Insertion of an NPVY sequence into the cytosolic domain of the erythropoietin receptor selectively affects erythropoietin-mediated signalling and function

2010 ◽  
Vol 427 (2) ◽  
pp. 305-312 ◽  
Author(s):  
Tamar Liron ◽  
Tal Nahari ◽  
Miriam C. Souroujon ◽  
Drorit Neumann
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Erythropoietin Receptor ◽  
Janus Kinase ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Erythroid Progenitor ◽  
Cytosolic Domain ◽  
Surface Receptor ◽  
Induced Apoptosis

EPO (erythropoietin), the major hormone regulating erythropoiesis, functions via activation of its cell-surface receptor (EPO-R) present on erythroid progenitor cells. One of the most striking properties of EPO-R is its low expression on the cell surface, as opposed to its high intracellular levels. The low cell-surface expression of EPO-R may thus limit the efficacy of EPO that is routinely used to treat primary and secondary anaemia. In a recent study [Nahari, Barzilay, Hirschberg and Neumann (2008) Biochem. J. 410, 409–416] we have shown that insertion of an NPVY sequence into the intracellular domain of EPO-R increases its cell-surface expression. In the present study we demonstrate that this NPVY EPO-R insert has a selective effect on EPO-mediated downstream signalling in Ba/F3 cells expressing this receptor (NPVY-EPO-R). This is monitored by increased phosphorylation of the NPVY-EPO-R (on Tyr479), Akt, JAK2 (Janus kinase 2) and ERK1/2 (extracellular-signal-regulated kinase 1/2), but not STAT5 (signal transducer and activator of transcription 5), as compared with cells expressing wild-type EPO-R. This enhanced signalling is reflected in augmented proliferation at low EPO levels (0.05 units/ml) and protection against etoposide-induced apoptosis. Increased cell-surface levels of NPVY-EPO-R are most probably not sufficient to mediate these effects as the A234E-EPO-R mutant that is expressed at high cell-surface levels does not confer an augmented response to EPO. Taken together, we demonstrate that insertion of an NPVY sequence into the cytosolic domain of the EPO-R confers not only improved maturation, but also selectively affects EPO-mediated signalling resulting in an improved responsiveness to EPO reflected in cell proliferation and protection against apoptosis.

Mutations in the WSAWSE and cytosolic domains of the erythropoietin receptor affect signal transduction and ligand binding and internalization

1992 ◽  
Vol 12 (10) ◽  
pp. 4553-4561
Author(s):  
D E Quelle ◽  
F W Quelle ◽  
D M Wojchowski
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Ligand Binding ◽  
Erythropoietin Receptor ◽  
Cell Surface Receptor ◽  
Erythroid Progenitor ◽  
Epo Receptor ◽  
Cytosolic Domain ◽  
Cytosolic Domains ◽  
Surface Receptor

The terminal development of erythroid progenitor cells is promoted in part through the interaction of erythropoietin (EPO) with its cell surface receptor. This receptor and a growing family of related cytokine receptors share homologous extracellular features, including a well-conserved WSXWS motif. To explore the functional significance of this motif in the murine EPO receptor, five WSAWSE mutants were prepared and their signal-transducing, ligand binding, and endocytotic properties were compared. EPO receptors mutated at tryptophan residues (W-232, W-235----G; W-235----G; W-235----F) failed to mediate EPO-induced growth or pp100 phosphorylation, while S-236----T and E-237----K mutants exhibited partial to full activity (50 to 100% of wild-type growth and induced phosphorylation). Ligand affinity was reduced for mutant receptors (two- to fivefold), yet expression at the cell surface for all receptors was nearly equivalent. Also, the ability of mutated receptors to internalize ligand was either markedly reduced or abolished (W-235----F), indicating a role for the WSAWSE region in hormone internalization. Interestingly, receptor forms lacking 97% of the cytosolic domain (no signal-transducing capacity; binding affinity reduced two- to threefold) internalized EPO efficiently. This and all WSAWSE receptor forms studied also mediated specific cross-linking of 125I-EPO to three accessory membrane proteins (M(r)s, 120,000, 105,000, and 93,000). These findings suggest that the WSAWSE domain of the EPO receptor is important for EPO-induced signal transduction and ligand internalization. In contrast, although the cytosolic domain is required for growth signaling, it appears nonessential for efficient endocytosis.

scholarly journals Mutations in the WSAWSE and cytosolic domains of the erythropoietin receptor affect signal transduction and ligand binding and internalization.

