Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex in smooth muscle cells

The plasmalemma of smooth muscle cells is periodically banded. This arrangement ensures efficient transmission of contractile activity, via the firm, actin-anchoring regions, while the more elastic caveolae-containing "hinge" regions facilitate rapid cellular adaptation to changes in cell length. Since cellular mechanics are undoubtedly regulated by components of the membrane and cytoskeleton, we have investigated the potential role played by annexins (a family of phospholipid- and actin-binding, Ca(2+)-regulated proteins) in regulating sarcolemmal organization. Stimulation of smooth muscle cells elicited a relocation of annexin VI from the cytoplasm to the plasmalemma. In smooth, but not in striated muscle extracts, annexins II and VI coprecipitated with actomyosin and the caveolar fraction of the sarcolemma at elevated Ca(2+) concentrations. Recombination of actomyosin, annexins, and caveolar lipids in the presence of Ca(2+) led to formation of a structured precipitate. Participation of all 3 components was required, indicating that a Ca(2+)-dependent, cytoskeleton-membrane complex had been generated. This association, which occurred at physiological Ca(2+) concentrations, corroborates our biochemical fractionation and immunohistochemical findings and suggests that annexins play a role in regulating sarcolemmal organization during smooth muscle contraction.     

Annexins in cell membrane dynamics. Ca(2+)-regulated association of lipid microdomains

The sarcolemma of smooth muscle cells is composed of alternating stiff actin-binding, and flexible caveolar domains. In addition to these stable macrodomains, the plasma membrane contains dynamic glycosphingolipid- and cholesterol-enriched microdomains, which act as sorting posts for specific proteins and are involved in membrane trafficking and signal transduction. We demonstrate that these lipid rafts are neither periodically organized nor exclusively confined to the actin attachment sites or caveolar regions. Changes in the Ca²⁺ concentration that are affected during smooth muscle contraction lead to important structural rearrangements within the sarcolemma, which can be attributed to members of the annexin protein family. We show that the associations of annexins II, V, and VI with smooth muscle microsomal membranes exhibit a high degree of Ca²⁺ sensitivity, and that the extraction of annexins II and VI by detergent is prevented by elevated Ca²⁺ concentrations. Annexin VI participates in the formation of a reversible, membrane–cytoskeleton complex (Babiychuk, E.B., R.J. Palstra, J. Schaller, U. Kämpfer, and A. Draeger. 1999. J. Biol. Chem. 274:35191–35195). Annexin II promotes the Ca²⁺-dependent association of lipid raft microdomains, whereas annexin V interacts with glycerophospholipid microcompartments. These interactions bring about a new configuration of membrane-bound constituents, with potentially important consequences for signaling events and Ca²⁺ flux.     

Formation of tightly bound forms of annexins V and VI in rabbit skeletal muscle membranes isolated in the presence of barium ions

After isolation of rabbit skeletal muscle membranes in the presence of Ba2+ or Ca2+, significant portions of annexin V and VI tightly bind to membranes and become inaccessible for chelating agents. Tightly bound annexin VI is virtually completely solubilized only after treatment with a buffer supplemented both with EGTA and detergent. The portion of tightly bound annexin V cannot be removed even by extraction with buffer containing both EGTA and detergent. In some cases, tightly bound annexin V or VI is detected even in the control (not treated with cations) membranes, thus indicating the possible formation of tight annexin--membrane complexes in situ. The addition of exogenous cations seems to promote only the accumulation of tightly bound annexins within the cell. After temperature-induced phase separation, annexin V and VI bound to the membranes isolated in the presence of Ba2+ or Ca2+ remains mainly in the aqueous phase, similarly to annexins isolated from the control membranes. Neither annexin partitions into the detergent-enriched phase. This indicates the absence of hydrophobicity change in comparison with the standard EGTA-soluble annexins.     

Retinoic acid stimulates annexin-mediated growth plate chondrocyte mineralization

