scholarly journals Functional cell surface expression of the anion transport domain of human red cell band 3 (AE1) in the yeast Saccharomyces cerevisiae.

1996 ◽  
Vol 93 (22) ◽  
pp. 12245-12250 ◽  
Author(s):  
J. D. Groves ◽  
P. Falson ◽  
M. le Maire ◽  
M. J. Tanner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Surface Expression ◽  
Band 3 ◽  
Anion Transport ◽  
Cell Surface Expression ◽  
Red Cell ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transport Domain

scholarly journals Red-cell glycophorin A–band 3 interactions associated with the movement of band 3 to the cell surface

2000 ◽  
Vol 350 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Mark T. YOUNG ◽  
Roland BECKMANN ◽  
Ashley M. TOYE ◽  
Michael J. A. TANNER
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Band 3 ◽  
Cell Surface Expression ◽  
Glycophorin A ◽  
Detergent Solution ◽  
Red Cell ◽  
Cell Clones ◽  
Absolute Requirement ◽  
Dimerization Interface

We have examined the mechanism by which glycophorin A (GPA) facilitates the movement of the human red-cell anion exchanger (band 3, AE1) to the cell surface. GPA itself forms stable dimers in membranes and detergent solution. Four mutants of human GPA with impaired dimerization were prepared (L75I, I76A, G79L and G83L). All four GPA mutants enhanced band 3 translocation to the Xenopus oocyte plasma membrane in the same way as wild-type GPA, showing that the GPA monomer is sufficient to mediate this process. Cell-surface expression of the natural band 3 mutant G701D has an absolute requirement for GPA. GPA monomers also rescued the cell-surface expression of this mutant band 3. Taking into account other evidence, we infer that the site of GPA interaction with band 3 is located outside the GPA dimerization interface but within the GPA transmembrane span. The results of examination of the cell-surface expression of GPA and band 3 in different K562 erythroleukaemia cell clones stably transfected with band 3 are consistent with the movement of GPA and band 3 to the cell surface together. We discuss the pathways by which band 3 moves to the cell surface in the presence and the absence of GPA, concluding that GPA has a role in enhancing the folding and maturation of band 3. We propose that GPA functions in erythroid cells to assist with the incorporation of large amounts of properly folded band 3 into the membrane within a limited time span during erythroid maturation.

Functional Cell Surface Expression of Band 3, the Human Red Blood Cell Anion Exchange Protein (AE1), in K562 Erythroleukemia Cells: Band 3 Enhances the Cell Surface Reactivity of Rh Antigens

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4428-4438 ◽  
Author(s):  
Roland Beckmann ◽  
Jonathan S. Smythe ◽  
David J. Anstee ◽  
Michael J.A. Tanner
Keyword(s):  
Cell Surface ◽  
Chloride Transport ◽  
K562 Cells ◽  
Surface Expression ◽  
Band 3 ◽  
Surface Reactivity ◽  
Cell Surface Expression ◽  
Anion Exchanger 1 ◽  
Erythroleukemia Cells ◽  
Antigen Activity

Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe retroviral vector. Stable K562 clones expressing band 3 were isolated by flow cytometry, and surface expression was quantified by immunoblotting. The function of band 3 expressed at the cell surface was demonstrated in chloride transport assays. K562 cells expressing band 3 also displayed high levels of the Wrb blood group antigen, confirming the role of band 3 in Wrb expression, and an increase in the low levels of endogenous Rh antigen activity. We also performed coexpression experiments with K562 clones that had previously been transduced with cDNAs encoding RhD or RhcE polypeptides. The transfection and expression of band 3 in these clones substantially increased the levels of RhD and cE antigen activity expressed on the cells and also increased the reactivity of the cells with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The increased reactivity of Rh antigens may result from cell surface or intracellular interactions of band 3 with the protein complex which contains the Rh polypeptides and RhGP, or from indirect effects of band 3 on the membrane environment. This work establishes a system for cell surface expression of band 3 in a mammalian cell line, which will enable further studies of the protein and its interactions with other membrane components.

