Two functionally different domains of rabphilin-3A, Rab3A p25/smg p25A-binding and phospholipid- and Ca(2+)-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 Ca(2+)-dependent manner. In the current study, we have investigated the functional domains or 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 GTP gamma S-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 Ca(2+)-binding domains.
Yamaguchi
T
T
Department of Biochemistry, Kobe University School of Medicine, Japan.
Shirataki
H
H
Kishida
S
S
Miyazaki
M
M
Nishikawa
J
J
Wada
K
K
Numata
S
S
Kaibuchi
K
K
Takai
Y
Y
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium; D019204:GTP-Binding Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010743:Phospholipids; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A