Evolutionary conservation and structural determinants of the calelectrins (annexins).

The calelectrins were originally characterized as a family of Ca?'-dependent membrane-binding proteins [ 1-41 which werc postulated t o be related to each other, and t o a previously described Torpedo protein [5-71, because they exhibited similar biochemical properties and shared immunologic epitopes. Sequences from several of the calelectrins [ 8, 01 revealed them to be homologous to a number of other proteins that were independently characterized in recent years by several investigators. Members of this class of proteins which can now be considered as a large multigene family have been described under the following names: lipocortins [ 101, p3S and p36, also named calpactins [ 111; synexin [ 121; endonexins I and 11, which were the 32.5 kDa and 35 kDa calelectrins [ I , 2 I renamed by Geisow er ul. [ 8 1; proteins I , 11, and I l l [ 131; lymphocyte Ca?+-binding protein p68 [ 141; chromobindins [ 151, and human placental anticoagulant protein [ 16). All these proteins were isolated from vertebrates. Although no specific function has been demonstrated for these proteins which is accepted by all investigators, recently a consensus was reached to name all proteins from this supergene family 'annexins'. The annexins are Ca2+-binding proteins that bind to negatively charged phospholipids in a Ca"-dependent manner [ I , 6, 17-20]. The amino acid sequences of all annexins predict the presence of four internal repeats with the 67 kDa calelectrin (annexin VI) containing a duplication of the four to eight such repeats [9, 141. When the sequences of different members of the annexin family are compared with each other, their internal repeats are found to be conserved better by position between distinct members of the family than within a given protein 191. The four repeats have differences in length that are conserved in all proteins with the third repeat being the longest (generally 8.5 residues), and the second the shortest (generally 72 residues). No sequences homologous to the EF-hand sequence of Ca2 + -binding proteins can be found in the primary structure of the annexins, suggesting that they contain a different Ca2'-binding structure. In addition to binding to negatively charged phospholipids in a Ca'+-dependent manner, annexins 1 and I 1 (lipocortins 1 and 2, also referred to as p35 and p36 or as calpactins 1 and 2 (2 1-23]) are tyrosine phosphorylated by growth-factor receptors in a stimulation-dependent manner [2 1-24]. The functional significance of the tyrosine phosphorylation is unknown, but the fact that annexins I and I1 are major substrates for the growth factor receptor tyrosine kinases indicates that they are intracellular proteins. The different members of the annexin protein family are differentially distributed in vertebrate tissues, with some proteins showing very restricted localizations. For example, annexin IV (32.5 kDa calelectrin) is almost exclusively localized to ductal epithelia, such as those of biliary and pancreatic ducts [25], while annexin I1 (lipocortin 2/p36) is preferentially found in microvilli [26]. On the other hand, annexin VI (67 kDa calelectrin), appears to be ubiquitously found in all cells examined, although it is more concentrated in endocrine tissues 1251. Several, sometimes conflicting hypotheses have been proposed regarding the functions o f the annexins. As lipocortins, they were thought to represent humoral mediators of the glucocorticoid-dependent regulation of inflammation [ 271; as calpactins, to represent Ca? + -dependent actin-binding proteins [23]; and as anticoagulants, to consist of circulating components of the coagulation system [ 161. Recently, an annexin was suggested to be identical with inositol 1,2-cyclic phosphate 2-phosphohydrolase [28]. None of these proposed functions is universally accepted, since it is controversial whether the annexins are secreted or intracellular proteins, and because most members of the protein family do not bind actin [ 11. We originally hypothesized that these proteins might be involved in regulating membrane traffic [ 1 J, and recent evidence has implicated annexin 1 (lipocortin I/calpactin) in regulating exocytosis in chromaffin cells [ 2Y 1. However, a distinct cellular function for this protein family remains to be established. Previously, the annexins have only been studied in vertebrates. Most recently, novel homologues of the annexins/ calelectrins have been characterized in Drosophilu melunogusrer 1301 and in Hydra [31]. Two novel annexins were identified by cDNA cloning from D. mefunoguster. The two Drosophilu annexins are 46% identical with each other, with most of the residues that are identical between the two Drosophilu annexins also being conserved in vertebrate annexins. A similarly high degree of homology relates Drosophilu annexins to all different vertebrate annexins ( 40-SOn% identity). Together these sequence comparisons demonstrate that the Drosophilu annexins are not the invertebrate homologues of particular mammalian annexins, but that they constitute novel members of the annexin gene family, and they were named annexins 1X and X in continuation with a recently established terminology. RNA blots indicate that the messages for the two Drosophilu proteins are differently expressed in development, with one message being expressed throughout development, whereas the other message is only found in early embryos and adult flies. I n siru hybridizations localized the two Drosophila genes to 93B and 19A-4,7. Considering the fact that vertebrate organisms contain at least eight annexins, it seems likely that Drosophilu contains additional members of this gene family. The two currently known Drosophilu annexins [30] were cloned from an adult