):  
Tumor Microenvironment ◽  
Cell Surface ◽  
Surface Expression ◽  
Receptor Internalization ◽  
Critical Parameters ◽  
Cell Surface Expression ◽  
Cell Surface Antigens ◽  
Drug Conjugates ◽  
Coefficients Of Variation

e22187 Background: One premise of antibody-drug conjugates (ADC) is that the bound mAb-antigen complex on the cell surface will internalize and be metabolized by lysosomal proteases to release the free drug. Thus, the efficacy of an ADC is dependent not only on the presence of cell surface antigens, but also intact delivery of the conjugated drug. In most cases, the biological mechanisms behind these processes have not been elucidated. Furthermore, the extracellular stability of the ADC may be affected by the activity of the target proteases in the tumor microenvironment outside of the lysozome. These concepts are especially important for CDx approaches, which would ideally account not only for the degree of cell surface expression of the target, but also receptor internalization and potential effects of tumor microenvironment. Although in vitro approaches exist to measure these attributes, there are no clinically amenable approaches to measure these critical parameters. Methods: Flagship Biosciences has invented several proprietary approaches for measuring critical properties of the therapeutic target on the cell surface or inside the cell, as well as properties of the TME which could be used to predict efficacy to an ADC using FFPE biopsies. These image analysis approaches have been designed specifically in context of ADC CDx programs to: 1) Accurately define cell surface target expression independent of cytoplasmic expression; 2) Assess critical factors in the TME which may affect delivery of the drug to the intracellular target; and 3) Multiplex these evaluations for an integrative answer to be derived from a typical clinical biopsy. Results: The measurement of cytoplasm/membrane localization was evaluated on several hundred tissue sections, and the methodology was found to be highly reproducible, with coefficients of variation lower than that observed by manual pathologist accessment. Conclusions: Our approach discretely measures endpoints of cell surface biomarker prevalence, biomarker membrane/cytoplasmic ratio, heterogeneity, stromal contribution, inflammatory environment, and other important cell-by-cell outputs from a single FFPE slide in the context of typical drug discovery.

Developmental acquisition of T3-sensitive Na-K-ATPase stimulation by rat alveolar epithelial cells

2007 ◽  
Vol 292 (1) ◽  
pp. L6-L14 ◽  
Author(s):  
Jianxun Lei ◽  
Christine H. Wendt ◽  
Daosheng Fan ◽  
Cary N. Mariash ◽  
David H. Ingbar
Keyword(s):  
Atpase Activity ◽  
Cell Line ◽  
Alveolar Epithelial Cell ◽  
Developmental Regulation ◽  
Fluid Secretion ◽  
Surface Expression ◽  
Alveolar Epithelial Cells ◽  
Cell Surface Expression ◽  
Alveolar Epithelial ◽  
Parallel Increase

Late in gestation, the developing air space epithelium switches from chloride and fluid secretion to sodium and fluid absorption. Absorption requires Na-K-ATPase acting in combination with apical sodium entry mechanisms. Hypothyroidism inhibits perinatal fluid resorption, and thyroid hormone [triiodothyronine (T3)] stimulates adult alveolar epithelial cell (AEC) Na-K-ATPase. This study explored the developmental regulation of Na-K-ATPase by T3 in fetal rat distal lung epithelial (FDLE) cells. T3 increased Na-K-ATPase activity in primary FDLE cells from gestational day 19 [both primary FDLE cells at embryonic day 19 (E19) and the cell line FD19 derived from FDLE cells at E19]. However, T3 did not increase the Na-K-ATPase activity in less mature FDLE cells, including primary E17 and E18 FDLE cells and the cell line FD18 (derived from FDLE cells at E18). Subsequent experiments assessed the T3 signal pathway to define whether it was similar in the late FDLE and adult AEC and to determine the site of the switch in responsiveness to T3. As in adult AEC, in the FD19 cell line, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin blocked the T3-induced increase in Na-K-ATPase activity and plasma membrane quantity. T3 caused a parallel increase in phosphorylation of Akt at Ser473 in FDLE cells from E19, but not from E17 or E18. In the FD18 cell line, transient expression of a constitutively active mutant of the PI3K catalytic p110 subunit significantly augmented the Na-K-ATPase activity and the cell surface expression of Na-K-ATPase α1 protein. In conclusion, FDLE cells from E17 and E18 lacked T3-sensitive Na-K-ATPase activity but acquired this response at E19. The developmental stimulation of Na-K-ATPase by T3 in rat FDLE cells requires activation of PI3K, and the acquisition of T3 responsiveness may be at PI3K or upstream in the signaling pathway.