Biomineralization is a highly regulated process that plays a major role during the development of skeletal tissues. Despite its obvious importance, little is known about its regulation. Previously, it has been demonstrated that retinoic acid (RA) stimulates terminal differentiation and mineralization of growth plate chondrocytes (Iwamoto, M., I.M. Shapiro, K. Yagumi, A.L. Boskey, P.S. Leboy, S.L. Adams, and M. Pacifici. 1993. Exp. Cell Res. 207:413-420). In this study, we provide evidence that RA treatment of growth plate chondrocytes caused a series of events eventually leading to mineralization of these cultures: increase in cytosolic calcium concentration, followed by up-regulation of annexin II, V, and VI gene expression, and release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles. Cotreatment of growth plate chondrocytes with RA and BAPTA-AM, a cell permeable Ca2+ chelator, inhibited the up-regulation of annexin gene expression and mineralization of these cultures. Interestingly, only matrix vesicles isolated from RA-treated cells that contained annexins, were able to take up Ca2+ and mineralize, whereas vesicles isolated from untreated or RA/BAPTA-treated cells, that contained no or only little annexins were not able to take up Ca2+ and mineralize. Cotreatment of chondrocytes with RA and EDTA revealed that increases in the cytosolic calcium concentration were due to influx of extracellular calcium. Interestingly, the novel 1,4-benzothiazepine derivative K-201, a specific annexin Ca2+ channel blocker, or antibodies specific for annexin II, V, or VI inhibited the increases in cytosolic calcium concentration in RA-treated chondrocytes. These findings indicate that annexins II, V, and VI form Ca2+ channels in the plasma membrane of terminally differentiated growth plate chondrocytes and mediate Ca2+ influx into these cells. The resulting increased cytosolic calcium concentration leads to a further up-regulation of annexin II, V, and VI gene expression, the release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles, and the initiation of mineralization by these vesicles.     

An activating factor (tropomyosin) for the superprecipitation of actomyosin prepared from scallop adductor muscles

An activating factor for the superprecipitation of actomyosin reconstructed from scallop smooth muscle myosin and rabbit skeletal muscle F-actin was purified from thin filaments of scallop smooth and striated muscles. Two components were obtained from the smooth muscle and one from the striated muscle. All three components similarly affected the actomyosin ATPase activity. According to the results of analysis involving double reciprocal plotting of the ATPase activity versus F-actin concentration, the activating factor for superprecipitation decreased the apparent dissociation constants of actomyosin about 30 to 110 times. The activation of the superprecipitation by the factor, therefore, may be due to the enhancement of the affinity between F-actin and myosin in the presence of ATP. The activating factor was identified as tropomyosin based on it mobility on polyacrylamide gel electrophoresis and on the recovery of the Ca2+-sensitivity of purified rabbit skeletal actomyosin in the presence of troponin.     

Troponin and its components from ascidian smooth muscle

Troponin was isolated from the thin filaments of ascidian smooth muscle and separated into three components by ion-exchange chromatography, the molecular weights of which were 33,000, 24,000, and 18,000, respectively. The three components were designated as troponin t (TN-T), troponin I (TN-I), and troponin C (TN-C) in order of molecular weight, since each component had properties similar to those of the respective components of vertebrate skeletal-muscle troponin. The ascidian troponin or the mixture of the three components conferred Ca2+-sensitivity on reconstituted rabbit actomyosin in the presence of tropomyosin. One of the characteristics of the ascidian troponin was Ca2+-dependent activation of actin-myosin interaction in collaboration with tropomyosin, whereas its inhibitory action on the actomyosin ATPase in the absence of Ca2+ was less remarkable. From this, it is concluded that in the ascidian smooth muscle actin-myosin interaction is regulated by an actin-linked troponin-tropomyosin system, but the ascidian troponin acts as a Ca2+-dependent activator of an actomyosin system.     

Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin.

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.     

Ca(2+)-dependent phospholipid and arachidonic acid binding by the placental annexins VI and IV.

Using an assay system in which phospholipids were immobilised on phenyl-Sepharose, we examined the affinities of the placental annexins VI and IV for binding to specific phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol at Ca2+ concentrations of 0.6, 0.4 and 3.5 microM, respectively, compared to values of 4.5, 4.5 and 20 microM Ca2+, respectively for purified annexin IV. These values did not change significantly in the presence of other proteins from the family. Neither annexin VI or IV bound to phosphatidylinositol bisphosphate and phosphatidylcholine, even at millimolar concentrations of Ca2+. However, both proteins bound to arachidonic acid, oleic acid and palmitic acid in a Ca(2+)-dependent manner, using the same assay system. The level of binding for both proteins was significantly increased when mixtures of phosphatidylcholine and arachidonic acid were examined. A dose-dependent inhibition of phospholipase A2 by both annexins VI and IV, at millimolar concentrations of Ca2+ was observed when phosphatidylcholine liposomes were used as a substrate. These results raise questions about the interpretation of experiments in which the release of arachidonic acid is used as a measure of lipase activity, and of the validity of the substrate-depletion model for the inhibition of phospholipases by the annexins.     

Caldesmon150 regulates the tropomyosin-enhanced actin-myosin interaction in gizzard smooth muscle

Using a reconstituted system in which myosin was preferentially phosphorylated, we examined the regulatory action of caldesmon150 on the smooth muscle actin-myosin interaction. Caldesmon150 inhibited the tropomyosin-enhanced actomyosin ATPase activity in a Ca2+-independent manner. This inhibitory effect of caldesmon150 was observed to be overcome by the addition of calmodulin in a Ca2+-dependent manner. In accordance with the observations of ATPase activity, we demonstrated evidence that the regulatory action of caldesmon150 on the actin site was mainly through control of the tropomyosin-enhanced actin-myosin interaction and calmodulin confers the Ca2+-sensitivity upon the caldesmon150 action determined by the cosedimentation method.     

