Identification of the Spectrin Subunit and Domains Required for Formation of Spectrin/Adducin/Actin Complexes*

Abstract

Adducin is an actin-binding protein that has been proposed to function as a regulated assembly factor for the spectrin/actin network. This study has addressed the question of the subunit and domains of spectrin required for formation of spectrin/adducin/actin complexes in in vitro assays. Quantitative evidence is presented that the β-spectrin N-terminal domain plus the first two α-helical domains are required for optimal participation of spectrin in spectrin/adducin/actin complexes. The α subunit exhibited no detectable activity either alone or following association with β-spectrin. The critical domains of β-spectrin involved in complex formation were determined using recombinant proteins expressed in bacteria. The N-terminal domain (residues 1–313) of β-spectrin associated with F-actin with a K_d of 26 μM, and promoted adducin binding to F-actin with half-maximal activation at 110 nM. Addition of the first α-helical domain (residues 1–422) lowered the K_d for F-actin by 4-fold to 6 μM, but also reduced the capacity by 3-fold and had no effect on interaction with adducin. Further addition of the second α-helical domain (residues 1–528) did not alter binding to F-actin but resulted in a 2-fold increased activity in promoting adducin binding with half-maximal activation at 50 nM. Addition of up to eight additional α-helical domains (residues 1-1388) resulted in no further change in F-actin binding or association with adducin. These results demonstrate an unanticipated role of the first repeat of β-spectrin in actin binding activity and of the second repeat in association with adducin/actin, and imply the possibility of an extended contact between adducin, spectrin, and actin involving several actin subunits.