scholarly journals Establishment and characterization of a chordoma cell line from the tissue of a patient with dedifferentiated-type chordoma

2016 ◽  
Vol 25 (5) ◽  
pp. 626-635 ◽  
Author(s):  
Jeong-Yub Kim ◽  
Jongsun Lee ◽  
Jae-Soo Koh ◽  
Myung-Jin Park ◽  
Ung-Kyu Chang
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Nude Mice ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wide Resection ◽  
Cell Cycle Distribution ◽  
Facs Analysis ◽  
Mesenchymal Transition

OBJECTIVE Chordoma is a rare bone tumor of the axial skeleton believed to originate from the remnants of the embryonic notochord. The available tumor cells are characteristically physaliferous and express brachyury, a transcription factor critical for mesoderm specification. Although chordomas are histologically not malignant, treatments remain challenging because they are resistant to radiation therapy and because wide resection is impossible in most cases. Therefore, a better understanding of the biology of chordomas using established cell lines may lead to the advancement of effective treatment strategies. The authors undertook a study to obtain this insight. METHODS Chordoma cells were isolated from the tissue of a patient with dedifferentiated-type chordoma (DTC) that had recurred. Cells were cultured with DMEM/F12 containing 10% fetal bovine serum and antibiotics (penicillin and streptomycin). Cell proliferation rate was measured by MTS assay. Cell-cycle distribution and cell surface expression of proteins were analyzed by fluorescence-activated cell sorting (FACS) analysis. Expression of proteins was analyzed by Western blot and immunocytochemistry. Radiation resistance was measured by clonogenic survival assay. Tumor formation was examined by injection of chordoma cells at hindlimb of nude mice. RESULTS The putative (DTC) cells were polygonal and did not have the conventional physaliferous characteristic seen in the U-CH1 cell line. The DTC cells exhibited similar growth rate and cell-cycle distribution, but they exhibited higher clonogenic activity in soft agar than U-CH1 cells. The DTC cells expressed high levels of platelet-derived growth factor receptor–β and a low level of brachyury and cytokeratins; they showed higher expression of stemness-related and epithelial to mesenchymal transition–related proteins than the U-CH1 cells. Intriguingly, FACS analysis revealed that DTC cells exhibited marginal surface expression of CD24 and CD44 and high surface expression of CXCR4 in comparison to U-CH1 cells. In addition, blockade of CXCR4 with its antagonist AMD3100 effectively suppressed the growth of both cell lines. The DTC cells were more resistant to paclitaxel, cisplatin, etoposide, and ionizing radiation than the U-CH1 cells. Injection of DTC cells into the hindlimb region of nude mice resulted in the efficient formation of tumors, and the histology of xenograft tumors was very similar to that of the original patient tumor. CONCLUSIONS The use of the established DTC cells along with preestablished cell lines of chordoma may help bring about greater understanding of the mechanisms underlying the chordoma that will lead to therapeutic strategies targeting chordomas.

scholarly journals Antibody-mediated inhibition of syndecan-4 dimerisation reduces interleukin (IL)-1 receptor trafficking and signalling

2020 ◽  
Vol 79 (4) ◽  
pp. 481-489 ◽  
Author(s):  
Lars Godmann ◽  
Miriam Bollmann ◽  
Adelheid Korb-Pap ◽  
Ulrich König ◽  
Joanna Sherwood ◽  
...  
Keyword(s):  
Core Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Independent Manner ◽  
Necrosis Factor Alpha ◽  
Matrix Metalloproteinase 3 ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Inflammatory Stimuli