1992 ◽  
Vol 12 (10) ◽  
pp. 4553-4561 ◽  
Author(s):  
D E Quelle ◽  
F W Quelle ◽  
D M Wojchowski
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Ligand Binding ◽  
Erythropoietin Receptor ◽  
Cell Surface Receptor ◽  
Erythroid Progenitor ◽  
Epo Receptor ◽  
Cytosolic Domain ◽  
Cytosolic Domains ◽  
Surface Receptor

The terminal development of erythroid progenitor cells is promoted in part through the interaction of erythropoietin (EPO) with its cell surface receptor. This receptor and a growing family of related cytokine receptors share homologous extracellular features, including a well-conserved WSXWS motif. To explore the functional significance of this motif in the murine EPO receptor, five WSAWSE mutants were prepared and their signal-transducing, ligand binding, and endocytotic properties were compared. EPO receptors mutated at tryptophan residues (W-232, W-235----G; W-235----G; W-235----F) failed to mediate EPO-induced growth or pp100 phosphorylation, while S-236----T and E-237----K mutants exhibited partial to full activity (50 to 100% of wild-type growth and induced phosphorylation). Ligand affinity was reduced for mutant receptors (two- to fivefold), yet expression at the cell surface for all receptors was nearly equivalent. Also, the ability of mutated receptors to internalize ligand was either markedly reduced or abolished (W-235----F), indicating a role for the WSAWSE region in hormone internalization. Interestingly, receptor forms lacking 97% of the cytosolic domain (no signal-transducing capacity; binding affinity reduced two- to threefold) internalized EPO efficiently. This and all WSAWSE receptor forms studied also mediated specific cross-linking of 125I-EPO to three accessory membrane proteins (M(r)s, 120,000, 105,000, and 93,000). These findings suggest that the WSAWSE domain of the EPO receptor is important for EPO-induced signal transduction and ligand internalization. In contrast, although the cytosolic domain is required for growth signaling, it appears nonessential for efficient endocytosis.

scholarly journals Role of Asparagine-Linked Glycosylation in Cell Surface Expression and Function of the Human Adrenocorticotropin Receptor (Melanocortin 2 Receptor) in 293/FRT Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (2) ◽  
pp. 660-670 ◽  
Author(s):  
Simon Roy ◽  
Benoît Perron ◽  
Nicole Gallo-Payet
Keyword(s):  
Cell Surface ◽  
Fold Increase ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Camp Production ◽  
Glycosylation Sites ◽  
Surface Receptor

Asparagine-linked glycosylation (N-glycosylation) of G protein-coupled receptors may be necessary for functions ranging from agonist binding, folding, maturation, stability, and internalization. Human melanocortin 2 receptor (MC2R) possesses putative N-glycosylation sites in its N-terminal extracellular domain; however, to date, the role of MC2R N-glycosylation has yet to be investigated. The objective of the present study is to examine whether N-glycosylation is essential or not for cell surface expression and cAMP production in native and MC2R accessory protein (MRAPα, -β, or -dCT)-expressing cells using 293/FRT transfected with Myc-MC2R. Western blot analyses performed with or without endoglycosidase H, peptide:N-glycosidase F or tunicamycin treatments and site-directed mutagenesis revealed that MC2R was glycosylated in the N-terminal domain at its two putative N-glycosylation sites (Asn12-Asn13-Thr14 and Asn17-Asn18-Ser19). In the absence of human MRAP coexpression, N-glycosylation of at least one of the two sites was necessary for MC2R cell surface expression. However, when MRAP was present, cell surface expression of MC2R mutants was either rescued entirely with the N17-18Q (QQNN) and N12-13Q (NNQQ) mutants or partially with the unglycosylated N12-13, 17-18Q (QQQQ) mutant. Functional and expression analyses revealed a discrepancy between wild-type (WT) and QQQQ cell surface receptor levels and maximal cAMP production with a 4-fold increase in EC50 values. Taken together, these results indicate that the absence of MC2R N-glycosylation abrogates to a large extent MC2R cell surface expression in the absence of MRAPs, whereas when MC2R is N-glycosylated, it can be expressed at the plasma membrane without MRAP assistance.

