Doc2, a Third Isoform of Double C2 Protein, Lacking Calcium-Dependent Phospholipid Binding Activity 1 Mitsunori Fukuda* ,2 and Katsuhiko Mikoshiba* , † *Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; and †Division of Molecular Neurobiology, Department of Basic Medical Science, The Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Received August 17, 2000 The Doc2 (double C2) family consists of two isoforms (Doc2 and Doc2) characterized by an N-terminal Munc13-1 interacting domain (Mid) and two C2 do- mains that interact with Ca 2 and phospholipid at the C-terminus. This Ca 2 -binding property is thought to be important to the regulation of neurotransmitter release. In this paper, we report a third isoform of mouse Doc2, named Doc2. Doc2 also contains a pu- tative Mid domain and two C2 domains, and it is 45.6 and 43.2% identical to mouse Doc2 and Doc2, respec- tively, at the amino acid level. In contrast to the other Doc2 isoforms, the C2 domains of Doc2 impair Ca 2 - dependent phospholipid binding activity. The highest expression of Doc2 mRNA was found in the heart, but occurs ubiquitously, the same as Doc2. These find- ings indicate that Doc2 may also function as an effec- tor for Munc13-1 and that it may be involved in the regulation of vesicular trafficking. © 2000 Academic Press Key Words: Doc2; synaptotagmin; rabphilin; C2 do- main; exocytosis; phospholipid binding. Neurotransmitter release from nerve terminals in the brain is regulated by several families of Ca 2+ - binding proteins having tandem C2 domains (C2A do- main and C2B domain) (reviewed in ref. 1). These domains originated from a C2 regulatory region of pro- tein kinase C, and they are thought to function as a Ca 2+ -dependent phospholipid binding site. Three pro- tein families, the synaptotagmin (Syt) family (1–3), rabphilin-3A (downstream target of rab3A) (4), and the Doc2 family (double C2 protein) (5), have been identi- fied on synaptic vesicles. Although their C2 domains show similar Ca 2+ -dependent phospholipid binding ac- tivity (5–9), they are involved in different steps of syn- aptic vesicle exocytosis (1). Syt I, an integral mem- brane protein of synaptic vesicles, is essential for the fusion step between the synaptic vesicle and the pre- synaptic plasma membrane, probably functioning as a Ca 2+ -sensor (10 –20). By contrast, the latter two pro- teins play a regulatory role in a step before the final fusion step of the synaptic vesicle with the presynaptic plasma membrane (21–24), because knock-out mice of these proteins showed a much milder phenotype (still exhibiting normal neurotransmission) than Syt I-knock-out mice (lacking a synchronous, fast compo- nent of neurotransmission and thereby dying shortly after birth) (10, 25, 26). Doc2 consists of two isoforms, Doc2 and Doc2 (5, 9, 27). Doc2 is specifically expressed in neuronal cells (5, 28), whereas Doc2 is expressed ubiquitously (9, 27, 29). Both isoforms of Doc2 interact with Munc 13-1 (30, 31) and Munc 18 (28), both of which are also important for neurotransmitter release (32). Munc13-1 binds the conserved amino terminal domain of Doc2 (Mid do- main, amino acid residues 13-37 of Doc2) (30), and this interaction is induced by binding of diacylglycerol or phorbol ester to the Munc13-1 C1 domain (30, 31). Thus, Munc13-1-Doc2 interaction is to thought to un- derlie the molecular mechanism of phorbol ester- enhancement of neurotransmitter release (33, 34). In this paper, we report a third isoform of Doc2, Doc2, which shows striking homology with the entire region of both Doc2 and Doc2. Doc2 also has a putative Mid domain (a potential Munc13-1 binding site), but unlike the other isoforms, it lacks Ca 2+ - The nucleotide sequence reported in this paper is deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under Accession No. AB046665. Abbreviations used: Doc2, double C2 protein; GST, glutathione S-transferase; Mid, Munc13-1 interacting domain; PAGE, poly- acrylamide gel electrophoresis; PC, phosphatidylcholine; PCR, polymerase chain reaction; PS, phosphatidylserine; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; Syt(s), synaptotagmin(s). 1 This work was supported in part by grants from the Science and Technology Agency to Japan (K.M.) and from the Ministry of Edu- cation, Science, and Culture of Japan (K.M. and M.F.; 11780571 and 12053274). 2 To whom correspondence should be addressed. Fax: +81-48-467- 9744. E-mail: mnfukuda@brain.riken.go.jp. Biochemical and Biophysical Research Communications 276, 626 – 632 (2000) doi:10.1006/bbrc.2000.3520, available online at http://www.idealibrary.com on 626 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.