Identification of the spectrin subunit and domains required for formation of spectrin/adducin/actin complexes.

Journal Article

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 beta-spectrin N-terminal domain plus the first two alpha-helical domains are required for optimal participation of spectrin in spectrin/adducin/actin complexes. The alpha subunit exhibited no detectable activity either alone or following association with beta-spectrin. The critical domains of beta-spectrin involved in complex formation were determined using recombinant proteins expressed in bacteria. The N-terminal domain (residues 1-313) of beta-spectrin associated with F-actin with a Kd of 26 microM, and promoted adducin binding to F-actin with half-maximal activation at 110 nM. Addition of the first alpha-helical domain (residues 1-422) lowered the Kdfor F-actin by 4-fold to 6 microM, but also reduced the capacity by 3-fold and had no effect on interaction with adducin. Further addition of the second alpha-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 alpha-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 beta-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.

Full Text

Duke Authors

Cited Authors

  • Li, X; Bennett, V

Published Date

  • June 28, 1996

Published In

Volume / Issue

  • 271 / 26

Start / End Page

  • 15695 - 15702

PubMed ID

  • 8663089

International Standard Serial Number (ISSN)

  • 0021-9258

Language

  • eng

Conference Location

  • United States