Copines are highly conserved, calcium-dependent membrane binding proteins found in a variety of eukaryotic organisms. Multiple copine homologs exist in each of Paramecium, Arabidopsis, C. elegans, mice, and humans. Copines are characterized as having two C2 domains at the N-terminal region followed by an "A domain" at the C-terminal region. The "A domain" is similar in sequence to the von Willebrand A (VWA) domain found in integrins. Following the A domain, copines have a variable length C-terminal domain, which may confer unique characteristics to the different copine family members .
The C2 domain is a calcium-dependent phospholipid-binding motif originally identified in protein kinase C. Single and multiple copies of C2 domains are found in a large number of eukaryotic proteins. Most proteins containing a single C2 domain are involved in signaling pathways; examples include protein kinases, lipid kinases, phospholipases, and GTPase activating proteins. In contrast, most proteins that have multiple C2 domains are involved in membrane trafficking. Some examples of multiple C2 domain proteins are synaptotagmin, rabphilin, DOC2, each of which have two C2 domains, and munc13, which has three C2 domains [2, 3]. The VWA domain is named from the von Willebrand Factor, a plasma and extracellular matrix protein. VWA domains have been studied in integrins and several extracellular matrix proteins and appear to function as protein-binding domains . Copines were the first intracellular proteins to be identified as having a VWA domain . However, a recent sequence database search for VWA domains revealed that VWA domains are found in several other intracellular proteins present in all eukaryotes .
Copines possess several characteristics that suggest they may have a role in membrane trafficking. As described above, copines have two C2 domains, similar to other membrane trafficking proteins. Biochemical studies have shown that copines, like other C2 domain containing proteins, bind to phospholipids in a calcium-dependent manner [1, 5–7]. In addition, the protein chromobindin 17, which binds to the secretory granules of chromaffin cells in the presence of calcium, has been identified as a copine . However, no functional data exists to indicate a role for copines in membrane trafficking.
Although copines possess two C2 domains, studies with human copines indicate that copines are involved in calcium-dependent signaling pathways and are therefore, more functionally related to the single C2 domain proteins [8, 9]. Using the A domain of several human copine proteins as bait in a yeast two-hybrid screening of a mouse embryo cDNA library, Tomsig et al.  isolated a wide variety of proteins, several of which are components of intracellular signaling pathways. Many of these interactions between the copine A domains and their target proteins were verified in in vitro pull-down assays. The authors hypothesized that copines may act to localize target proteins to a particular membrane in response to calcium. To test this idea, they used an in vitro assay to show that full-length copines were able to recruit target proteins to membranes in a calcium-dependent manner. In an in vivo assay, Tomsig et al.  used a dominant negative mutant copine construct consisting of only the A domain to inhibit signaling from the TNF-α receptor in human embryonic kidney 293 cells.
Copine mutants have been isolated in both Arabidopsis [7, 10, 11] and C. elegans . Arabidopsis plants with loss of function alleles of one of the copines, CPN1/BON1, exhibit mutant phenotypes only under certain environmental conditions. Copine mutant plants are miniature at 22°C, but grow normally at 28°C. The miniature phenotype is due to a reduction in both the size and the number of cells in the plant . In low humidity conditions, copine mutant plants are also smaller and display abnormal regulation of cell death, with small necrotic lesions on the leaves, an accelerated programmed cell death response, and increased resistance to pathogens .
In C. elegans, mutations in nem-4, which encodes a copine, are capable of suppressing loss of function alleles of gon-2. Suppression of gon-2 by nem-4 requires a low level of GON-2 activity. GON-2 is a cation channel required for postembryonic gonadal cell divisions and loss of function mutations in GON-2 lead to a sterile phenotype. The nem-4 mutant strains do not exhibit any obvious phenotype . The data from these copine mutant studies in Arabidopsis and C. elegans suggest that copines may function in a wide variety of calcium-mediated signaling pathways that control processes such as cell growth, cell division, and cell death.
To further investigate the function of copines, we have chosen to study copines in the simple eukaryote Dictyostelium discoideum and have identified six copines genes in the Dictyostelium genome. Dictyostelium provides an ideal system for studying copine function for several reasons. First, although Dictyostelium lives as a single-celled amoeba, it contains multiple copine homologs and a comparison of each of the Dictyostelium copines with the other five indicates that they share only 28–60% identity in amino acid sequence. Therefore, the Dictyostelium copine genes are diverse in sequence and may carry out distinct functions. To study the function of each copine gene, single and multiple gene knockout mutants can be created by homologous recombination. Second, Dictyostelium are highly motile, phagocytic cells, possessing organelles and membrane trafficking pathways similar to mammalian cells. Therefore, Dictyostelium serves as a good model for studying membrane trafficking and a particularly good model for many of the phagocytic cells found in human tissues, in which copines are highly expressed [13, 14]. Third, Dictyostelium executes a simple 24-hour developmental program to form multicellular fruiting bodies and thus, Dictyostelium provides a simplistic model to study copine function in programmed cell death and development.
Our studies have focused on cpnA, the most abundant copine gene cDNA in the Dictyostelium cDNA Sequencing Project database . To characterize CpnA in Dictyostelium, we have determined the protein expression pattern of CpnA during development, examined the calcium-dependent membrane binding properties of CpnA, and expressed a GFP-tagged version of CpnA in wildtype Dictyostelium to determine the intracellular location of CpnA.