scholarly journals Trafficking defects of the Southeast Asian ovalocytosis deletion mutant of anion exchanger 1 membrane proteins

2005 ◽  
Vol 392 (3) ◽  
pp. 425-434 ◽  
Author(s):  
Joanne C. Cheung ◽  
Emmanuelle Cordat ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Cell Surface ◽  
Anion Exchanger ◽  
Mdck Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intercalated Cells ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Human AE1 (anion exchanger 1) is a membrane glycoprotein found in erythrocytes and as a truncated form (kAE1) in the BLM (basolateral membrane) of α-intercalated cells of the distal nephron, where they carry out electroneutral chloride/bicarbonate exchange. SAO (Southeast Asian ovalocytosis) is a dominant inherited haematological condition arising from deletion of Ala400–Ala408 in AE1, resulting in a misfolded and transport-inactive protein present in the ovalocyte membrane. Heterozygotes with SAO are able to acidify their urine, without symptoms of dRTA (distal renal tubular acidosis) that can be associated with mutations in kAE1. We examined the effect of the SAO deletion on stability and trafficking of AE1 and kAE1 in transfected HEK-293 (human embryonic kidney) cells and kAE1 in MDCK (Madin–Darby canine kidney) epithelial cells. In HEK-293 cells, expression levels and stabilities of SAO proteins were significantly reduced, and no mutant protein was detected at the cell surface. The intracellular retention of AE1 SAO in transfected HEK-293 cells suggests that erythroid-specific factors lacking in HEK-293 cells may be required for cell-surface expression. Although misfolded, SAO proteins could form heterodimers with the normal proteins, as well as homodimers. In MDCK cells, kAE1 was localized to the cell surface or the BLM after polarization, while kAE1 SAO was retained intracellularly. When kAE1 SAO was co-expressed with kAE1 in MDCK cells, kAE1 SAO was largely retained intracellularly; however, it also co-localized with kAE1 at the cell surface. We propose that, in the kidney of heterozygous SAO patients, dimers of kAE1 and heterodimers of kAE1 SAO and kAE1 traffic to the BLM of α-intercalated cells, while homodimers of kAE1 SAO are retained in the endoplasmic reticulum and are rapidly degraded. This results in sufficient cell-surface expression of kAE1 to maintain adequate bicarbonate reabsorption and proton secretion without dRTA.

scholarly journals Palmitoylation is not required for trafficking of human anion exchanger 1 to the cell surface

2004 ◽  
Vol 378 (3) ◽  
pp. 1015-1021 ◽  
Author(s):  
Joanne C. CHEUNG ◽  
Reinhart A. F. REITHMEIER
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Palmitic Acid ◽  
Anion Exchanger ◽  
Cell Surface Expression ◽  
Co2 Removal ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.

scholarly journals MUPP1 complexes renal K+ channels to alter cell surface expression and whole cell currents

2009 ◽  
Vol 297 (1) ◽  
pp. F36-F45 ◽  
Author(s):  
Aleksandra Sindic ◽  
Chunfa Huang ◽  
An-Ping Chen ◽  
Yaxian Ding ◽  
William A. Miller-Little ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Pdz Domain ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Kidney Cortex ◽  
Whole Cell ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

We previously found that the Ca2+-sensing receptor (CaR) interacts with and inactivates the inwardly rectifying K+ channel Kir4.2 that is expressed in the kidney cortex and that has a COOH-terminal PDZ domain. To identify potential scaffolding proteins that could organize a macromolecular signaling complex involving the CaR and Kir4.2, we used yeast two-hybrid cloning with the COOH-terminal 125 amino acids (AA) of Kir4.2 as bait to screen a human kidney cDNA library. We identified two independent partial cDNAs corresponding to the COOH-terminal 900 AA of MUPP1, a protein containing 13 PDZ binding domains that is expressed in the kidney in tight junctions and lateral borders of epithelial cells. When expressed in human embryonic kidney (HEK)-293 cells, Kir4.2 coimmunoprecipitates reciprocally with MUPP1 but not with a Kir4.2 construct lacking the four COOH-terminal amino acids, Kir5.1, or the CaR. MUPP1 and Kir4.2 coimmunoprecipitate reciprocally from rat kidney cortex extracts. Coexpression of MUPP1 with Kir4.2 in HEK-293 cells leads to reduced cell surface expression of Kir4.2 as assessed by cell surface biotinylation. Coexpression of MUPP1 and Kir4.2 in Xenopus oocytes results in reduced whole cell currents compared with expression of Kir4.2 alone, whereas expression of Kir4.2ΔPDZ results in minimal currents and is not affected by coexpression with MUPP1. Immunofluorescence studies of oocytes demonstrate that MUPP1 reduces Kir4.2 membrane localization. These results indicate that Kir4.2 interacts selectively with MUPP1 to affect its cell surface expression. Thus MUPP1 and Kir4.2 may participate in a protein complex in the nephron that could regulate transport of K+ as well as other ions.