scholarly journals PDZK1 binding and serine phosphorylation regulate subcellular trafficking of organic anion transport protein 1a1

2011 ◽  
Vol 300 (3) ◽  
pp. G384-G393 ◽  
Author(s):  
Jo H. Choi ◽  
John W. Murray ◽  
Allan W. Wolkoff
Keyword(s):  
Cell Surface ◽  
Subcellular Distribution ◽  
Organic Anion ◽  
Surface Expression ◽  
Anion Transport ◽  
Transport Protein ◽  
Serine Phosphorylation ◽  
Cell Surface Expression ◽  
Organic Anion Transport ◽  
Huh7 Cells

Although perturbation of organic anion transport protein (oatp) cell surface expression can result in drug toxicity, little is known regarding mechanisms regulating its subcellular distribution. Many members of the oatp family, including oatp1a1, have a COOH-terminal PDZ consensus binding motif that interacts with PDZK1, while serines upstream of this site (S634 and S635) can be phosphorylated. Using oatp1a1 as a prototypical member of the oatp family, we prepared plasmids in which these serines were mutated to glutamic acid [E634E635 (oatp1a1EE), phosphomimetic] or alanine [A634A635 (oatp1a1AA), nonphosphorylatable]. Distribution of oatp1a1AA and oatp1a1EE was largely intracellular in transfected human embryonic kidney (HEK) 293T cells. Cotransfection with a plasmid encoding PDZK1 revealed that oatp1a1AA was now expressed largely on the cell surface, while oatp1a1EE remained intracellular. To quantify these changes, studies were performed in HuH7 cells stably transfected with these oatp1a1 plasmids. These cells endogenously express PDZK1. Surface biotinylation at 4°C followed by shift to 37°C showed that oatp1a1EE internalizes quickly compared with oatp1a1AA. To examine a physiological role for phosphorylation in oatp1a1 subcellular distribution, studies were performed in rat hepatocytes exposed to extracellular ATP, a condition that stimulates serine phosphorylation of oatp1a1 via activity of a purinergic receptor. Internalization of oatp1a1 under these conditions was rapid. Thus, although PDZK1 binding is required for optimal cell surface expression of oatp1a1, phosphorylation provides a mechanism for fast regulation of the distribution of oatp1a1 between the cell surface and intracellular vesicular pools. Identification of the proteins and motor molecules that mediate these trafficking events represents an important area for future study.

Functional Cell Surface Expression of Band 3, the Human Red Blood Cell Anion Exchange Protein (AE1), in K562 Erythroleukemia Cells: Band 3 Enhances the Cell Surface Reactivity of Rh Antigens

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4428-4438 ◽  
Author(s):  
Roland Beckmann ◽  
Jonathan S. Smythe ◽  
David J. Anstee ◽  
Michael J.A. Tanner
Keyword(s):  
Cell Surface ◽  
Chloride Transport ◽  
K562 Cells ◽  
Surface Expression ◽  
Band 3 ◽  
Surface Reactivity ◽  
Cell Surface Expression ◽  
Anion Exchanger 1 ◽  
Erythroleukemia Cells ◽  
Antigen Activity

Abstract Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe retroviral vector. Stable K562 clones expressing band 3 were isolated by flow cytometry, and surface expression was quantified by immunoblotting. The function of band 3 expressed at the cell surface was demonstrated in chloride transport assays. K562 cells expressing band 3 also displayed high levels of the Wrb blood group antigen, confirming the role of band 3 in Wrb expression, and an increase in the low levels of endogenous Rh antigen activity. We also performed coexpression experiments with K562 clones that had previously been transduced with cDNAs encoding RhD or RhcE polypeptides. The transfection and expression of band 3 in these clones substantially increased the levels of RhD and cE antigen activity expressed on the cells and also increased the reactivity of the cells with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The increased reactivity of Rh antigens may result from cell surface or intracellular interactions of band 3 with the protein complex which contains the Rh polypeptides and RhGP, or from indirect effects of band 3 on the membrane environment. This work establishes a system for cell surface expression of band 3 in a mammalian cell line, which will enable further studies of the protein and its interactions with other membrane components.

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.

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