scholarly journals Mutational analysis of the cytoplasmic domain of  1,4-galactosyltransferase I: influence of phosphorylation on cell surface expression

2003 ◽  
Vol 116 (21) ◽  
pp. 4319-4330 ◽  
Author(s):  
H. J. Hathaway
Keyword(s):  
Cell Surface ◽  
Mutational Analysis ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Cell Surface Expression ◽  
Galactosyltransferase I

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 Mutational analysis of the N-linked glycosylation sites of the human insulin receptor

2000 ◽  
Vol 347 (3) ◽  
pp. 771-779 ◽  
Author(s):  
Thomas C. ELLEMAN ◽  
Maurice J. FRENKEL ◽  
Peter A. HOYNE ◽  
Neil M. MCKERN ◽  
Leah COSGROVE ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mutational Analysis ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Insulin Binding ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Triple Mutant ◽  
Glycosylation Sites

Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into α and β subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined. The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624+730+743+881 showed normal processing and ligand binding but exhibited a constitutively active tyrosine kinase as judged by autophosphorylation. Three higher-order mutants were constructed, two of which, 16+337+418+730+743+881 (∆6) and 16+295+337+418+730+743+881 (∆7a), seemed normal. The third construct, 16+337+418+514+730+743+881 (∆7b), was expressed at high levels on the cell surface, essentially as uncleaved proreceptor with only the small proportion of ∆7b that was correctly processed showing insulin-stimulated autophosphorylation. The mutations of ∆6 and ∆7a were incorporated into soluble ectodomains, which had affinities for insulin that were 4-fold that of wild-type ectodomain. The ∆6 ectodomain expressed in Lec8 cells was produced in quantity in a bioreactor for subsequent structural analysis.

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 Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1

1998 ◽  
Vol 331 (1) ◽  
pp. 257-264 ◽  
Author(s):  
Ana M. PAJOR ◽  
Ning SUN ◽  
Heidi G. VALMONTE
Keyword(s):  
Cell Surface ◽  
Mutational Analysis ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Membrane Expression ◽  
Histidine Residues ◽  
Cell Surface Biotinylation

Succinate transport by the rabbit Na+/dicarboxylate co-transporter, NaDC-1, expressed in Xenopusoocytes was inhibited by the histidyl-selective reagent diethyl pyrocarbonate (DEPC). Therefore the role of histidine residues in the function of NaDC-1 was examined by site-directed mutagenesis. All 11 histidine residues in NaDC-1 were converted to alanine, but only mutant H106A exhibited a decrease in succinate transport. Additional mutations of NaDC-1 at position 106 showed that aspartic acid and asparagine, but not arginine, can substitute for histidine. Examination of succinate and citrate kinetics of H106A revealed a decrease in Vmax with no change in Km. Cell surface biotinylation experiments showed that the transport activity of all four mutants at position 106 was correlated with the amount of cell surface expression, suggesting a role of His-106 in membrane expression rather than function. Two of the histidine mutants, H153A and H569A, exhibited insensitivity to inhibition by DEPC, indicating that these residues are involved in binding DEPC. Neither of these residues is required for transport activity; thus DEPC probably inhibits NaDC-1 function by hindrance of the mobility of the carrier. We conclude that histidine residues are not critical for transport function in NaDC-1, although His-106 might be involved in determining protein expression or stability in the membrane.

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 gp25L/emp24/p24 Protein Family Members of the cis-Golgi Network Bind Both COP I and II Coatomer

1998 ◽  
Vol 140 (4) ◽  
pp. 751-765 ◽  
Author(s):  
Michel Dominguez ◽  
Kurt Dejgaard ◽  
Joachim Füllekrug ◽  
Sophie Dahan ◽  
Ali Fazel ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Cell Surface Expression ◽  
Binding Motif ◽  
Steady State Distribution ◽  
P24 Protein ◽  
Golgi Network

Abstract. Five mammalian members of the gp25L/ emp24/p24 family have been identified as major constituents of the cis-Golgi network of rat liver and HeLa cells. Two of these were also found in membranes of higher density (corresponding to the ER), and this correlated with their ability to bind COP I in vitro. This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members. A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II). This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif. In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER. Surprisingly, upon expression of mutated members, steady-state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligomeric properties.

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