Characterization of mammalian heart annexins with special reference to CaBP33 (annexin V)

Porcine heart was observed to express annexins V (CaBP33) and VI in large amounts, and annexins III and IV in much smaller amounts. Annexin V (CaBP33) in porcine heart was examined in detail by immunochemistry. Homogenization and further processing of heart in the presence of EGTA resulted in the recovery of annexin V (CaBP33) in the cytosolic fraction and in an EGTA-resistant, Triton X-100-soluble fraction from cardiac membranes. Including Ca2+ in the homogenization medium resulted in a significant decrease in the annexin V (CaBP33) content of the cytosolic fraction with concomitant increase in the content of this protein in myofibrils, mitochrondria, the sarcoplasmic reticulum and the sarcolemma. The amount of annexin V (CaBP33) in each of these subfractions depended on the free Ca2+ concentration in the homogenizing medium. At the lowest free Ca2+ concentration tested, 0.8 microM, only the sarcolemma appeared to contain bound annexin V (CaBP33). Membrane-bound annexins V (CaBP33) and VI partitioned in two fractions, one EGTA-resistant and Triton X-100-extractable, and one Triton X-100-resistant and EGTA-extractable. Altogether, these data suggest that annexins V and VI are involved in the regulation of membrane-related processes.     

Regulation of the sarcoplasmic reticulum Ca(2+)-release channel requires intact annexin VI.

Annexin VI has eight highly conserved repeated domains; all other annexins have four. Díaz-Mu?oz et al. (J Biol Chem 265:15894, 1990) reported that annexin VI alters the gating properties of the ryanodine-sensitive Ca(2+)-release channel isolated from sarcoplasmic reticulum. The investigate the domain structure of rat annexin VI (67 kDa calcimedin) required for this channel regulation, various proteolytic digestions were performed. In each case, protease-resistant core polypeptides were produced. Annexin VI was digested with V8 protease and two core polypeptides were purified by Ca(2+)-dependent phospholipid binding followed by HPLC. The purified fragments were shown to be derived from the N- and C-terminal halves of annexin VI, and demonstrated differential immunoreactivity with monoclonal antibodies to rat annexin VI. While both core polypeptides retained their ability to bind phospholipids in a Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-release channel as did intact annexin VI.     

The roles of annexins and types II and X collagen in matrix vesicle-mediated mineralization of growth plate cartilage

Annexins II, V, and VI are major components of matrix vesicles (MV), i.e. particles that have the critical role of initiating the mineralization process in skeletal tissues. Furthermore, types II and X collagen are associated with MV, and these interactions mediated by annexin V stimulate Ca(2+) uptake and mineralization of MV. However, the exact roles of annexin II, V, and VI and the interaction between annexin V and types II and X collagen in MV function and initiation of mineralization are not well understood. In this study, we demonstrate that annexin II, V, or VI mediate Ca(2+) influx into phosphatidylserine (PS)-enriched liposomes, liposomes containing lipids extracted from authentic MV, and intact authentic MV. The annexin Ca(2+) channel blocker, K-201, not only inhibited Ca(2+) influx into fura-2-loaded PS-enriched liposomes mediated by annexin II, V, or VI, but also inhibited Ca(2+) uptake by authentic MV. Types II and X collagen only bound to liposomes in the presence of annexin V but not in the presence of annexin II or VI. Binding of these collagens to annexin V stimulated its Ca(2+) channel activities, leading to an increased Ca(2+) influx into the liposomes. These findings indicate that the formation of annexin II, V, and VI Ca(2+) channels in MV together with stimulation of annexin V channel activity by collagen (types II and X) binding can explain how MV are able to rapidly take up Ca(2+) and initiate the formation of the first crystal phase.     

Intracellular localization of endothelial cell annexins is differentially regulated by oxidative stress

Annexins are calcium-dependent phospholipid binding proteins that are implicated in the regulation of both intracellular and extracellular thrombostatic mechanisms in the vascular endothelium. Tight control of annexin gene expression and targeting of annexin proteins is therefore of importance in maintaining the health of the endothelium. Because annexins are abundant in vascular endothelial cells and could be either dysregulated by or contribute to anomalies in Ca2+ signaling, we investigated annexin gene expression and subcellular localization in human umbilical vein endothelial cells (HUVEC) in a model of chronic oxidative stress. HUVEC were cultured under mild hyperoxic conditions in a custom-built chamber to induce oxidative stress over a period of 12 days. Although annexin expression levels did not change significantly in response to hyperoxic stress, immunofluorescence analysis revealed striking effects on the subcellular localization of certain annexins, including the redistribution of annexins 5 and 6 from the cytosol to the nucleus. In addition, oxidative stress modulated the responses of certain annexins to stimulation with a range of pharmacological and physiological Ca2+-mobilizing agonists, in a manner that suggested that annexin localization is regulated via the complex integration of both Ca2+ and intracellular signaling pathways. These results show that differential regulation of annexin localization by oxidative stress may have a causative role in the cellular pathophysiology of vascular endothelial cell disease.     

