scholarly journals The N-Terminal Domain of Janus Kinase 2 Is Required for Golgi Processing and Cell Surface Expression of Erythropoietin Receptor

2001 ◽  
Vol 8 (6) ◽  
pp. 1327-1338 ◽  
Author(s):  
Lily Jun-shen Huang ◽  
Stefan N. Constantinescu ◽  
Harvey F. Lodish
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Erythropoietin Receptor ◽  
Janus Kinase ◽  
Cell Surface Expression ◽  
Janus Kinase 2 ◽  
Terminal Domain

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.

scholarly journals Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1

1996 ◽  
Vol 315 (1) ◽  
pp. 217-225 ◽  
Author(s):  
R. A. Jeffrey McILHINNEY ◽  
Elek MOLNÁR
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Glutamate Receptor ◽  
Cell Membranes ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Receptor Subunit ◽  
Binding Studies ◽  
Terminal Domain ◽  
Glutamate Receptor Subunit

To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510 and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2 and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [3H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([3H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.

scholarly journals Janus Kinase 2 Determinants for Growth Hormone Receptor Association, Surface Assembly, and Signaling

2003 ◽  
Vol 17 (11) ◽  
pp. 2211-2227 ◽  
Author(s):  
Kai He ◽  
Xiangdong Wang ◽  
Jing Jiang ◽  
Ran Guan ◽  
Kenneth E. Bernstein ◽  
...  
Keyword(s):  
Growth Hormone Receptor ◽  
Surface Expression ◽  
Kinase Domain ◽  
Janus Kinase ◽  
Cytokine Receptors ◽  
Cell Surface Expression ◽  
Janus Kinase 2 ◽  
Functional Interaction ◽  
Receptor Surface Expression ◽  
Computational Analyses

Abstract GH signaling depends on functional interaction of the GH receptor (GHR) and the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), which possesses a C-terminal kinase domain, a catalytically inactive pseudokinase domain just N-terminal to the kinase domain, and an N-terminal half shown by us and others to harbor elements for GHR association. Computational analyses indicate that JAKs contain in their N termini (∼450 residues) divergent FERM domains. FERM domains (or subdomains within them) in JAKS may be important for associations with cytokine receptors. For some cytokine receptors, JAK interaction may be required for receptor surface expression. We previously demonstrated that a JAK2 mutant devoid of its N-terminal 239 residues (JAK2-Δ1–239) did not associate with GHR and could not mediate GH- induced signaling. In this report we employ a JAK2-deficient cell line to further define N-terminal JAK2 regions required for physical and functional association with the GHR. We also examine whether JAK2 expression affects cell surface expression of the GHR. Our results suggest that FERM motifs play an important role in the interaction of GHR and JAK2. While JAK2 expression is not required for detectable surface GHR expression, an increased JAK2 level increases the fraction of GHRs that achieves resistance to deglycosylation by endoglycosidase H, suggesting that the GHR-JAK2 association may enhance either the receptor’s efficiency of maturation or its stability. Further, we report evidence for the existence of a novel GH-inducible functional interaction between JAK2 molecules that may be important in the mechanism of GH-triggered JAK2 signaling.

scholarly journals The Amino Terminal Lectin-Like Domain of Thrombomodulin Is Required for Constitutive Endocytosis

Blood ◽  
1997 ◽  
Vol 89 (2) ◽  
pp. 652-661 ◽  
Author(s):  
Edward M. Conway ◽  
Saskia Pollefeyt ◽  
Desiré Collen ◽  
Marta Steiner-Mosonyi
Keyword(s):  
Cell Surface ◽  
Protein C ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Immunogold Electron Microscopy ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Cos Cells ◽  
Amino Terminal ◽  
Terminal Domain

Abstract Thrombomodulin (TM) is a multidomain protein that serves as a cofactor in a major natural anticoagulant system. To further characterize the structure-function of TM, we have transfected COS cells with different truncated forms of TM. In the first form, COS cells expressing TM that lacks the putative signal peptide (17 residues); the lectin-like, hydrophobic N-terminal domain (226 residues); and 12 residues of the first epidermal growth factor (EGF )-like repeat (COSdel.238 cells) were found to function normally with respect to TM transport to the cell surface and thrombin-dependent protein C activation. However, in contrast to wild-type TM, as visually studied by immunofluorescence and immunogold electron microscopy, the COSdel.238 cells did not constitutively internalize anti-TM–TM or thrombin-TM complexes. To identify the region responsible for mediating the endocytic process, deletant forms of TM lacking either the lectin-like region (residues 2-155) or the hydrophobic region of the N-terminal domain (residues 161-202) were expressed in COS cells (COSdel.2-155 and COSdel.161-202, respectively). Protein C cofactor activity was maintained in both cells. Although the COSdel.161-202 cells behaved similarly to wild-type TM-transfected cells, visual studies showed a lack of constitutive internalization of thrombin-TM or anti-TM–TM complexes in the COSdel.2-155 cells. We conclude that the lectin-like domain of human TM serves to regulate cell surface expression of TM via the endocytic route and therefore may also play a major physiologic role in controlling intracellular and extracellular accumulation of thrombin in a variety of biologic systems.

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