P1-485: Phosphorylation of the cytoplasmic domain of neprilysin and its role in cell surface expression and activity

2008 ◽  
Vol 4 ◽  
pp. T361-T362
Author(s):  
Sathish H.S. Kumar ◽  
Martin Siepmann ◽  
Jochen Walter
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Cell Surface Expression ◽  
Expression And Activity

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 Cyclic AMP Increases Cell Surface Expression of Functional Na,K-ATPase Units in Mammalian Cortical Collecting Duct Principal Cells

2001 ◽  
Vol 12 (2) ◽  
pp. 255-264 ◽  
Author(s):  
Sandrine Gonin ◽  
Georges Deschênes ◽  
Frank Roger ◽  
Marcelle Bens ◽  
Pierre-Yves Martin ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Cell Surface ◽  
Atpase Activity ◽  
Cultured Cells ◽  
Collecting Duct ◽  
Brefeldin A ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cortical Collecting Duct ◽  
Expression And Activity

Cyclic AMP (cAMP) stimulates the transport of Na+ and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCDc14 collecting duct cells. db-cAMP (10−3 M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of 86Rb+ uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20°C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca2+chelator bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

Regulation of tissue factor cytoplasmic domain phosphorylation by palmitoylation

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 3998-4005 ◽  
Author(s):  
Andrea Dorfleutner ◽  
Wolfram Ruf
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Tissue Factor ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Golgi Compartment ◽  
Expression Site ◽  
Surface Location

Abstract The tissue factor (TF)–initiated coagulation pathway plays important roles in hemostasis, inflammation, metastasis, and angiogenesis. Phosphorylation of the TF cytoplasmic domain is functionally relevant in metastasis. How TF cytoplasmic domain phosphorylation downstream of protein kinase C (PKC) activation is regulated in primary vascular cells remains poorly understood. Here, phosphorylation of Ser258, rather than the PKC consensus site Ser253, is identified as the major conformational switch required for recognition by a phosphorylation-specific antibody. With this novel reagent, we demonstrate that the TF cytoplasmic domain is primarily unphosphorylated in confluent endothelial cells. TF cytoplasmic domain phosphorylation can occur in the absence of the autologous TF transmembrane and extracellular domains but requires maturation of TF in the Golgi compartment and cell surface expression. Site-directed mutagenesis and 2-bromopalmitate treatment provide evidence that palmitoylation of the cytoplasmic Cys245 is a negative regulatory mechanism of Ser258 phosphorylation. Profiling with PKC-selective inhibitors identifies PKCα as important for TF cytoplasmic domain phosphorylation. Mutagenesis of protein kinase consensus sites are consistent with a model in which PKC-dependent phosphorylation of Ser253 enhances subsequent Ser258 phosphorylation by a Pro-directed kinase. Thus, cell surface location–dependent phosphorylation of the TF cytoplasmic domain is regulated at multiple levels.

scholarly journals VXFD in the Cytoplasmic Domain of Macrophage Scavenger Receptors Mediates Their Efficient Internalization and Cell-Surface Expression.

1999 ◽  
Vol 22 (10) ◽  
pp. 1022-1026 ◽  
Author(s):  
Kazunobu MORIMOTO ◽  
Youichiro WADA ◽  
Jun-ichi HINAGATA ◽  
Takeshi IMANISHI ◽  
Tatsuhiko KODAMA ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Cell Surface Expression ◽  
Scavenger Receptors

scholarly journals Modulation of Env Content in Virions of Simian Immunodeficiency Virus: Correlation with Cell Surface Expression and Virion Infectivity

2004 ◽  
Vol 78 (13) ◽  
pp. 6775-6785 ◽  
Author(s):  
Eloísa Yuste ◽  
Jacqueline D. Reeves ◽  
Robert W. Doms ◽  
Ronald C. Desrosiers
Keyword(s):  
Cell Surface ◽  
Simian Immunodeficiency Virus ◽  
Stop Codon ◽  
Transmembrane Protein ◽  
Fold Increase ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Cell Surface Expression ◽  
Molar Ratio ◽  
Immunodeficiency Virus

ABSTRACT Specific mutations were created in the cytoplasmic domain of the gp41 transmembrane protein of simian immunodeficiency virus strain 239 (SIV239). The resultant strains included a mutant in which Env residue 767 was changed to a stop codon, a double mutant in which positions 738 and 739 were changed to stop codons, another mutant in which a prominent endocytosis motif was changed from YRPV to GRPV by the substitution of tyrosine 721, and a final combination mutant bearing Q738stop, Q739stop, and Y721G mutations. The effects of these mutations on cell surface expression, on Env incorporation into virions, and on viral infectivity were examined. The molar ratio of Gag to gp120 of 54:1 that we report here for SIV239 virions agrees very well with the ratio of 60:1 reported previously by Chertova et al. (E. Chertova, J. W. Bess, Jr., B. J. Crise, R. C. Sowder II, T. M. Schaden, J. M. Hilburn, J. A. Hoxie, R. E. Benveniste, J. D. Lifson, L. E. Henderson, and L. O. Arthur, J. Virol. 76:5315-5325, 2002), although they were determined by very different methodologies. Assuming 1,200 to 2,500 Gag molecules per virion, this corresponds to 7 to 16 Env trimers per SIV239 virion particle. Although all of the mutations increased Env levels in virions, E767stop had the most dramatic effect, increasing the Env content per virion 25- to 50-fold. Increased levels of Env content in virions correlated strictly with higher levels of Env expression on the cell surface. The increased Env content with the E767stop mutation also correlated with an increased infectivity, but the degree of change was not proportional: the 25- to 50-fold increase in Env content only increased infectivity 2- to 3-fold. All of the mutants replicated efficiently in the CEMx174 and Rh221-89 cell lines. Although some of these findings have been reported previously, our findings show that the effects of the cytoplasmic domain of gp41 on the Env content in virions can be dramatic, that the Env content in virions correlates strictly with the levels of cell surface expression, and that the Env content in virions can determine infectivity; furthermore, our results define a particular change with the most dramatic effects.

scholarly journals Interactions of the Cytoplasmic Domains of Human and Simian Retroviral Transmembrane Proteins with Components of the Clathrin Adaptor Complexes Modulate Intracellular and Cell Surface Expression of Envelope Glycoproteins

1999 ◽  
Vol 73 (2) ◽  
pp. 1350-1361 ◽  
Author(s):  
Clarisse Berlioz-Torrent ◽  
Barbara L. Shacklett ◽  
Lars Erdtmann ◽  
Lelia Delamarre ◽  
Isabelle Bouchaert ◽  
...  
Keyword(s):  
Cell Surface ◽  
Virus Type ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Major Determinant ◽  
Cell Surface Expression ◽  
Envelope Glycoproteins ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXØ, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXØ motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXØ motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXØ motifs interact with the μ1 and μ2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the β2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXØ-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.

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