ObjectiveSyndecan-4 (sdc4) is a cell-anchored proteoglycan that consists of a transmembrane core protein and glucosaminoglycan (GAG) side chains. Binding of soluble factors to the GAG chains of sdc4 may result in the dimerisation of sdc4 and the initiation of downstream signalling cascades. However, the question of how sdc4 dimerisation and signalling affects the response of cells to inflammatory stimuli is unknown.MethodsSdc4 immunostaining was performed on rheumatoid arthritis (RA) tissue sections. Interleukin (IL)-1 induced extracellular signal-regulated kinases (ERK) phosphorylation and matrix metalloproteinase-3 production was investigated. Il-1 binding to sdc4 was investigated using immunoprecipitation. IL-1 receptor (IL1R1) staining on wild-type, sdc4 and IL1R1 knockout fibroblasts was performed in fluorescence-activated cell sorting analyses. A blocking sdc4 antibody was used to investigate sdc4 dimerisation, IL1R1 expression and the histological paw destruction in the human tumour necrosis factor-alpha transgenic mouse.ResultsWe show that in fibroblasts, the loss of sdc4 or the antibody-mediated inhibition of sdc4 dimerisation reduces the cell surface expression of the IL-1R and regulates the sensitivity of fibroblasts to IL-1. We demonstrate that IL-1 directly binds to sdc4 and in an IL-1R-independent manner leads to its dimerisation. IL-1-induced dimerisation of sdc4 regulates caveolin vesicle-mediated trafficking of the IL1R1, which in turn determines the responsiveness to IL-1. Administration of antibodies (Ab) against the dimerisation domain of sdc4, thus, strongly reduces the expression IL1R1 on arthritic fibroblasts both in vitro and an animal model of human RA.ConclusionCollectively, our data suggest that Ab that specifically inhibit sdc4 dimerisation may support anti-IL-1 strategies in diseases such as inflammatory arthritis.

scholarly journals RMA/S cells present endogenously synthesized cytosolic proteins to class I-restricted cytotoxic T lymphocytes.

1992 ◽  
Vol 175 (1) ◽  
pp. 163-168 ◽  
Author(s):  
F Esquivel ◽  
J Yewdell ◽  
J Bennink
Keyword(s):  
T Lymphocytes ◽  
Cell Surface ◽  
Cytotoxic T Lymphocytes ◽  
Antigen Processing ◽  
Mutant Cell ◽  
Surface Expression ◽  
Class I ◽  
Cell Surface Expression ◽  
Antigenic Peptides ◽  
Infected Cells

RMA/S is a mutant cell line with decreased cell surface expression of major histocompatibility complex class I molecules that has been reported to be deficient in presenting endogenously synthesized influenza virus nucleoprotein (NP) to cytotoxic T lymphocytes (CTL). In the present study we show that RMA/S cells can present vesicular stomatitis virus nucleocapsid protein, and, under some conditions, NP, to Kb-and Db-restricted CTL, respectively. Antigen presentation results from processing of cytosolic pools of endogenously synthesized proteins, and not the binding to cell surface class I molecules of antigenic peptides present in the virus inoculum or released from infected cells. Antigen processing of RMA/S differs, however, from processing by wild-type cells in requiring greater amounts of antigen, longer times to assemble or transport class I-peptide complexes, and in being more sensitive to blocking by anti-CD8 antibody. Thus, the antigen processing deficit in RMA/S cells is of a partial rather than absolute nature.

Defects of the Tpo-Receptor, Mpl, Caused by Mutations Found in CAMT Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2181-2181
Author(s):  
Marloes R. Tijssen ◽  
Franca di Summa ◽  
Sonja Van den Oudenrijn ◽  
Carlijn Voermans ◽  
C.Ellen Van der Schoot ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Amino Acid ◽  
Cell Surface ◽  
Cell Line ◽  
Mrna Level ◽  
K562 Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Amino Acid Substitutions ◽  
Bhk Cells

