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 relea se, If either rabphilin 3A or Doc2(alpha) plays a role in membrane tra fficking, like the synaptotagmins, then non-neural forms should be pre sent, Here we describe the isolation of a mouse cDNA which encodes a n ovel Doc2 homologue (Doc2(beta)) that is present in all tissues, The e ncoded protein, which is highly homologous to human Doc2(alpha) (70% i dentity), is composed of 412 amino acids with a calculated relative mo lecular mass (M(r)) of 45,837, The sequence identity is especially hig h 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 line s and tissues tested, Ca2+-dependent phospholipid binding assaying of recombinant fusion proteins revealed that the single C2A domain, but n ot the C2B domain, of Doc2(beta) binds phosphatidycholine and phosphat idylserine (2.5:1, w/w) liposomes, The binding is Ca2+-dependent, with an EC(50) value of approximately 1 mu M and a Hill coefficient of app roximately 3, which are comparable to those of synaptotagmins, rabphil in 3A and Doc2(alpha). Our results suggest that Doc2(beta) is involved in constitutive membrane trafficking.