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.
Molecular Neurobiology Laboratory, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Ibaraki.
T
T
Kojima
M
M
Fukuda
J
J
Aruga
K
K
Mikoshiba
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