Human kanadaptin and kidney anion exchanger 1 (kAE1) do not interact in transfected HEK 293 cells

2004 ◽  
Vol 21 (6) ◽  
pp. 395-402 ◽  
Author(s):  
Saranya Kittanakom ◽  
Thitima Keskanokwong ◽  
Varaporn Akkarapatumwong ◽  
Pa-thai Yenchitsomanus ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Anion Exchanger ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Essential role for Ca2+in regulation of IL-1β secretion by P2X7nucleotide receptor in monocytes, macrophages, and HEK-293 cells

2003 ◽  
Vol 285 (2) ◽  
pp. C286-C299 ◽  
Author(s):  
Lalitha Gudipaty ◽  
Jonathan Munetz ◽  
Philip A. Verhoef ◽  
George R. Dubyak
Keyword(s):  
Essential Role ◽  
Signal Sequence ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Nucleotide Receptors ◽  
Hek 293 ◽  
External Stimulation ◽  
Hek 293 Cells ◽  
293 Cells

Interleukin (IL)-1β is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1β in macrophages and monocytes can occur via stimulation of P2X7nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1β remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+to potentiate secretion of IL-1β via the P2X7receptors. Using HEK-293 cells engineered to coexpress P2X7receptors with mature IL-1β (mIL-1β), we show that activation of P2X7receptors results in a rapid secretion of mIL-1β by a process(es) that is dependent on influx of extracellular Ca2+and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+attenuates ∼90% of P2X7receptor-mediated IL-1β secretion but has no effect on enzymatic processing of precursor IL-1β (proIL-1β) to mIL-1β by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+in P2X7receptor-mediated secretion of IL-1β. In addition, we report that cell surface expression of P2X7receptors in the absence of external stimulation also results in enhanced release of IL-1β and that this can be repressed by inhibitors of P2X7receptors. We clarify an essential role for Ca2+in ATP-induced IL-1β secretion and indicate an additional role of P2X7receptors as enhancers of the secretory apparatus by which IL-1β is released.

Dominant-negative effect of Southeast Asian ovalocytosis anion exchanger 1 in compound heterozygous distal renal tubular acidosis

2008 ◽  
Vol 410 (2) ◽  
pp. 271-281 ◽  
Author(s):  
Saranya Kittanakom ◽  
Emmanuelle Cordat ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Cell Surface ◽  
Blood Cell ◽  
Renal Tubular Acidosis ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Compound Heterozygous ◽  
Intercalated Cells ◽  
Renal Tubular

The human chloride/bicarbonate AE1 (anion exchanger) is a dimeric glycoprotein expressed in the red blood cell membrane, and expressed as an N-terminal (Δ1–65) truncated form, kAE1 (kidney AE1), in the basolateral membrane of α-intercalated cells in the distal nephron. Mutations in AE1 can cause SAO (Southeast Asian ovalocytosis) or dRTA (distal renal tubular acidosis), an inherited kidney disease resulting in impaired acid secretion. The dominant SAO mutation (Δ400–408) that results in an inactive transporter and altered eythrocyte shape occurs in many dRTA families, but does not itself result in dRTA. Compound heterozygotes of four dRTA mutations (R602H, G701D, ΔV850 and A858D) with SAO exhibit dRTA and abnormal red blood cell properties. Co-expression of kAE1 and kAE1 SAO with the dRTA mutants was studied in polarized epithelial MDCK (Madin–Darby canine kidney) cells. Like SAO, the G701D and ΔV850 mutants were predominantly retained intracellularly, whereas the R602H and A858D mutants could traffic to the basolateral membrane. When co-expressed in transfected cells, kAE1 WT (wild-type) and kAE1 SAO could interact with the dRTA mutants. MDCK cells co-expressing kAE1 SAO with kAE1 WT, kAE1 R602H or kAE1 A858D showed a decrease in cell-surface expression of the co-expressed proteins. When co-expressed, kAE1 WT co-localized with the kAE1 R602H, kAE1 G701D, kAE1 ΔV850 and kAE1 A858D mutants at the basolateral membrane, whereas kAE1 SAO co-localized with kAE1 WT, kAE1 R602H, kAE1 G701D, kAE1 ΔV850 and kAE1 A858D in MDCK cells. The decrease in cell-surface expression of the dRTA mutants as a result of the interaction with kAE1 SAO would account for the impaired expression of functional kAE1 at the basolateral membrane of α-intercalated cells, resulting in dRTA in compound heterozygous patients.

