Absence of high-affinity band 4.1 binding sites from membranes of glycophorin C- and D-deficient (Leach phenotype) erythrocytes.
We investigated the role of glycophorins C and D in the association of band 4.1 with the erythrocyte membrane by measuring the binding of band 4.1 to erythrocyte inside-out vesicles stripped of endogenous band 4.1. Vesicles were prepared from either normal erythrocytes or erythrocytes completely lacking glycophorins C and D (Leach phenotype). Band 4.1 binding to vesicles from normal erythrocytes gave rise to a nonlinear Scatchard plot, indicative of two classes of binding sites: a low-capacity, high-affinity class of sites (about 10% of the total) and a high-capacity, low-affinity class of sites. Vesicles prepared from Leach erythrocytes had a binding capacity for band 4.1 that was, on average, 32% lower than that of vesicles from normal erythrocytes. This difference was caused by the complete absence of the high-affinity binding sites as well as by a decrease in the number of low-affinity binding sites. Reduction of membrane phosphatidylinositol 4,5-biphosphate (PIP2) content by adenosine triphosphate depletion or activation of phosphoinositidase C resulted in a decrease in band 4.1 binding capacity to a similar extent in both control and Leach vesicles. The principal effect of PIP2 depletion was a reduction in the number of low-affinity band 4.1 binding sites in control and Leach vesicles. The fact that PIP2 depletion induced a decrease in band 4.1 binding to Leach vesicles shows that glycophorin C or D is not required for the formation of PIP2-sensitive band 4.1 binding sites, and may not be involved in PIP2-sensitive band 4.1 binding sites even when they are present. Our studies give new insights into the involvement of glycophorins and of PIP2 in modulating cytoskeletal-membrane interactions.
Gascard
P
P
Department of Biomedical Research, St Elizabeth's Medical Center, Boston, MA 02135.
Cohen
C M
CM
D000255:Adenosine Triphosphate / Q000097:blood; D003598:Cytoskeletal Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology / Q000378:metabolism*; D006021:Glycophorins / Q000172:deficiency*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008563:Membrane Lipids / Q000097:blood / Q000302:isolation & purification; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009479:Neuropeptides; D010743:Phospholipids / Q000097:blood / Q000302:isolation & purification; D010727:Phosphoric Diester Hydrolases / Q000097:blood
D003598:Cytoskeletal Proteins; D006021:Glycophorins; D008563:Membrane Lipids; D008565:Membrane Proteins; D009479:Neuropeptides; D010743:Phospholipids; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D000255:Adenosine Triphosphate; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase