C 2 domains are primarily found in signal transduction proteins such as protein kinase C, which generally contain a single C 2 domain, and in membrane trafficking proteins such as synaptotagmins, which generally contain multiple C 2 domains. In both classes of proteins, C 2 domains usually regulate the respective protein's function by forming Ca 2؉ -dependent or Ca 2؉ -independent phospholipid complexes. We now describe MCTPs (multiple C 2 domain and transmembrane region proteins), a novel family of evolutionarily conserved C 2 domain proteins with unusual Ca 2؉ -dependent properties. MCTPs are composed of a variable N-terminal sequence, three C 2 domains, two transmembrane regions, and a short C-terminal sequence. The invertebrate organisms Caenorhabditis elegans and Drosophila melanogaster express a single MCTP gene, whereas vertebrates express two MCTP genes (MCTP1 and MCTP2) whose primary transcripts are extensively alternatively spliced. Most of the MCTP sequences, in particular the C 2 domains, are highly conserved. All MCTP C 2 domains except for the second C 2 domain of MCTP2 include a perfect Ca

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Arfophilin was first identified as a target protein for GTP-ARF5. The N-terminus of ARF5 (amino acids 2-17), which is distinct from that of class I or class III ARFs, is essential for binding to the C-terminus of arfophilin (amino acids 612-756). This study using GST fusion proteins in pulldown experiments in CHO-K1 cell lysates showed that, unexpectedly, ARF6 also bound to full-length arfophilin or the C-terminus of arfophilin (amino acids 612-756) in a GTP-dependent manner. Studies with ARF1/ARF6 chimeras further showed that the amino acid sequence of residues 37-80 of ARF6, which is different from the corresponding sequences in class I and class II ARFs, was essential for binding to arfophilin. Both GTP-ARF5 and GTP-ARF6 bound to arfophilin in CHO-K1 cell lysates, while GTP-ARF1 did not bind. In contrast, all three forms of ARF bound to arfaptin 2, with ARF1 showing the strongest binding. Yeast two-hybrid studies with wild-type, dominant negative, and constitutively active forms of ARF1, -5, and -6 and with ARF1/ARF6 chimeras confirmed these results, except that constitutively active ARF6 was autoactivating. Our findings suggest that both class II and III ARFs may influence the same cellular pathways through arfophilin as a common downstream effector.

AbbreviationsAMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; Cpx: Complexin (also known as Synaphin); GST: glutathione S-transferase; IGF-1: insulin-like growth factor-1; KD: knock-down; KO: knock-out; LTP: long-term potentiation; SNAP-23 or -25: synaptosomal-associated protein 23 or 25; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SNARE complex: a ternary complex composed of Syntaxin, Synaptobrevin, and either SNAP-23 or SNAP-25; Syb2: Synaptobrevin 2 (also known as vesicle-associated membrane protein 2, VAMP2); Syt1: Synaptotagmin 1; Syt10: Synaptotagmin 10; WT: wild-type. INTRODUCTION Ca2+ -triggered exocytosis is an essential cellular process that mediates transmitter release to the extracellular space for intercellular communications particularly in neurons [1][2][3] . The same or similar processes are used for intracellular trafficking to deliver membrane patches or proteins to the plasma membrane [4,5] . Stimulations of neurons cause an increase in cytosolic Ca 2+ concentrations and trigger fusions of synaptic vesicles into the plasma membrane to release transmitters to the extracellular space [1][2][3] . Synaptotagmin 1 -dependent manner [6,7] . Synaptobrevin 2 (Syb2; also known as vesicle-associated membrane protein 2, VAMP2), a vesicular SNARE protein, forms a ternary SNARE complex with Syntaxin and SNAP-25 that are located in the target plasma membrane [8] . ABSTRACTComplexin (Cpx), which is expressed mainly in the nervous system, binds efficiently to the SNARE complex that is composed of either Syntaxin 1 or Syntaxin 3. Both Syntaxin 1 and Syntaxin 3 are involved in mediating synchronous neurotransmitter release in the nervous system. Cpx stabilizes the SNARE complex upon binding, and potentiates the efficacy of synchronous exocytotic process. Cpx is consisted of N-terminal, accessory α-helix, central α-helix, and C-terminal domains. Each domain of Cpx has a distinct function that is differentially involved in the regulation of priming, clamping, and activating in the exocytotic process. These functions of Cpx domains coordinately potentiate the efficacy of synchronous exocytotic process.

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