scholarly journals Protein Kinase C Activation Regulates Human Serotonin Transporters in HEK-293 Cells via Altered Cell Surface Expression

1997 ◽  
Vol 17 (1) ◽  
pp. 45-57 ◽  
Author(s):  
Yan Qian ◽  
Aurelio Galli ◽  
Sammanda Ramamoorthy ◽  
Stefania Risso ◽  
Louis J. DeFelice ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Hek 293 ◽  
Kinase C ◽  
293 Cells ◽  
Altered Cell ◽  
Protein Kinase C Activation

Partial expression defect for the SCN5A missense mutation G1406R depends on splice variant background Q1077 and rescue by mexiletine

2006 ◽  
Vol 291 (4) ◽  
pp. H1822-H1828 ◽  
Author(s):  
Bi-Hua Tan ◽  
Carmen R. Valdivia ◽  
Chunhua Song ◽  
Jonathan C. Makielski
Keyword(s):  
Cell Surface ◽  
Current Density ◽  
Splice Variant ◽  
Splice Variants ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Loss Of Function ◽  
Mutant Channel ◽  
Hek 293 ◽  
293 Cells

Mutations in the cardiac Na+ channel gene SCN5A cause loss of function and underlie arrhythmia syndromes. SCN5A in humans has two splice variants, one lacking a glutamine at position 1077 (Q1077del) and one containing Q1077. We investigated the effect of splice variant background on loss of function and rescue for G1406R, a mutation reported to cause Brugada syndrome. Mutant and wild-type (WT) channels in both backgrounds were transfected into HEK-293 cells and incubated for up to 72 h with and without mexiletine. At 8 h, neither current nor cell surface expression was observed for the mutant in either background, but both were present in WT channels. At 24 h, small (<10% compared with WT) currents were noted and accompanied by cell surface expression. At 48 h, current density was ∼40% of WT channels for the mutant in the Q1077del variant background but remained at <10% of WT channels in Q1077. Current levels were stable by 72 h. Coexpression with β1- or β3-subunits or insertion of the polymorphism H558R in the background did not significantly affect current expression. Mexiletine restored current density of the mutant channel in both backgrounds to nearly WT levels. The mutant channels also showed a negative shift in inactivation, slower recovery, and enhanced slow inactivation, consistent with a loss of function phenotype. These data show that a trafficking defect may be partial and time dependent and may differ with the splice variant background. Also, expression defects and gating abnormalities may contribute to loss of function for the same mutation.

Expression and interaction of two compound heterozygous distal renal tubular acidosis mutants of kidney anion exchanger 1 in epithelial cells

2006 ◽  
Vol 291 (6) ◽  
pp. F1354-F1361 ◽  
Author(s):  
Emmanuelle Cordat ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Cell Surface ◽  
Renal Tubular Acidosis ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Compound Heterozygous ◽  
Intercalated Cells ◽  
Affinity Resin ◽  
Renal Tubular

Kidney AE1 (kAE1) is a glycoprotein responsible for the electroneutral exchange of chloride for bicarbonate, promoting the reabsorption of bicarbonate into the blood by α-intercalated cells of the collecting tubule. Mutations occurring in the gene encoding kAE1 can induce defects in urinary acidification resulting in distal renal tubular acidosis (dRTA). We expressed two kAE1 dRTA mutants, A858D, a mild dominant mutation, and ΔV850, a recessive mutation, in epithelial Madin-Darby canine kidney (MDCK) cells. Individuals heterozygous with wild-type (WT) kAE1 either did not display any symptoms of dRTA (ΔV850/WT) or displayed a mild incomplete form of dRTA (A858D/WT), while compound heterozygotes (ΔV850/A858D) had dRTA. We found that the A858D mutant was slightly impaired in the endoplasmic reticulum (ER) exit but could target to the basolateral membrane of polarized MDCK cells. Despite an altered binding to an inhibitor affinity resin, anion transport assays showed that the A858D mutant was functional at the cell surface. The ΔV850 mutant showed altered binding to the affinity resin but was predominantly retained in the ER, resulting in undetectable AE1 expression at the basolateral membrane. When coexpressed in MDCK cells, the WT protein, and to a lesser extent the A858D mutant, enhanced the cell surface expression of the ΔV850 mutant. The ΔV850 mutant also affected the cell surface expression of the A858D mutant. Compound heterozygous (A858D/ΔV850) patients likely possess a decreased amount of functional anion exchangers at the basolateral membrane of their α-intercalated cells, resulting in impaired bicarbonate transport into the blood and defective acid transport into the urine.

