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 Mutational analysis of histidine residues in human organic anion transporter 4 (hOAT4)

2004 ◽  
Vol 384 (1) ◽  
pp. 87-92 ◽  
Author(s):  
Fanfan ZHOU ◽  
Zui PAN ◽  
Jianjie MA ◽  
Guofeng YOU
Keyword(s):  
Mutational Analysis ◽  
Organic Anion ◽  
The Body ◽  
Organic Anion Transporter ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Membrane Expression ◽  
Histidine Residues ◽  
Anion Transporter

Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, antitumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. hOAT4-mediated transport of the organic anion oestrone sulphate in COS-7 cells was inhibited by the histidine-modifying reagent DEPC (diethyl pyrocarbonate). Therefore the role of histidine residues in the function of hOAT4 was examined by site-directed mutagenesis. All five histidine residues of hOAT4 were converted into alanine, singly or in combination. Single replacement of His-47, or simultaneous replacement of His-47/52/83 or His-47/52/83/305/469 (H-less) led to a 50–80% decrease in transport activity. The decreased transport activity of these mutants was correlated with a decreased amount of cell-surface expression, although the total cell expression of these mutants was similar to that of wild-type hOAT4. These results suggest that mutation at positions 47, 47/52/83 and 47/52/83/305/469 impaired membrane expression rather than function. We also showed that, although most of the histidine mutants of hOAT4 were sensitive to inhibition by DEPC, H469A (His-469→Ala) was completely insensitive to inhibition by this reagent. Therefore modification of His-469 is responsible for the inhibition of hOAT4 by DEPC.

Role ofN-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3

2012 ◽  
Vol 302 (5) ◽  
pp. C781-C795 ◽  
Author(s):  
Hisayoshi Hayashi ◽  
Yukari Yamashita
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Tryptic Digestion ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Biochemical Modification ◽  
Glycosylation Sites ◽  
Increased Susceptibility

SLC26A3 is a Cl−/HCO3−exchanger that plays a major role in Cl−absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.

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.

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 Is Phosphorylation of the α1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester–activated Protein Kinase C?

2000 ◽  
Vol 11 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Eric Féraille ◽  
Pascal Béguin ◽  
Maria-Luisa Carranza ◽  
Sandrine Gonin ◽  
Martine Rousselot ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Wild Type ◽  
Α1 Subunit ◽  
Stimulation Of

The α1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufoα1 subunits. In cells incubated at 37°C, phorbol 12,13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally (∼20–30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A2, and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18°C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing α1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase α1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.

scholarly journals Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 1038-1047 ◽  
Author(s):  
Isabelle Mouro-Chanteloup ◽  
Anne Marie D'Ambrosio ◽  
Pierre Gane ◽  
Caroline Le Van Kim ◽  
Virginie Raynal ◽  
...  
Keyword(s):  
Cell Surface ◽  
K562 Cells ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Expression Vectors ◽  
Cell Surface Expression ◽  
Membrane Expression ◽  
Cmv Promoter ◽  
Transfected Cells

Abstract In most cases, the lack of Rh in Rhnull red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter–based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.

scholarly journals Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 1038-1047 ◽  
Author(s):  
Isabelle Mouro-Chanteloup ◽  
Anne Marie D'Ambrosio ◽  
Pierre Gane ◽  
Caroline Le Van Kim ◽  
Virginie Raynal ◽  
...  
Keyword(s):  
Cell Surface ◽  
K562 Cells ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Expression Vectors ◽  
Cell Surface Expression ◽  
Membrane Expression ◽  
Cmv Promoter ◽  
Transfected Cells

In most cases, the lack of Rh in Rhnull red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter–based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.

Role of glycosylation in cell surface expression and stability of HERG potassium channels

2002 ◽  
Vol 283 (1) ◽  
pp. H77-H84 ◽  
Author(s):  
Qiuming Gong ◽  
Corey L. Anderson ◽  
Craig T. January ◽  
Zhengfeng Zhou
Keyword(s):  
Cell Surface ◽  
Protein Trafficking ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Herg Channel ◽  
Cell Surface Expression ◽  
Delayed Rectifier ◽  
Channel Stability ◽  
Herg Channels

The human ether-à-go-go-related gene (HERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability.

scholarly journals Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region

2021 ◽  
Vol 22 (19) ◽  
pp. 10207
Author(s):  
Julien Vitry ◽  
Guillaume Paré ◽  
Andréa Murru ◽  
Xavier Charest-Morin ◽  
Halim Maaroufi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Activation ◽  
Secretory Pathway ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Inhibitory Receptor ◽  
Lectin Receptors ◽  
Stalk Domain

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor’s expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.

scholarly journals Analysis of the Role of N-Linked Glycosylation in Cell Surface Expression, Function, and Binding Properties of SARS-CoV-2 Receptor ACE2

2021 ◽  
Author(s):  
Raymond Rowland ◽  
Alberto Brandariz-Nuñez
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Receptor Interaction ◽  
Minimal Effect ◽  
Binding Properties ◽  
Virus Receptor

Understanding the role of glycosylation in the virus-receptor interaction is important for developing approaches that disrupt infection. In this study, we showed that deglycosylation of both ACE2 and S had a minimal effect on the spike-ACE2 interaction.

Sign in / Sign up

Export Citation Format

Share Document