Protein 4.1R-dependent multiprotein complex: new insights into the structural organization of the red blood cell membrane

Abstract

Protein 4.1R (4.1R) is a multifunctional component of the red cell membrane. It forms a ternary complex with actin and spectrin, which defines the nodal junctions of the membrane-skeletal network, and its attachment to the transmembrane protein glycophorin C creates a bridge between the protein network and the membrane bilayer. We now show that deletion of 4.1R in mouse red cells leads to a large diminution of actin accompanied by extensive loss of cytoskeletal lattice structure, with formation of bare areas of membrane. Whereas band 3, the preponderant transmembrane constituent, and proteins known to be associated with it are present in normal or increased amounts, glycophorin C is missing and XK, Duffy, and Rh are much reduced in the 4.1R-deficient cells. The inference that these are associated with 4.1R was borne out by the results of in vitro pull-down assays. Furthermore, whereas Western blot analysis showed normal levels of band 3 and Kell, flow cytometric analysis using an antibody against the extracellular region of band 3 or Kell revealed reduction of these two proteins, suggesting a conformational change of band 3 and Kell epitopes. Taken together, we suggest that 4.1R organizes a macromolecular complex of skeletal and transmembrane proteins at the junctional node and that perturbation of this macromolecular complex not only is responsible for the well characterized membrane instability but may also remodel the red cell surface.

Fig. 1.

Immunoblots of membrane skeletal proteins in red cells of 4.1R^+/+ and 4.1R^−/− mice. Blots of SDS/PAGE of total membrane protein were probed with antibodies against the indicated proteins. Note the absence of 4.1R, as well as p55 in the 4.1R-deficient cells, the reduced actin concentration, and the elevated tropomyosin and adducin.

Fig. 2.

Electron micrographs of membrane skeletons of red cells of 4.1R^+/+ and 4.1R^−/− mice. (Left) Membrane skeleton of wild-type cells. (Right) Membrane skeleton of 4.1R-deficient cells. Note deficient membrane junctions in the mutant cells and large bare areas.

Fig. 3.

Immunoblots of transmembrane proteins in red cells of 4.1R^+/+ and 4.1R^−/− mice. Blots of SDS/PAGE of total membrane protein were probed with antibodies against the indicated proteins. Note the disappearance of GPC and diminution of Rh, XK, and Duffy proteins in the 4.1R-deficient mice and the increase of GPA, LW, and NHE1 expression.

Fig. 4.

Flow cytometric analysis of red cell membrane proteins of 4.1R^+/+ and 4.1R^−/− mice. The ordinate measures the number of cells displaying the fluorescent intensity given by the abscissa. Black lines, 4.1R^+/+; gray lines, 4.1R^−/−; dotted lines, negative control.

Fig. 5.

Direct interaction of 4.1R with Rh, XK, and Duffy. The binding of 4.1R 80-kDa and its functional domains to cytoplasmic tails of XK, Duffy, and Rh was assessed by streptavidin pull-down assay. The binding of 4.1R, GST-tagged domains of 4.1R, and MBP-tagged subdomains of 30-kDa to the cytoplasmic domains of XK, Duffy, and Rh was detected by using anti-4.1R antibody, anti-GST antibody, and anti-MBP antibody, respectively. Note the binding of all three proteins to 30-kDa, the binding of Rh to lobe A, and the binding of XK and Duffy to lobe B.

Fig. 6.

Schematic representation of two types of multiprotein complexes in the red cell membrane. (Left) Protein complex attached to spectrin near the center of the tetramer (dimer–dimer interaction site). Tetrameric band 3 is bound to ankyrin, which is bound to spectrin. The membrane skeletal protein 4.2 has binding sites for band 3 and for ankyrin. Transmembrane glycoproteins GPA, Rh, and RhAG bind to band 3, and CD47 and LW associate with Rh/RhAG. The two cytoplasmic domains of band 3 contain binding sites for soluble proteins, the short C-terminal domain for CA II, the large N-terminal domain for deoxyhemoglobin and for glycolytic enzymes, aldolase, phosphofructokinase (PFK), and glyceraldehyde 3′-phosphate dehydrogenase (GAPDH). (Right) Protein complex at membrane skeletal junctions. The junctions contain the ternary complex of spectrin, F-actin, and 4.1R, as well as the actin-binding proteins tropomyosin, tropomodulin, adducin, and dematin. 4.1R enters into an additional ternary interaction with the transmembrane protein GPC and p55 and is taken also to bind to band 3, in the form of a dimer, which also carries GPA. Rh, Kell, and XK also have binding sites on 4.1R. Note, however, that the copy numbers of all transmembrane proteins except GPA and GPC are low and therefore will not be present on all complexes.