Interaction of anion exchanger 1 and glycophorin A in human erythroleukaemic K562 cells

2009 ◽  
Vol 421 (3) ◽  
pp. 345-356 ◽  
Author(s):  
Allison J. Pang ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Cell Surface ◽  
Anion Exchanger ◽  
K562 Cells ◽  
Surface Expression ◽  
Protein Band ◽  
Band 3 ◽  
Cell Surface Expression ◽  
Glycophorin A ◽  
Anion Exchanger 1 ◽  
Integral Proteins

AE1 [anion exchanger 1, also known as SLC4A1 (solute carrier family 4, anion exchanger, member 1) and band 3 (erythrocyte membrane protein band 3)] is a major membrane glycoprotein expressed in human erythrocytes where it mediates the exchange of chloride and bicarbonate across the plasma membrane. Glycophorin A (GPA) is a sialoglycoprotein that associates with AE1 in erythrocytes forming the Wrb (Wright b) blood group antigen. These two integral proteins may also form a complex during biosynthesis, with GPA facilitating the cell surface expression of AE1. This study investigates the interaction of GPA with AE1 in K562 cells, a human erythroleukaemic cell line that expresses GPA, and the role of GPA in the cell surface expression of AE1. In K562 cells, GPA was dimeric and N- and O-glycosylated similar to erythroid GPA. GPA was localized at the cell surface, but also localized to the Golgi. AE1 expressed in K562 cells contained both complex and high-mannose oligosaccharides, and co-localized with GPA at the cell surface and in the endoplasmic reticulum (ER). The Wrb antigen was detected at the cell surface of AE1-transfected K562 cells, indicating the existence of an AE1–GPA complex. Immunofluorescence and co-immunoprecipitation studies using AE1 and an ER-localized hereditary spherocytosis mutant (R760Q AE1) showed that GPA and AE1 could interact in the ER. GPA knockdown by shRNAs (small-hairpin RNAs), however, had no effect on the level of cell surface expression of AE1. The results indicate that AE1 and GPA form a complex in the ER of human K562 cells, but that both proteins can also traffic to the cell surface independently of each other.

scholarly journals Co-expression of metabotropic glutamate receptor type 1α with Homer-1a/Vesl-1S increases the cell surface expression of the receptor

1999 ◽  
Vol 341 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Francisco CIRUELA ◽  
Mikhail M. SOLOVIEV ◽  
R. A. Jeffrey McILHINNEY
Keyword(s):  
Cell Surface ◽  
Metabotropic Glutamate Receptor ◽  
Inositol Phosphates ◽  
Immunofluorescent Staining ◽  
Cell Surface Expression ◽  
Immunochemical Analysis ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Metabotropic Glutamate ◽  
293 Cells

Homer-1a is a 30 kDa protein that forms part of a family of conserved Homer-related proteins that interact with the C-termini of the metabotropic glutamate receptors mGluR1α and mGluR5a. Analysis of HEK-293 cells by PCR showed that they contained mRNA coding for members of the Homer family with the predominant form being Homer-1b, which is consistent with the immunochemical analysis of these cells. Homer-1a could not be detected by immunochemical analysis. To examine the function of Homer-1a, HEK-293 cells were transfected with cDNA encoding mGluR1α or Homer-1a or co-transfected with both cDNAs. When cells were co-transfected with the cDNAs for both proteins, immunofluorescent staining and biotinylation of cell surface molecules revealed a significant increase in the amount of receptor present at the cell surface in contrast to cells transfected with mGluR1α cDNA alone. This finding was consistent with a concomitant increase in the production of inositol phosphates after treatment of the doubly transfected cells with agonist. Intracellular immunostaining for both proteins revealed that they were co-localized and underwent a redistribution into a large vesicular compartment when they were co-expressed.

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