© BioMed Central Ltd 2004
Published: 31 March 2004
Annexins are traditionally thought of as calcium-dependent phospholipid-binding proteins, but recent work suggests a more complex set of functions. More than a thousand proteins of the annexin superfamily have been identified in major eukaryotic phyla, but annexins are absent from yeasts and prokaryotes. The unique annexin core domain is made up of four similar repeats approximately 70 amino acids long, each of which usually contains a characteristic 'type 2' motif for binding calcium ions. Animal and fungal annexins also have non-homologous amino-terminal domains of varying length and sequence, which are responsible for the distinct localizations and specialized functions of the proteins through post-translational modification and binding to other proteins. Annexins interact with various cell-membrane components that are involved in the structural organization of the cell, intracellular signaling by enzyme modulation and ion fluxes, growth control, and they can act as atypical calcium channels. Analysis of site-specific conservation in the core domain suggests a role for certain buried residues in the calcium-channel activity of vertebrate annexins and in the structural stability of their core domains. Evolutionarily significant differences between subfamilies are preferentially localized to accessible sites on the protein surface that determine membrane binding and interactions with cytosolic proteins.
Gene organization and evolutionary history
Annexins were discovered approximately 25 years ago. The first to be described as an isolated, purified protein was human annexin A7 (then known as synexin) , and the first to be cloned were human annexins A1 and A2 (formerly known as lipocortin and calpactin respectively) [2, 3]. The name 'annexin' was proposed for the superfamily in 1990, and the 12 annexins common to vertebrates were recently classified in the annexin A family and named as annexins A1-A13 (or ANXA1-ANXA13), leaving A12 unassigned in the official nomenclature . Annexins outside vertebrates are classified into families B (in invertebrates), C (in fungi and some groups of unicellular eukaryotes), D (in plants), and E (in protists); at least 40 additional subfamilies await formal classification into these families.
Annexin genes in different groups of organisms
Number of genes
Western clawed frog
African clawed frog
Basidiomycetes (mushrooms, rusts)
Gymnosperms (cycads, gingkoes, conifers)
The annexin C family consists of diverse members in unicellular organisms, represented by fungi, mycetozoa (slime molds) and the newly defined kingdom of chromalveolates (a grouping of chromist stramenopiles, including brown algae and diatoms, and alveolates, including ciliates and dinoflagellates). Individual species in these groups may have no annexins (yeasts), one to three (other fungi), or up to six (potato rot). Members of the annexin B family, found in both protostome and deuterostome invertebrates, have also undergone many lineage-specific duplications, leading to more than 20 subfamilies whose gene organization, protein structures and chromosomal maps differ between clades and from vertebrate annexins. Insect annexins exemplify the complex pattern of duplication and loss in individual lineages: tsetse flies and mosquitoes have four annexins, whereas Drosophila, honeybees and silkmoths have only three, of which only one or two are clear orthologs between species. The early-branching deuterostomes - sea urchins, tunicates and lancelets - have 5-12 annexins; these include close relatives of annexins A13, A7 and A11, the founder genes of vertebrate annexins [8, 9]. Although this establishes the invertebrate ancestry of vertebrate annexins, none of the 12 annexins in the vertebrate A family have (yet) been assigned a true invertebrate ortholog.
The vertebrate A family includes the 12 annexins that have been confirmed to make up the complete family in mammals, but the number of annexins may vary in other classes of vertebrates as genes have been gained and lost. Ancient polyploidization events in bony fish, and more recent genome duplications in pseudotetraploid frogs (Xenopus), have duplicated many of the annexin genes. Thus, annexin A1 has undergone two successive duplications to yield up to four copies in some fish, amphibians and birds. Mammalian ANXA6 is a compound gene, probably derived from the fusion of duplicated ANXA5 and ANXA10 genes in early vertebrate evolution (the two halves of the encoded protein are indicated as 5'ANX6 and 3'ANX6 in Figure 1). Annexins A7, A8 and A10 have not yet been detected in fish, although genes similar to annexin A7 have been found in earlier-diverging species such as the sea urchin, the earthworm and Hydra. The reasons for the tendency of annexin genes (or their chromosomal regions) to duplicate, their successful preservation, and the extent to which they contribute to vertebrate complexity are as yet unknown.
Characteristic structural features
All annexins share a core domain made up of four similar repeats, each approximately 70 amino acids long. Each repeat is made up of five α helices and usually contains a characteristic 'type 2' motif for binding calcium ions with the sequence 'GxGT-[38 residues]-D/E' (in the single-letter amino-acid code; see Figure 2b). Animal and fungal annexins also have variable amino-terminal domains.
