Rabphilin-3A binds to a M(r) 115,000 polypeptide in a phosphatidylserine- and Ca(2+)-dependent manner.
Rabphilin-3A is a putative target protein for Rab3A/Smg 25A, which is a member of the Ras-related small GTP-binding protein and implicated in neurotransmitter release from the synapse. Rabphilin-3A is composed of two functionally different domains: the N-terminal Rab3A-binding and the C-terminal phosphatidylserine- and Ca(2+)-binding domains. The C-terminal domain has two copies of an internal repeat that are homologous to the C2 domains of protein kinase C, synaptotagmin, and phospholipase A2 and C-gamma 1, which are known to bind phosphatidylserine and Ca2+. In this study, we attempted to identify the Rabphilin-3A-interacting molecule in bovine brain by use of an overlay assay technique. The 32P-labeled C-terminal fragment of Rabphilin-3A (281-704 amino acids) bound to a protein molecule with a M(r) of about 115 kDa which was immobilized on a nitrocellulose sheet. This protein was highly purified and characterized. The binding of the 32P-labeled C-terminal fragment to this protein was dependent on both phosphatidylserine and Ca2+, and inhibited by an excess amount of the C-terminal fragment and the C2 domain fragment (396-704 amino acids) but not by the N-terminal fragment (1-280 amino acids). These results indicate that Rabphilin-3A binds to a protein molecule with a M(r) of 115 kDa through the C2 domain in the presence of phosphatidylserine and Ca2+.
Miyazaki
M
M
Department of Biochemistry, Kobe University School of Medicine, Japan.
Kaibuchi
K
K
Shirataki
H
H
Kohno
H
H
Ueyama
T
T
Nishikawa
J
J
Takai
Y
Y
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008055:Lipids; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D010455:Peptides; D012694:Serine / Q000378:metabolism*; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium; D002352:Carrier Proteins; D019204:GTP-Binding Proteins; D008055:Lipids; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D010455:Peptides; D012694:Serine; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000097624:Rabphilin-3A