Phosphorylation in skeletal myosin light chain modulates the actin--myosin interaction in the presence of regulatory proteins in vitro

The effect of phosphorylation in skeletal myosin light chain (LC2) on the actomyosin and acto-heavymeromyosin (HMM) ATPase activities was investigated in the presence or absence of regulatory proteins (tropomyosin-troponin complex). Phosphorylation in LC2 did not modulate the actin-myosin and actin-HMM interactions over a wide range of KCl concentrations from 30 to 150 mM without regulatory proteins. In the presence of regulatory proteins, phosphorylation in myosin LC2 enhanced the ATPase activity of actomyosin with calcium ions, but the removal of calcium ions made little difference in the ATPase activity between phosphorylated and dephosphorylated myosins. Ca2+-sensitivity of the regulated actomyosin was slightly changed by phosphorylation in myosin LC2. However, both the ATPase activity and Ca2+-sensitivity of the regulated acto-HMM were unaffected by phosphorylation in HMM LC2.     

Myosin and actin from ascidian smooth muscle and their interaction

Myosin and actin were purified from ascidian smooth muscle. Ascidian myosin contained two classes of light chains and the pH dependence of Ca2+-activated ATPase and the KCl dependence of actin-activated ATPase of ascidian myosin differed from those of vertebrate skeletal myosin. Troponin-tropomyosin complex from ascidian increased the ATPase activity of ascidian reconstituted actomyosin in a Ca2+-dependent manner. Ascidian myosin provided the reconstituted actomyosin with the responsiveness to calcium ions. Two actin isoforms were present in ascidian, which were distinguished by isoelectric points.     

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.     

Troponin C-like protein in chicken gizzard muscle.

1. A troponin C-like protein was prepared from frozen chicken gizzard by preparative polyacrylamide gel electrophoresis and its apparent molecular weight was estimated to be about 15,500 daltons. 2. In urea gel electrophoresis, the mobility of the troponin C-like protein increased slightly in the presence of Ca2+, like that of skeletal muscle troponin C. On the other hand, the mobility of the the troponin C-like protein in glycerol gel electrophoresis, unlike that of skeletal muscle troponin C, was significantly decreased by Ca2+. 3. In alkaline gel electrophoresis, the troponin C-like protein formed a Ca2+-dependent complex with troponin I or troponin T from skeletal muscle. 4. The troponin C-like protein could neutralize the inhibitory effect of skeletal muscle troponin I on the Mg2+-activated ATPase of actomyosin from rabbit skeletal muscle, but could not confer Ca2+-sensitivity on the actomyosin in the presence of troponin I and troponin T from skeletal muscle.     

Calcium-sensitivity of pig-carotid-actomyosin ATPase in relation to phosphorylation of the regulatory light chain

Ca2+-dependent phosphorylation of the 20000-Mr regulatory light chain was found to be a necessary condition for the Ca2+-sensitivity of the Mg2+-dependent ATPase activity and superprecipitation of pig carotid actomyosin. Actin-myosin interaction independent of phosphorylation and Ca2+ (ATPase activity and superprecipitation) were demonstrated in aged actomyosin preparations and in preparations from which the regulatory light chains were removed by papain digestion.     

GTPase activity and biochemical characterization of a recombinant cotton fiber annexin

A cDNA encoding annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA was expressed in Escherichia coli, and the resultant recombinant protein was purified. We then investigated some biochemical properties of the recombinant annexin based on the current understanding of plant annexins. An "add-back experiment" was performed to study the effect of the recombinant annexin on beta-glucan synthase activity, but no effect was found. However, it was found that the recombinant annexin could display ATPase/GTPase activities. The recombinant annexin showed much higher GTPase than ATPase activity. Mg2+ was essential for these activities, whereas a high concentration of Ca2+ was inhibitory. A photolabeling assay showed that this annexin could bind GTP more specifically than ATP. The GTP-binding site on the annexin was mapped into the carboxyl-terminal fourth repeat of annexin from the photolabeling experiment using domain-deletion mutants of this annexin. Northern-blot analysis showed that the annexin gene was highly expressed in the elongation stages of cotton fiber differentiation, suggesting a role of this annexin in cell elongation.