Proteins that interact with vertebrate annexins
Epithelial growth factor receptor, formyl peptide receptor, selectin, actin, integrin A4
Tissue plasminogen activator, angiostatin, insulin receptors, tenascin C, caveolin 1
Lectins, glycoprotein 2
Collagen type 2, vascular endothelial growth factor receptor2, integrin B5, protein kinase C, cellular modulator of immune recognition (MIR), G-actin, helicase, DNA (cytosine-5-)methyltransferase 1 (DNMT1)
Calcium-responsive heat stable protein-28 (CRHSP-28), ras GTPase activating protein, chondroitin, actin
Programmed cell death 6 (PDCD6), sorcin
Neural precursor cell expressed, developmentally down-regulated 4 (NEDD4)
Localization and function
Annexins are generally cytosolic proteins, with pools of both a soluble form and a form stably or reversibly associated with components of the cytoskeleton or proteins that mediate interactions between the cell and the extracellular matrix (matricellular proteins). Some, such as annexins A11 and A2, have been found in the nucleus under particular circumstances [25, 26]. In certain instances, annexins may be expressed at the cell surface, despite the absence of any secretory signal peptide; for example, annexin A1 translocates from the cytosol to the cell surface following exposure of cells to glucocorticoids , and annexin A2 is constitutively expressed at the surface of vascular endothelial cells where it functions in the regulation of blood clotting . The expression level and tissue distribution of annexins span a broad range, from abundant and ubiquitous (annexins A1, A2, A4, A5, A6, A7, A11) to selective (such as annexin A3 in neutrophils and annexin A8 in the placenta and skin) or restrictive (such as annexin A9 in the tongue, annexin A10 in the stomach and annexin A13 in the small intestine).
The presence of multiple annexins in all higher eukaryotic cell types suggests fundamental roles in cell biology , even though prokaryotes and yeasts appear to tolerate their absence, but the apparent functional diversity within the family remains perplexing. The development of knockout mice has provided insight into the functions of annexins A1, A2, A5, A6 and A7. Loss of ANXA1 leads to changes in the inflammatory response and the effects of glucocorticoids , whereas the ANXA2 knockout mouse has defects in neovascularization and fibrin homeostasis . The ANXA5 and ANXA6 knockout mice have subtler phenotypes and need further investigation [31, 32], and two independently derived ANXA7 null mutant mouse strains are either embryonic lethal  or show changes in calcium homeostasis . The diversity of phenotype in the annexin knockout mice is consistent with these proteins having largely independent functions. Roles for annexins that have been established from studies using cultured cells are not always reflected in phenotypic abnormalities in the corresponding knockout mice, suggesting that functional redundancy may, in some instances, obscure the full range of functions of these multifunctional proteins. In mice that lack an overt phenotype, there is now the opportunity to test molecular theories of annexin function, such as the proposed calcium channel activity of annexin A5.
The definition of the biological processes in which annexins are involved has progressed through the use of gene knockouts and imaging. On the basis of studies using live cell imaging and targeted gene disruption, roles have now been unequivocally established for annexin A1 in inflammation, annexin A2 in vesicle traffic and annexin A7 in regulation of cell growth. The ubiquity and stability of annexins suggest some fundamental role of the unique core domain in cellular physiology, possibly involving adhesion mechanics, membrane traffic, signal transduction and/or developmental processes. To the extent that annexins may have adapted to the particular needs of their host species, molecular-evolution studies offer some insight into which structural changes may be responsible for their functional diversity, but biological data remain scant for nonvertebrate annexins. Transcript expression studies using microarrays and RNA interference offer new experimental approaches that could implicate annexins in some defined cellular process or pathway.
Long-standing problems also remain to be addressed. Do individual annexins have different functions in different cell types? How are annexins secreted? Can annexins be classified into groups with integrated functions, or are they functionally independent of each other? These and many other questions, and perhaps most importantly the need to understand mechanism, will occupy annexin biologists for years to come. The discovery of annexins with negligible calcium-binding capacity and growing evidence for interactions with other proteins may make the traditional definition of annexins as calcium-dependent phospholipid-binding proteins superfluous in the near future.
This work was supported by grant number BMC2002-00827 from the Ministry of Science and Technology, Spain, and by the Wellcome Trust, UK.
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