Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation.
Several studies suggest that the Rh complex represents a major interaction site between the membrane lipid bilayer and the red cell skeleton, but little is known about the molecular basis of this interaction. We report here that ankyrin-R is capable of interacting directly with the C-terminal cytoplasmic domain of Rh ... and RhAG polypeptides. We first show that the primary defect of ankyrin-R in normoblastosis (nb/nb) spherocytosis mutant mice is associated with a sharp reduction of RhAG and Rh polypeptides. Secondly, our flow cytometric analysis of the Triton X-100 extractability of recombinant fusion proteins expressed in erythroleukemic cell lines suggests that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid membrane skeleton. Using the yeast two-hybrid system, we demonstrate a direct interaction between the cytoplasmic tails of Rh and RhAG and the second repeat domain (D2) of ankyrin-R. This finding is supported by the demonstration that the substitution of Asp-399 in the cytoplasmic tail of RhAG, a mutation associated with the deficiency of the Rh complex in one Rhnull patient, totally impaired interaction with domain D2 of ankyrin-R. These results identify the Rh/RhAG-ankyrin complex as a new interaction site between the red cell membrane and the spectrin-based skeleton, the disruption of which might result in the stomato-spherocytosis typical of Rhnull red cells.
Mesh Terms:
Animals, Ankyrins, Blood Proteins, Blotting, Western, Calmodulin-Binding Proteins, Cytoplasm, Detergents, Electrophoresis, Polyacrylamide Gel, Erythrocytes, Flow Cytometry, Glutathione Transferase, Humans, K562 Cells, Lipid Bilayers, Membrane Glycoproteins, Mice, Mice, Inbred BALB C, Mice, Mutant Strains, Models, Biological, Octoxynol, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, Rh-Hr Blood-Group System, Transfection, Two-Hybrid System Techniques
Animals, Ankyrins, Blood Proteins, Blotting, Western, Calmodulin-Binding Proteins, Cytoplasm, Detergents, Electrophoresis, Polyacrylamide Gel, Erythrocytes, Flow Cytometry, Glutathione Transferase, Humans, K562 Cells, Lipid Bilayers, Membrane Glycoproteins, Mice, Mice, Inbred BALB C, Mice, Mutant Strains, Models, Biological, Octoxynol, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, Rh-Hr Blood-Group System, Transfection, Two-Hybrid System Techniques
J. Biol. Chem.
Date: Jul. 11, 2003
PubMed ID: 12719424
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