2 FUNCTIONALLY DIFFERENT DOMAINS OF RABPHILIN-3A, RAB3A P25 SMG P25A-BINDING AND PHOSPHOLIPID-BINDING AND CA2+-BINDING DOMAINS/

Rabphilin-3A is a putative target molecule for rab3A p25/smg p25A, whi ch is a member of a ras p21-related small GTP-binding protein and impl icated in neurotransmitter release from the synapse. Rabphilin-3A has two copies of an internal repeat that are homologous to the C2 domains of protein kinase C, synaptotagmin, and phospholipase A2, which are k nown to bind to phospholipid in a Ca2+-dependent manner. In the curren t study, we have investigated the functional domains of rabphilin-3A b y use of three recombinant proteins as follows: full rabphilin-3A (1-7 04 amino acids), an N-terminal fragment (1-280 amino acids), and a C-t erminal fragment containing the C2 domains (281-704 amino acids). Both rabphilin-3A and the C-terminal fragment bound to phospholipid in the presence of Ca2+, but the N-terminal fragment did not bind to phospho lipid. Ca-45(2+) bound to rabphilin-3A and the C-terminal fragment onl y in the presence of phospholipid but did not bind to the N-terminal f ragment. The GTPgammaS-bound form of rab3A p25 bound to both rabphilin -3A and the N-terminal fragment but did not bind to the C-terminal fra gment. These results indicate that rabphilin-3A has at least two funct ionally different domains, the N-terminal rab3A p25-binding and C-term inal phospholipid- and Ca2+-binding domains.