Two functionally different domains of rabphilin-3A, rab3A p25/smg p25A- binding and phospholipid- and Ca²⁺-binding domains

Rabphilin-3A is a putative target molecule for rab3A p25/smg p25A, which is a member of a ras p21-related small GTP-binding protein and implicated 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 known to bind to phospholipid in a Ca²⁺-dependent manner. In the current study, we have investigated the functional domains of rabphilin-3A by use of three recombinant proteins as follows: full rabphilin-3A (1-704 amino acids), an N-terminal fragment (1- 280 amino acids), and a C-terminal 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 phospholipid. ⁴⁵Ca²⁺ bound to rabphilin-3A and the C-terminal fragment only in the presence of phospholipid but did not bind to the N- terminal fragment. The GTPγS-bound form of rab3A p25 bound to both rabphilin-3A and the N-terminal fragment but did not bind to the C-terminal fragment. These results indicate that rabphilin-3A has at least two functionally different domains, the N-terminal rab3A p25-binding and C- terminal phospholipid- and Ca²⁺-binding domains.