Flow cytometric analysis of the association between blood group‐related proteins and the detergent‐insoluble material of K562 cells and erythroid precursors

Abstract

Summary. 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, DIMassociated. 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.

Rh-RhAG/Ankyrin-R, a New Interaction Site between the Membrane Bilayer and the Red Cell Skeleton, Is Impaired by Rhnull-associated Mutation

“…It is noteworthy that the cytoplasmic tails of Rh and RhAG are particularly well conserved across species as compared with the other domains of these 12 TM proteins (37) and are capable of mediating the attachment of Lu-fusion transmembrane proteins to the cytoskeleton of K562 cells to the same extent as the whole Rh and RhAG proteins (our present results and Ref. 16). We therefore assume that interaction between these domains and ankyrin-R plays a major role in the attachment of Rh and RhAG to the red cell skeleton.…”

“…Lu-positive cells were detected by flow cytometry using the anti-Lu b mAb. The linkage between each fusion protein and the DIM was assessed by comparing the anti-Lu b antibody binding capacity of intact and Triton X-100 treated cells, as described previously (16).…”

“…2) by the DIM obtained after Triton X-100 extraction of the lipid bilayer from K562 erythroleukemic cells. Using this approach, we have estimated that 54 and 79% of Rh and RhAG molecules were DIM-associated in K562 cells, respectively (16). To evaluate the specific role of the C-terminal tails of the 12 transmembrane domain (TM) Rh and RhAG proteins in the association with the DIM of K562 cells, we have constructed fusion proteins in which the 29 and 26 C-terminal residues of Rh and RhAG, respectively, were directly fused to the single extracellular and transmembrane domains of the Lu adhesion molecule (Fig.…”

“…However, Rh and RhAG are associated with the skeleton of K562 cells and early erythroid precursors (16) (39). Potential interaction between CD47 and protein 4.2 is suggested from the deficiency of CD47 in 4.2(Ϫ)HS red cells (21,22).…”

B‐CAM/LU expression and the role of B‐CAM/LU activation in binding of low‐ and high‐density red cells to laminin in sickle cell disease

“…Therefore, to determine if the cytoskeleton is involved in regulation of B-CAM/LUmediated cell adhesion to laminin, we performed adhesion studies under both static and continuousflow conditions using MEL cells expressing B-CAM with a cytoplasmic tail truncated to five amino acids (B-CAM-ST5/MEL). This shortened cytoplasmic tail of five amino acids ( 570 CVRRK) is similar to one ( 570 CVRREF) previously shown to result in reduced B-CAM binding to the cytoskeleton [31]. Nevertheless, under static conditions, B-CAM-ST5/MEL adhered to laminin as strongly as did MEL cells expressing the entire B-CAM protein, with CSS > 10 dynes/cm 2 ( Fig.…”