Interaction of anion exchanger 1 and glycophorin A in human erythroleukaemic K562 cells.
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.
Department of Biochemistry, University of Toronto, 1 King's College Circle, Medical Sciences Building, Toronto, Canada, M5S 1A8.
Allison J
AJ
Pang
Reinhart A F
RA
Reithmeier
D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D006021:Glycophorins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport
D001457:Anion Exchange Protein 1, Erythrocyte; D006021:Glycophorins