Identification of the spectrin subunit and domains required for formation of spectrin/adducin/actin complexes.
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.
Li
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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Bennett
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D000199:Actins / Q000737:chemistry*; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002417:Cattle; D002645:Chickens; D046911:Macromolecular Substances; D011485:Protein Binding; D011817:Rabbits; D013049:Spectrin / Q000737:chemistry*; D013329:Structure-Activity Relationship; D000099291:Adducins
D000199:Actins; D002148:Calmodulin-Binding Proteins; D046911:Macromolecular Substances; D013049:Spectrin; D000099291:Adducins