Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta spectrin: regulation of the interactions by phosphatidylinositol-4,5-bisphosphate.

The ternary complex of spectrin, F-actin, and protein 4.1R defines the erythrocyte membrane skeletal network, which governs the stability and elasticity of the membrane. It has been shown that both 4.1R and actin bind to the N-terminal region (residues 1-301) of the spectrin beta chain, which contains two calponin homology domains, designated CH1 and CH2. Here, we show that 4.1R also binds to the separate CH1 and CH2 domains. Unexpectedly, truncation of the CH2 domain by its 20 amino acids, corresponding to its N-terminal alpha helix, was found to greatly enhance its binding to 4.1R. The intact N terminus and the CH1 but not the CH2 domain bind to F-actin, but again, deletion of the first 20 amino acids of the latter exposes an actin-binding activity. As expected, the polypeptide 1-301 inhibits the binding of spectrin dimer to actin and formation of the spectrin-actin-4.1R ternary complex in vitro. Furthermore, the binding of 4.1R to 1-301 is greatly enhanced by PIP(2), implying the existence of a regulatory switch in the cell.