Calmodulin-Binding Domain of Recombinant Erythrocyte β-Adducin

• Published in: Proceedings of the National Academy of Sciences - PNAS 1993-04, Vol.90 (8), p.3398-3402
• Main Authors: Scaramuzzino, Dominick A., Morrow, Jon S.
• Format: Article
• Language: English
• Subjects: Actins; Amino Acid Sequence; Amino acids; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; beta -adducin; binding; Binding Sites; Biological and medical sciences; Blood Proteins - chemistry; Blood Proteins - genetics; Blood Proteins - metabolism; calcium; Calmodulin - metabolism; Calmodulin-Binding Proteins - chemistry; Calmodulin-Binding Proteins - genetics; Calmodulin-Binding Proteins - metabolism; Calpain; Cattle; Cloning, Molecular; Complementary DNA; Cytoskeleton; DNA - genetics; DNA - isolation & purification; domains; Erythrocytes; Erythrocytes - metabolism; Fundamental and applied biological sciences. Psychology; Half lives; Kinetics; Macromolecular Substances; Miscellaneous; Molecular Sequence Data; Molecules; Oligodeoxyribonucleotides; Pests; Phosphorylation; Protein Structure, Secondary; Proteins; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Restriction Mapping; sensitivity; Trypsin
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.8.3398

Adducin is a 200-kDa heterodimeric protein of the cortical cytoskeleton of mammalian erythrocytes. Analogs are also abundant in brain and several other tissues. In vitro, adducin bundles F-actin and enhances the binding of spectrin to actin. Previous studies have established that the β subunit of adducin binds calmodulin (CaM) in a Ca2+-dependent fashion with intermediate affinity (≈200 nM) and that this activity is destroyed by proteolysis. We have confirmed the trypsin sensitivity of CaM binding by β-adducin and the existence of a 38- to 39-kDa protease-resistant core. Calpain I digestion generates a larger core fragment (49 kDa) that is also devoid of CaM-binding activity. Use of recombinant β-adducin peptides generated from partial cDNA clones identified strong CaM-binding activity within the protease-sensitive domain in residues 425-461: KQQKEKTRWLNTPNTYLRVNVADEVQRNMGSPRPKTT in single-letter amino acid codes. This region of the molecule is highly conserved between mouse, rat, and human and shares structural features with CaM-binding sequences in other proteins. Multiple flanking PEST sequences (sequences rich in proline, glutamic acid, serine, and threonine residues that enhance proteolytic sensitivity) may contribute to the protease sensitivity of this region. Consensus sequences for phosphorylation by cAMP-dependent kinases and by protein kinase C (or CaM-dependent kinase) are also found within or near this CaM-binding domain. Collectively, these data suggest a structural basis for the regulation of adducin by Ca2+-dependent CaM binding and possibly by covalent phosphorylation and calpain I-mediated proteolysis as well.