Flow cytometric analysis of the association between blood group-related proteins and the detergent-insoluble material of K562 cells and erythroid precursors.
The linkage between blood group-related cell surface proteins and the detergent-insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody-binding capacity of intact and Triton-X100-treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton-insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton-insoluble. Glycophorin A (GPA), however, behaved as Triton-soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton-insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM-associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIM-associated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.
Gane
P
P
INSERM U76, Institut National de la Transfusion Sanguine, Paris, France.
Le Van Kim
C
C
Bony
V
V
El Nemer
W
W
Mouro
I
I
Nicolas
V
V
Colin
Y
Y
Cartron
J P
JP
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D000953:Antigens, Protozoan; D000954:Antigens, Surface / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism; D051928:CD47 Antigen; D002352:Carrier Proteins / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D003902:Detergents / Q000378:metabolism*; D004375:Duffy Blood-Group System; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006021:Glycophorins / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells / Q000378:metabolism*; D008188:Lutheran Blood-Group System; D008565:Membrane Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D017830:Octoxynol / Q000378:metabolism*; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface / Q000378:metabolism; D012204:Rh-Hr Blood-Group System; D012995:Solubility
C473745:ACKR1 protein, human; D001457:Anion Exchange Protein 1, Erythrocyte; D015703:Antigens, CD; D000953:Antigens, Protozoan; D000954:Antigens, Surface; C090228:BCAM protein, human; D001798:Blood Proteins; D051928:CD47 Antigen; C497894:CD47 protein, human; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D003902:Detergents; D004375:Duffy Blood-Group System; C066001:Duffy antigen binding protein, Plasmodium; D006021:Glycophorins; D008188:Lutheran Blood-Group System; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; D012204:Rh-Hr Blood-Group System; C041106:kell-active proteins; D017830:Octoxynol