Carl E. Creutz

Annexins: linking Ca2+ signalling to membrane dynamics

Eukaryotic cells contain various Ca2+-effector proteins that mediate cellular responses to changes in intracellular Ca2+ levels. A unique class of these proteins — annexins — can bind to certain membrane phospholipids in a Ca2+-dependent manner, providing a link between Ca2+ signalling and membrane functions. By forming networks on the membrane surface, annexins can function as organizers of membrane domains and membrane-recruitment platforms for proteins with which they interact. These and related properties enable annexins to participate in several otherwise unrelated events that range from membrane dynamics to cell differentiation and migration.

Calcium-dependent Binding of Sorcin to the N-terminal Domain of Synexin (Annexin VII)

The annexins are characterized by their ability to bind phospholipid membranes in a Ca2+-dependent manner. Sequence variability between the N-terminal domains of the family members may contribute to the specific cellular function of each annexin. To identify proteins that interact with the N-terminal domain of synexin (annexin VII), a fusion protein was constructed composed of glutathioneS-transferase fused to amino acids 1–145 of human synexin. Affinity chromatography using this construct identified sorcin as a Ca2+-dependent synexin-binding protein. Overlay assays confirmed the interaction. The glutathioneS-transferase construct associates with recombinant sorcin over the range of pCa2+ = 4.7–3.1 with no binding observed at pCa2+ = 5.4. Overlay assays using deletion constructs of the synexin N-terminal domain mapped the sorcin binding site to the N-terminal 31 amino acids of the synexin protein. Additionally, synexin forms a complex with sorcin and recruits this protein to chromaffin granule membranes in a Ca2+-dependent manner. Sorcin is able to inhibit synexin-mediated chromaffin granule aggregation in a manner saturable with increasing sorcin concentrations, but does not influence the Ca2+ sensitivity of synexin-mediated granule aggregation. Therefore, the interaction between sorcin and synexin may serve to regulate the functions of these proteins on membrane surfaces in a Ca2+-dependent manner.

p60c-src activity detected in the chromaffin granule membrane

Using monoclonal antibodies specific for p60c-src we have detected high levels of this kinase in adrenal medullary chromaffin tissue and in highly purified chromaffin granule (secretory vesicle) membranes. An immune complex kinase assay was applied to fractions of adrenal medullary tissue resolved on sucrose density gradients. Thirty-seven per cent of the total tissue p60c-src activity was found in association with chromaffin granule or granule membrane markers. Localization of a significant fraction of total cellular p60c-src activity to this secretory vesicle membrane suggests that the kinase may function in the regulation of neurotransmitter release.

Crystal structure of bovine annexin VI in a calcium-bound state

The crystal structure of a calcium-bound form of bovine annexin VI has been determined with X-ray diffraction data to 2.9 A by molecular replacement. Six Ca2+ ions were found, five in AB loops, one in a DE loop. Two loops (II-AB, which binds calcium, and V-AB, which does not) have conformations that differ significantly from those in calcium-free, human recombinant annexin VI. There are only small differences between the calci- and the apo-annexin VI in the rest of the molecule. Calcium by itself does not promote a major conformational change.

Calcium-dependent self-association of synaptotagmin I.

Abstract: Synaptotagmin I, an integral membrane protein of secretory vesicles, appears to have an essential role in calcium‐triggered hormone and neurotransmitter release. The large cytoplasmic domain of synaptotagmin I has two C2 domains that are thought to mediate calcium and phospholipid binding. A recombinant protein (p65 1–5) comprised of the cytoplasmic domain was previously shown to aggregate purified chromaffin granules and artificial phospholipid vesicles in a calcium‐dependent manner. p65 1–5 may be able to aggregate membrane vesicles by a self‐association reaction. This hypothesis led us to investigate the ability of synaptotagmin I protein fragments to multimerize in vitro. We found that p65 1–5, in the absence of membranes, was able to self‐associate to form large aggregates in a calcium‐dependent manner as shown by light‐scattering assays and electron microscopy. In addition, a recombinant protein comprised of only the second half of the cytoplasmic domain, including the second C2 domain, was also able to self‐associate and aggregate phospholipid vesicles in a calcium‐dependent manner. A recombinant protein comprised of only the first C2 domain was not able to self‐associate or aggregate vesicles. These results suggest that synaptotagmin I is able to bind calcium in the absence of membranes and that the second half of the cytoplasmic domain is able to bind calcium and mediate its multimerization in a calcium‐dependent manner. The ability of synaptotagmin I protein fragments to multimerize in a calcium‐dependent manner in vitro suggests that multimerization may have an important function in vivo.

Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine.

