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Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation.

http://www.ncbi.nlm.nih.gov/pubmed/12719424

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.

Pubmed ID: 12719424 RIS Download

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

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