Calcium-dependent phospholipid binding to the C2A domain of a ubiquitous form of double C2 protein (Doc2 beta).
Rabphilin 3A and Doc2 alpha are synaptic vesicle-associated proteins, and are thought to function as Ca2+ sensors in neurotransmitter release. If either rabphilin 3A or Doc2 alpha plays a role in membrane trafficking, like the synaptotagmins, then non-neural forms should be present. Here we describe the isolation of a mouse cDNA which encodes a novel Doc2 homologue (Doc2 beta) that is present in all tissues. The encoded protein, which is highly homologous to human Doc2 alpha (70% identity), is composed of 412 amino acids with a calculated relative molecular mass (M(r)) of 45,837. The sequence identity is especially high in two C2 domains (74% in C2A and 84% in C2B). Northern and Western blot analyses have shown that Doc2 beta is expressed in all cell lines and tissues tested. Ca(2+)-dependent phospholipid binding assaying of recombinant fusion proteins revealed that the single C2A domain, but not the C2B domain, of Doc2 beta binds phosphatidycholine and phosphatidylserine (2.5:1, w/w) liposomes. The binding is Ca(2+)-dependent, with an EC50 value of approximately 1 microM and a Hill coefficient of approximately 3, which are comparable to those of synaptotagmins, rabphilin 3A and Doc2 alpha. Our results suggest that Doc2 beta is involved in constitutive membrane trafficking.
Kojima
T
T
Molecular Neurobiology Laboratory, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Ibaraki.
Fukuda
M
M
Aruga
J
J
Mikoshiba
K
K
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D002531:Cerebellum / Q000378:metabolism*; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium; D002135:Calcium-Binding Proteins; D019204:GTP-Binding Proteins; D008081:Liposomes; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000097624:Rabphilin-3A; C091524:DOC2A protein, human; C490420:DOC2B protein, human; C490418:Doc2a protein, mouse