scholarly journals A Homozygous Microdeletion in Helix 7 of the Luteinizing Hormone Receptor Associated with Familial Testicular and Ovarian Resistance Is Due to Both Decreased Cell Surface Expression and Impaired Effector Activation by the Cell Surface Receptor

1998 ◽  
Vol 12 (3) ◽  
pp. 442-450 ◽  
Author(s):  
Ana C. Latronico ◽  
Yaohui Chai ◽  
Ivo J.P. Arnhold ◽  
Xuebo Liu ◽  
Berenice B. Mendonca ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Helix ◽  
External Genitalia ◽  
Surface Expression ◽  
Luteinizing Hormone Receptor ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Binding Assays ◽  
Mutant Receptor ◽  
Surface Receptor

Abstract In this report, the genomic DNA was examined from two siblings with gonadal LH resistance. A 46,XY pseudohermaphrodite presented with female external genitalia and his 46,XX sister exhibited menstrual irregularities (oligoamenorrhea) and infertility. Exons 1–11 of the LH receptor (LHR) gene were amplified by the PCR using different sets of intronic primers and were directly sequenced. Sequencing revealed that both individuals carried a deletion of nucleotides 1822–1827, resulting in the deletion of Leu-608 and Val-609 within the seventh transmembrane helix. This mutation was introduced into a recombinant human (h) LHR cDNA. Transfections of 293 cells with hLHR(wt) vs. hLHR(ΔL608,V609) revealed that very little of the mutant receptor was expressed at the cell surface. This was due to both a decrease in the total amount of receptor expressed as well as to an increased intracellular retention of the mutant receptor. In spite of the decreased cell surface expression of the mutant, sufficient amounts were present to allow for assessment of its functions. Equilibrium binding assays showed that the cell surface hLHR(ΔL608,V609) binds hCG with an affinity comparable to that of the wild-type receptor. However, the cells expressing the hLHR(ΔL608,V609) exhibit only a 1.5- to 2.4-fold stimulation of cAMP production in response to hCG. In contrast, cells expressing comparably low levels of hLHR(wt) responded to hCG with 11- to 30-fold increases of cAMP levels. Therefore, the testicular and ovarian unresponsiveness to LH in these patients appears to be due to a mutation of the hLHR gene in which Leu-608 and Val-609 are deleted. As a consequence, the majority of the mutant receptor is retained intracellularly. The small percentage of mutant receptor that is expressed at the cell surface binds hormone normally but is unable to activate Gs.

scholarly journals MEK1/2 activity modulates TREM2 cell surface recruitment

2020 ◽  
pp. jbc.RA120.014352
Author(s):  
Jason Schapansky ◽  
Yelena Y Grinberg ◽  
David M Osiecki ◽  
Emily A Freeman ◽  
Stephen G Walker ◽  
...  
Keyword(s):  
Cell Surface ◽  
Myeloid Cells ◽  
Surface Expression ◽  
Mek Inhibitor ◽  
Therapeutic Benefit ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Loss Of Function ◽  
Downstream Effector ◽  
Surface Receptor

Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer’s disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface Inhibitors of the kinases MEK1/2 displayed the strongest and most consistent increases in cell surface TREM2 protein, identifying a previously unreported pathway for TREM2 regulation. Unexpectedly, inhibitors of the downstream effector ERK kinases did not have the same effect, suggesting that non-canonical MEK signaling regulates TREM2 trafficking. In addition, siRNA knockdown experiments confirmed that decreased MEK1 and MEK2 was required for this recruitment. In iPSC-derived microglia, MEK inhibition increased cell surface TREM2 only modestly, so various cytokines were used to alter iPSC microglia phenotype, making cells more sensitive to MEK inhibitor-induced TREM2 recruitment. Of those tested, only IFN-gamma priming prior to MEK inhibitor treatment resulted in greater TREM2 recruitment. These data identify the first known mechanisms for increasing surface TREM2 protein and TREM2-regulated function in human myeloid cells, and are the first to show a role for MEK1/MEK2 signaling in TREM2 activity.