The role of copines in regulating signalling from the TNF-alpha (tumour necrosis factor-alpha) receptor was probed by the expression of a copine dominant-negative construct in HEK293 (human embryonic kidney 293) cells. The construct was found to reduce activation of the transcription factor NF-kappaB (nuclear factor-kappaB) by TNF-alpha. The introduction of calcium into HEK293 cells either through the activation of muscarinic cholinergic receptors or through the application of the ionophore A23187 was found to enhance TNF-alpha-dependent activation of NF-kappaB. This effect of calcium was completely blocked by the copine dominant-negative construct. TNF-alpha was found to greatly enhance the expression of endogenous copine I, and the responsiveness of the TNF-alpha signalling pathway to muscarinic stimulation increased in parallel with the increased copine I expression. The copine dominant-negative construct also inhibited the TNF-alpha-dependent degradation of IkappaB, a regulator of NF-kappaB. All of the effects of the dominant-negative construct could be reversed by overexpression of full-length copine I, suggesting that the construct acts specifically through competitive inhibition of copine. One of the identified targets of copine I is the NEDD8-conjugating enzyme UBC12 (ubiquitin C12), that promotes the degradation of IkappaB through the ubiquitin ligase enzyme complex SCF(betaTrCP). Therefore the copine dominant-negative construct might inhibit TNF-alpha signalling by dysregulation or mislocalization of UBC12. Based on these results, a hypothesis is presented for possible roles of copines in regulating other signalling pathways in animals, plants and protozoa.

Evaluation of the annexins as potential mediators of membrane fusion in exocytosis

Membrane fusion is a central event in the process of exocytosis. It occurs between secretory vesicle membranes and the plasma membrane and also among secretory vesicle membranes themselves during compound exocytosis. In many cells the fusion event is regulated by calcium. Since the relevant membranes do not undergo fusion in vitro when highly purified, much attention has been paid to possible protein mediators of these calcium-dependent fusion events. The annexins comprise a group of calcium-dependent membrane-aggregating proteins, of which synexin is the prototype, which can initiate contacts between secretory vesicle membranes which will then fuse if the membranes are further perturbed by the addition of exogenous free fatty acids. This review discusses the secretory pathway and the evidence obtained fromin vitro studies that suggests the annexins may be mediators or regulators of membrane fusion in exocytosis.

Differential subcellular distribution of p36 (the heavy chain of calpactin I) and other annexins in the adrenal medulla.

Abstract: The annexins are a group of highly related Ca2+‐dependent membrane‐binding proteins that are present in a wide variety of cells and tissues. We have examined the subcellular distribution of five members of the annexin family in the adrenal medulla. Bovine adrenal medullary tissue was homogenized in buffers containing EGTA and fractionated on sucrose gradients. p36 (the large subunit of calpactin I) was found to be predominantly membrane associated, with ∼20% present in fractions enriched in chromaffin granules. In contrast, lipocortin I was localized primarily to the cytosol, with only a small proportion found in plasma membrane‐containing fractions. Like lipocortin I, endonexin I was found to be present almost entirely in the soluble fractions. The 67‐kDa calelectrin was localized primarily to the plasma membrane fractions, with a small amount present in the chromaffin granule and cytoplasmic fractions. Synexin was present in both membranous and cytoplasmic fractions. p36 appeared to be a peripherally associated granule membrane protein in that it was dissociated from the membrane by addition of base and it partitioned with the aqueous phase when granule membranes were treated with Triton X‐114. Antiserum against p10 (the small subunit of calpactin I) reacted with a protein of 19 kDa that is specifically localized in chromaffin granule membrane fractions. The differences in subcellular distributions of the annexins suggest that these proteins have distinct cellular functions. The finding that p36 is associated with chromaffin granule and plasma membrane fractions provides further support for a possible role of calpactin in exocytosis.

Calcium dependence of the binding of synexin to isolated chromaffin granules

The calcium dependence of the binding of synexin to isolated chromaffin granules has been investigated. The calcium dependence was found to be pH sensitive, binding occurring at higher Ca2+ concentrations at lower values of pH. At pH 7.2 half-maximal binding occurred at 4 microM Ca2+. This is a lower Ca2+ concentration than the 200 microM that is required to give half-maximal self-association of synexin or membrane aggregation by synexin. The data therefore suggest that in the chromaffin cell stimulated to release catecholamines and proteins by exocytosis synexin first binds to membranes and then associates with itself to draw membranes together in preparation for fusion.

Annexin-chromaffin granule membrane interactions: a comparative study of synexin, p32 and p67

The chromaffin granule membrane binding and aggregating properties of three annexins, synexin, p32 and p67, have been studied and compared. Each protein was activated to bind and aggregate membranes with a biphasic Ca2+ dependence, with one phase titrating between pCa 5.0-3.5 and the second at higher levels of calcium (pCa less than 3.5). cis-Unsaturated free fatty acids lowered these Ca2+ requirements by approximately one log unit. Barium and strontium were able to partially substitute for calcium, with the order of sensitivity Ca2+ greater than Sr2+ greater than Ba2+. The proteins appeared to bind to distinct but overlapping populations of receptor sites, and did so in a manner displaying positive cooperativity at the higher Ca2+ levels. The maximal efficacy of the proteins as membrane aggregators differed with synexin being 1-2-fold more efficacious than p32, which in turn was 7-fold more efficacious than p67. In combination, p67 was an effective inhibitor of granule aggregation induced by synexin or p32, while p32 was able to both promote and inhibit synexin-induced granule aggregation in a manner which varied with synexin concentration. The complexity of these annexin-membrane interactions may be a reflection of the multidomain structure of the annexins and may have implications for the differential functions of these proteins in cells.