ROLES OF INDIVIDUAL DOMAINS OF ANNEXIN-I IN ITS VESICLE BINDING AND VESICLE AGGREGATION - A COMPREHENSIVE MUTAGENESIS STUDY

TO understand the mechanism by which annexin I induces membrane aggreg ation, a comprehensive mutagenesis of all six Ca2+-binding sites was p erformed. When the cap residues of type II Ca2+-binding sites were sys tematically mutated to Ala, a type II site in domain II was shown to b e essential for Ca2+-dependent vesicle binding of annexin I. Domain II was not, however, directly involved in vesicle aggregation. Instead, type II sites in domains III and IV, respectively, and type III sites in domains I and IV were involved in vesicle aggregation. When all typ e II sites were deactivated, three type III sites provided residual ve sicle binding and aggregating activities. Their contributions to these activities in the presence of type II sites were, however, relatively insignificant. To further investigate the role of each domain harbori ng a type II site, a set of mutants containing only a specific type II site(s) were generated and their activities measured. These measureme nts again underscored the importance of domain II in vesicle binding o f annexin I and the involvement of domains III and IV in vesicle aggre gation. The roles of individual domains in vesicle binding and aggrega tion can be accounted for by the conformational change of membrane-bou nd annexin I involving modular rotation of domains (I/IV) following th e initial membrane adsorption of domains (II/III). In conjunction with mutagenesis studies on other annexins, these results show that indivi dual domains of annexins, although structurally homologous, have disti nct functions and that different annexins might interact with membrane s via different domains.