Adducin Forms a Bridge between the Spectrin-Actin Junctional Complex and Band 3.

The classical model of the human erythrocyte membrane (RBCM) shows two bridges connecting the lipid bilayer to the membrane skeleton: 1) a bridge attaching the cytoplasmic domain of band 3 (CDB3) to ankyrin, which in turn binds β-spectrin, and 2) a bridge linking the cytoplasmic domain of glycophorin C to protein 4.1, which in turn binds the spectrin-actin junctional complex. Recent data, however, suggest while disruption of the band 3-ankyrin- β-spectrin interaction profoundly alters membrane stability, disruption of the glycophorin C-protein 4.1R linkage has no effect on membrane mechanical properties. In a search for additional bridges between the junctional complex and the bilayer, we discovered that adducin, a component of the spectrin-actin junctional complex, binds to band 3. Evidence for this interaction derives from a number of different experimental strategies. We have been able to show that: i) photoactivation of sulfo-SBED-labeled adducin reconstituted onto the RBCM leads to label transfer to band 3, ii) adducin binds to KI-stripped IOVs with a Kd of 280 nM and this binding is inhibited by an antibody to CDB3 (as well as by unlabeled adducin), iii) IOVs derived from normal erythrocytes retain adducin, whereas similar IOVs prepared from erythrocytes deficient in band 3 retain very little adducin, iv) the tail domain (but not the headpiece domain) of β-adducin binds to KI-stripped IOVs and this binding is competed by both anti-CDB3 and intact adducin, v) GST-labeled β-adducin tail domain can pull down band 3 in co-pelleting studies and this co-precipitation is blocked by anti-CDB3, and vi) the tail domain of β-adducin directly binds CDB3. Because adducin is an important structural component of the junctional complex, these data suggest that the junctional complex is linked to the RBCM via CDB3 and that part of the band 3 population must be located adjacent to the junctional complex. This putative new bridge between the RBCM and the spectrin-actin skeleton may also help explain why β-adducin knockout mice have unstable erythrocyte membranes.