Biochemical Analysis of Potential Sites for Protein 4.1-mediated Anchoring of the Spectrin-Actin Skeleton to the Erythrocyte Membrane

Abstract

Erythrocyte protein 4.1 has been hypothesized to link the spectrin-actin junctional complex directly to the cytoplasmic domain of glycophorin C, but this bridging function has never been directly demonstrated. Because an alternative protein-mediated bridge between the junctional complex and the cytoplasmic domain of band 3 is also plausible, we have undertaken to characterize the membrane sites to which protein 4.1 can anchor the spectrin and actin skeleton. We demonstrate that proteolytic removal of the cytoplasmic domain of band 3 has minimal effect on the ability of protein 4.1 to promote 125 I-labeled spectrin and actin binding to KI-stripped erythrocyte membrane vesicles. We also show that quantitative blockade of all band 3 sites with either monoclonal or polyclonal antibodies to band 3 is equally ineffective in preventing protein 4.1-mediated association of spectrin and actin with the membrane. In contrast, obstruction of protein 4.1 binding to its docking site on the cytoplasmic pole of glycophorin C is demonstrated to reduce the same protein 4.1 bridging function by ϳ85%. We conclude from these data that (i) glycophorin C contributes the primary anchoring site of the protein 4.1-mediated bridge to the spectrin-actin skeleton; (ii) band 3 is incapable of serving the same function; and (iii) additional minor protein 4.1 bridging sites may exist on the human erythrocyte membrane.

Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant

“…After further extensive washes, the blots were visualized using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Piscataway, NJ). ANK-1 antibodies were kindly provided by Connie Birkenmeier; ␤1 spectrin was purchased from Abcam (ab2808; Cambridge, MA); and actin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) from Santa Cruz Biotechnology (sc-1616; Santa Cruz, CA). All other antibodies were generously provided by Xiuli An.…”

“…2,3 In the erythrocyte, tetramers of ␣-spectrin and ␤-spectrin heterodimers are linked to the lipid bilayer through associations with 2 multiprotein complexes: the ankyrin, band 3, and protein 4.2 complex; and the protein 4.1, p55, and glycophorin C complex. [4][5][6][7][8][9] These associations are required for red cells to maintain their shape, and to withstand the physical forces imposed on them during circulation. Disruption of these interactions leads to fragmentation, accumulation of dysmorphic red cells in the spleen, and hemolytic anemia.…”

Identification of quantitative trait loci that modify the severity of hereditary spherocytosis in wan, a new mouse model of band-3 deficiency

“…[1][2][3] There are 2 major interactions between membrane skeleton components and integral membrane proteins that attach the spectrin array to the plasma membrane: (1) band 3-spectrin-ankyrin-protein 4.2 and (2) protein 4.1-p55-glycophorin C linkages. [4][5][6][7][8] The band 3-spectrin-ankyrin-protein 4.2 interactions are critical in the pathogenesis of HS. Defects in all of these proteins cause HS in humans and in mice.…”

CASK and Protein 4.1 Support F-actin Nucleation on Neurexins

“…This trimeric complex therefore provides two binding sites for protein 4.1, one found on the cytosolic domain of glycophorin C, the other on p55, which binds the extreme C terminus of glycophorin C via its PDZ domain (26). As a result, the majority of protein 4.1 associates with the plasma membrane at sites where glycophorin C is localized (25,27). Interestingly, erythrocytes lacking protein 4.1 show a secondary loss of p55, hDlg, and glycophorin C (28,29).…”