In vitro proteolysis of brain spectrin by calpain I inhibits association of spectrin with ankyrin-independent membrane binding site(s).
This report demonstrates that specific proteolysis of brain spectrin by a calcium-dependent protease, calpain I, abolishes association of brain spectrin with the ankyrin-independent binding site(s) in brain membranes. Calpain I cleaves the beta subunit of spectrin at the N-terminal end leaving a 218-kDa fragment and cleaves the alpha subunit in the midregion to produce 150- and 130-kDa fragments. Calpain-proteolyzed spectrin almost completely loses the capacity to displace binding of intact spectrin to membranes. Spectrin digested by calpain I under conditions that almost completely destroyed membrane-binding remained associated as a tetramer and retained about 60% of the ability to associate with actin filaments. Cleavage of spectrin occurred at sites distinct from the membrane-binding site which is located on the beta subunit since the isolated 218-kDa fragment of the beta subunit as well as a reconstituted complex of alpha and 218-kDa beta subunit fragment partially regained binding activity. Moreover, cleavage of the alpha subunit alone reduced the affinity of spectrin for membranes by 2-fold. A consequence of distinct sites for calpain I cleavage and membrane-binding is that calpain I can digest spectrin while spectrin is complexed with other proteins and therefore has the potential to mediate disassembly of a spectrin-actin network from membranes.
Hu
R J
RJ
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
Bennett
V
V
D000199:Actins / Q000378:metabolism; D000818:Animals; D017487:Ankyrins; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D013049:Spectrin / Q000378:metabolism* / Q000648:ultrastructure
D000199:Actins; D017487:Ankyrins; D001798:Blood Proteins; D008565:Membrane Proteins; D013049:Spectrin; D002154:Calpain