Identification of two regions of beta G spectrin that bind to distinct sites in brain membranes.
This study analyzed the complex interactions of intact spectrin with bovine brain membranes by evaluating membrane associations of defined regions of beta G spectrin, the subunit responsible for high affinity membrane binding. Two regions of beta G spectrin were expressed in bacteria and demonstrated to contain fully functional binding site(s) for a subset of spectrin-binding sites in brain membranes depleted of peripheral proteins. One region, located near the NH2 terminus, was comprised of 106-residue repeats and required repeats 2-7 for full activity. The other binding domain was located at the COOH terminus, which is the most variable between beta G and beta R spectrins, is distinct from the 106-residue repeats, and contains a pleckstrin homology domain. NH2-terminal beta spectrin polypeptides interacted with a membrane site(s) that recognized both brain and erythrocyte isoforms of spectrin, was inhibited by calcium/calmodulin, and was not blocked by the COOH-terminal polypeptide. The COOH-terminal region associated with a membrane site(s) that was specific for brain spectrin, was not inhibited by calcium/calmodulin, and was not blocked by the NH2-terminal polypeptide. These observations demonstrate membrane association of spectrin with at least two independent sites, which differ with regard to regulation by calcium/calmodulin and in selectivity for spectrin isoforms.
Davis
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LH
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
Bennett
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D000818:Animals; D017487:Ankyrins / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D066298:In Vitro Techniques; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013049:Spectrin / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D017487:Ankyrins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013049:Spectrin