Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.
In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.
Shirataki
H
H
Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Japan.
Kaibuchi
K
K
Sakoda
T
T
Kishida
S
S
Yamaguchi
T
T
Wada
K
K
Miyazaki
M
M
Takai
Y
Y
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005796:Genes; D008562:Membrane Glycoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D050857:Synaptotagmins; D014018:Tissue Distribution; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D004247:DNA; D019204:GTP-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A