Characterization of the Ca2+-binding sites of annexin II tetramer.
Annexin II heterotetramer (AIIt) is a multifunctional Ca(2+)-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains three type II and two type III Ca(2+)-binding sites which are thought to regulate the interaction of AIIt with anionic phospholipid, F-actin, and heparin. In the present study we utilized site-directed mutagenesis to create AIIt mutants with inactive type III (TM AIIt), type II (CM AIIt), and both type II and III Ca(2+)-binding sites (TCM AIIt). Surprisingly, we found that in the presence of Ca(2+), the TM, CM, and TCM AIIt bound phospholipid and F-actin with similar affinity to the wild type AIIt (WT AIIt). Furthermore, the TCM mutant, and to a lesser extent the TM and CM AIIt displayed dose-dependent Ca(2+)-independent phospholipid aggregation and binding. While the TM and CM AIIt demonstrated Ca(2+)-dependent binding to F-actin, the binding of the TCM AIIt was Ca(2+)-independent. These results suggest that the type II or type III Ca(2+)-binding sites do not directly participate in anionic phospholipid or F-actin binding. We therefore propose that in the absence of Ca(2+), the type II and type III Ca(2+)-binding sites of AIIt stabilize a conformation of AIIt that is unfavorable for binding phospholipid and F-actin. Ca(2+) binding to these sites, or the inactivation of these Ca(2+)-binding sites by site-directed mutagenesis, results in a conformational change that promotes binding to anionic phospholipid and F-actin. Since the TM, CM, and TCM AIIt require Ca(2+) for binding to heparin, we also propose that novel Ca(2+)-binding sites regulate this binding event.
Filipenko
N R
NR
Cancer Biology Research Group, Departments of Biochemistry & Molecular Biology and Oncology, University of Calgary, Calgary, Alberta, Canada.
Kang
H M
HM
Waisman
D M
DM
D000199:Actins / Q000378:metabolism*; D019943:Amino Acid Substitution; D017306:Annexin A2 / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D017306:Annexin A2; D008081:Liposomes; D046911:Macromolecular Substances; D010743:Phospholipids; D021122:Protein Subunits; D011994:Recombinant Proteins; D002118:Calcium