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.
INSERM U76, Institut National de la Transfusion Sanguine, 6 Rue Alexandre Cabanel, 75015 Paris, France.
Virginie
V
Nicolas
Caroline
C
Le Van Kim
Pierre
P
Gane
Connie
C
Birkenmeier
Jean-Pierre
JP
Cartron
Yves
Y
Colin
Isabelle
I
Mouro-Chanteloup
D000818:Animals; D017487:Ankyrins / Q000737:chemistry* / Q000378:metabolism*; D001798:Blood Proteins; D015153:Blotting, Western; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D008051:Lipid Bilayers / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008817:Mice, Mutant Strains; D008954:Models, Biological; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012204:Rh-Hr Blood-Group System / Q000737:chemistry; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000099291:Adducins
D017487:Ankyrins; D001798:Blood Proteins; D002148:Calmodulin-Binding Proteins; D003902:Detergents; D005982:Glutathione Transferase; D008051:Lipid Bilayers; D008562:Membrane Glycoproteins; D017830:Octoxynol; D011993:Recombinant Fusion Proteins; D012204:Rh-Hr Blood-Group System; D000099291:Adducins; C110202:ANK1 protein, human; C491707:Ank1 protein, mouse; C498523:RHAG protein, human; C401210:Rhag protein, mouse