Abstract Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder that presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. The disease may develop into bone marrow aplasia. In vitro, CD34-positive hematopoietic progenitor cells from CAMT patients did not show any megakaryocyte formation in a Tpo-driven expansion culture. We and others found genetic defects in the gene encoding the Tpo receptor, c-mpl (Van den Oudenrijn et al., Br J Haematol.2002, 117: 390–398 and Ballmaier et al., Ann N Y Acad Sci.2003, 996: 17–25). In our patients, we found four mutations that predicted amino-acid substitutions, of which three in the extracellular domain; Arg102Pro, Pro136His and Arg257Cys, and one in the intracellular signaling domain (Pro635Leu), which may result in either defective Tpo-binding and/or signaling. To investigate this, we transfected full-length Mpl (wt and mutants) into the erythroleukemic cell line K562 and truncated Mpl (encompassing the extracellular domain; wt and mutants) into Baby Hamster Kidney (BHK) cells. In the K562 cells, the mRNA level (RQ-PCR) of the Pro136His mutant was severely decreased compared to the wt transfectant, while the mRNA level of the other mutants was comparable to that of wt. On Western blot, wt Mpl migrated as two, presumably differently glycosylated, bands of 75 kD and 72 kD. The mutants showed an altered migration pattern, which might result from differences in glycosylation. With the Pro635Leu mutant lower signals were obtained when equal amounts of total protein were loaded. Since the Mpl mRNA level was comparable to that of wt, this suggests a higher level of protein degradation. Upon transfection of the Arg102Pro and the Arg257Cys mutants in BHK cells, we observed that these mutants did not gain endo-H resistency, which suggests an aberrant processing of these mutant Mpls through the Golgi apparatus and retention in the ER. However, in cell fractionation experiments with surface-biotinylated K562 cells, biotinylated wt Mpl and mutant Mpl (except Pro136His) could be detected. Apparently, in K562 cells, the amino-acid substitutions do not impair membrane expression completely. To examine whether the mutant receptors were still able to signal after Tpo incubation, K562 cells were serum-starved and subsequently stimulated with 50 ng/ml rhTpo for 5 to 30 minutes. All mutants, including Pro136His, showed increased ERK phosphorylation after 5 minutes. To summarize, the Pro136His mutant is hardly expressed in the K562 expression model, presumably because of instability of the mRNA, but is still able to induce signaling. In contrast to the results obtained in the BHK model, the Arg102Pro and Arg257Cys mutants, showed cell-surface expression in the K562 cell line. The obtained cell-surface expression in the K562 model may have been significantly increased compared to the in vivo situation on hematopoietic stem cells, because of artificially induced efficient expression. Finally, with a super-physiological concentration of rhTpo, we obtained evidence that all Mpl mutants were able to signal upon Tpo binding. Whether impaired signaling by the Mpl mutants in the presence of physiological levels of Tpo may contribute to the development of CAMT, will be investigated.

CD81-Dependent Trafficking of CD19: Restoration of CD19 Surface Expression in Human B Cells Harboring A CD81 Mutation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1049-1049
Author(s):  
Shoshana Levy ◽  
Chiung-Chi Kuo ◽  
Yael Sagi ◽  
Homer Chen ◽  
Neta Kela-Madar ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
B Cell ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Antibody Deficiency ◽  
Cell Surface Expression ◽  
Dominant Negative Effect ◽  
Proximity Ligation Assay

Abstract Abstract 1049 Introduction: A 6-year-old girl, who was diagnosed with a primary antibody deficiency, had B cells lacking surface CD19. However, both her CD19 alleles were normal and the impairment was actually caused by a homozygous exon splice site mutation in CD81 (1). The patient's B cells also lacked surface CD81 and produced an immature glycosylated CD19 protein that was retained intracellularly. Interestingly, this human deficiency differed from that of CD81 knockout mice as the latter still express a low level of CD19 on their B cells. Methods: We used an EBV-transformed B cell line from this patient to better understand i) the difference between the human and mouse CD81 deficiencies and ii) how CD81 controls the trafficking of CD19 to the cell surface. We reasoned that the truncated human CD81 mutant (CD81mut) protein might be expressed intracellularly. Indeed, whereas most anti-CD81 mAbs did not recognize CD81mut, we identified one that bound the mutated form and used it in this study. We also expressed the human CD81mut in a CD81-deficient mouse B cell line to determine if it could negatively regulate CD19 surface expression. Results: We show that the CD81mut protein is indeed expressed intracellularly in the patient's EBV-transformed B cells. We then used a proximity ligation assay to demonstrate that the truncated CD81mut protein interacts intracellularly with CD19. However, this interaction with the CD81mut protein abrogated carbohydrate maturation and the trafficking of CD19 to cell surface. We therefore expressed the CD81mut in CD81KO mouse B cells, which still express low levels of surface CD19, and found that it did not exert a dominant negative effect on CD19 surface expression. Finally, we used this reconstitution system to identify specific CD81 domains that restored carbohydrate maturation and cell surface expression of the CD19 molecule in the patient's B cells. Conclusion: This specific case of antibody deficiency was manifested because of lack of surface expression of CD19, an important B cell signaling molecule. However, the maturation of CD19 and its trafficking to the cell surface require the presence of specific domains of the tetraspanin CD81 molecule. Disclosures: No relevant conflicts of interest to declare.

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