scholarly journals Dectin-1-Mediated Production of Pro-Inflammatory Cytokines Induced by Yeast β-Glucans in Bovine Monocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana R. V. Pedro ◽  
Tânia Lima ◽  
Ricardo Fróis-Martins ◽  
Bárbara Leal ◽  
Isabel C. Ramos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Inflammatory Cytokines ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Feed Supplements ◽  
Surface Receptor ◽  
Dose Dependent ◽  
Pro Inflammatory Cytokines

Yeast-derived products containing β-glucans have long been used as feed supplements in domesticated animals in an attempt to increase immunity. β-glucans are mainly recognized by the cell surface receptor CLEC7A, also designated Dectin-1. Although the immune mechanisms elicited through Dectin-1 activation have been studied in detail in mice and humans, they are poorly understood in other species. Here, we evaluated the response of bovine monocytes to soluble and particulate purified β-glucans, and also to Zymosan. Our results show that particulate, but not soluble β-glucans, can upregulate the surface expression of costimulatory molecules CD80 and CD86 on bovine monocytes. In addition, stimulated cells increased production of IL-8 and of TNF, IL1B, and IL6 mRNA expression, in a dose-dependent manner, which correlated positively with CLEC7A gene expression. Production of IL-8 and TNF expression decreased significantly after CLEC7A knockdown using two different pairs of siRNAs. Overall, we demonstrated here that bovine monocytes respond to particulate β-glucans, through Dectin-1, by increasing the expression of pro-inflammatory cytokines. Our data support further studies in cattle on the induction of trained immunity using dietary β-glucans.

Effect of aging on CD11b and CD69 surface expression by vesicular insertion in human polymorphonuclear leucocytes

1999 ◽  
Vol 97 (3) ◽  
pp. 323-329 ◽  
Author(s):  
J. M. NOBLE ◽  
G. A. FORD ◽  
T. H. THOMAS
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Elderly Subjects ◽  
Polymorphonuclear Leucocytes ◽  
Cd11b Expression ◽  
Cd69 Expression ◽  
Vesicle Exocytosis

The exocytosis of intracellular vesicles is an important function of the plasma membrane, which is responsible for hormone secretion, cell surface expression of antigens, ion transporters and receptors, and intracellular and intercellular signalling. Human aging is associated with many physiological and cellular changes, many of which are due to alterations in plasma membrane functioning. Alterations in vesicle externalization with age could account for many of these changes. We investigated whether alterations in vesicle exocytosis occur with increasing age by flow-cytometric determination of CD11b and CD69 expression on the surface of human polymorphonuclear leucocytes (PMN) stimulated with phorbol myristate acetate (PMA), a tumour promoter which binds to and activates protein kinase C (PKC) directly, or with formyl-Met-Leu-Phe (fMLP), which activates PKC indirectly via interactions with a cell surface receptor and G-protein, and subsequent inositol phosphate hydrolysis. Following stimulation with PMA, a decrease in the proportion of PMN expressing CD69 at high levels was observed in elderly compared with young subjects (young, 55.3%; elderly, 43.9%; P = 0.01). No aging-related differences in the proportion of PMN expressing CD11b (young, 73.7%; elderly, 68.4%; P = 0.15), or in the number of molecules of CD69 or CD11b expressed per cell, were observed. Stimulation with fMLP or low PMA concentrations resulted in full CD11b expression but minimal CD69 expression in both young and elderly subjects. Cells which expressed CD69 had no CD11b expression, while those cells expressing CD11b had minimal CD69 expression. Thus the PMA-induced expression of CD11b and CD69 in human PMN represents two separate processes, only one of which is affected in aging. CD11b expression appears to require a lesser degree of PKC stimulation compared with that required for CD69 expression. The age-associated reduction in PMA-stimulated CD69 expression may occur either at or distal to PKC activation. Such a decrease may contribute to the age-associated impairments in PMN function that contribute, in turn, to immunosenescence.

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