Interaction of HRC (histidine-rich Ca(2+)-binding protein) and triadin in the lumen of sarcoplasmic reticulum

HRC (histidine-rich Ca(2+) binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca(2+) with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca(2+)-sensitive. Our data suggest that HRC may play a role in the regulation of Ca(2+) release from the sarcoplasmic reticulum by interaction with triadin.     

Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.

Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.     

cDNA cloning of a neural visinin-like Ca(2+)-binding protein.

A 21,000-dalton Ca(2+)-binding protein (Walsh, M.P., Valentine, K.A., Ngai, P.K., Carruthers, C.A., and Hollengerg, M.D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca(2+)-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca(2+)-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain.     

Identification of the Ca(2+)-dependent calmodulin-binding region of chromogranin A.

Chromogranin A (CGA), the most abundant protein in bovine adrenal chromaffin granules, is a high-capacity, low-affinity Ca(2+)-binding protein found in most neuroendocrine cells, and binds calmodulin (CaM) in a Ca(2+)-dependent manner. The binding of chromogranin A to calmodulin was determined by measuring the intrinsic tryptophan fluorescence of chromogranin A in the presence and absence of Ca2+. Binding was specifically Ca(2+)-dependent; neither Mg2+ nor Mn2+ could substitute for Ca2+. Chelation of Ca2+ by EGTA completely eliminated the chromogranin A-calmodulin interaction. CaM binding was demonstrated by a synthetic CGA peptide representing residues 40-65. When the CGA peptide and CaM were mixed in the presence of 15 mM CaCl2, the intrinsic tryptophan fluorescence emission underwent a substantial blue-shift, shifting from 350 to 330 nm. Like the intact CGA, the peptide-CaM binding was specifically Ca(2+)-dependent, and neither Mg2+ nor Mn2+ could induce the binding. Calmodulin bound both to CGA and to the synthetic CGA peptide with a stoichiometry of one to one. The dissociation constants (Kd) determined by fluorometric titration were 13 nM for the peptide-CaM binding and 17 nM for intact CGA-CaM binding. The Kd values are comparable to those (approximately 10(-9) M) of other CaM-binding proteins and peptides, demonstrating a tight binding of CaM by CGA. The CaM-binding CGA residues 40-65 are 100% conserved among all the sequenced CGAs in contrast to 50-60% conservation found in the entire sequence, implying essential roles of this region.     

Regulation of protein kinase C by Zn(2+)-dependent interaction with actin.

Zn2+ influences diverse cellular processes by poorly understood mechanisms. Some of these effects may be mediated by the protein kinase C (PKC) family of enzymes, since an influx of Zn2+ greatly increases their binding of regulatory ligand phorbol ester and induces their translocation from cytosol to the cytoskeleton. Using a model with purified components, we now show that Zn2+ acts by forming a phospholipid-dependent complex of PKC with filamentous actin, which results in expression of new binding sites for phorbol ester and phosphorylation of actin. These results provide a basis for the observed localization of PKC at actin-membrane junctions, in-vivo.     

Identification of a novel interaction between the Ca(2+)-binding protein S100A11 and the Ca(2+)- and phospholipid-binding protein annexin A6

S100A11 is a member of the S100 family of EF-hand Ca²⁺-binding proteins, which is expressed in smooth muscle and other tissues. Ca²⁺ binding to S100A11 induces a conformational change that exposes a hydrophobic surface for interaction with target proteins. Affinity chromatography with immobilized S100A11 was used to isolate a 70-kDa protein from smooth muscle that bound to S100A11 in a Ca²⁺-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca²⁺-dependent interaction between S100A11 and annexin A6 was confirmed by affinity chromatography of the purified bacterially expressed proteins, by gel overlay of annexin A6 with purified S100A11, by chemical cross-linking, and by coprecipitation of S100A11 with annexin A6 bound to liposomes. The expression of S100A11 and annexin A6 in the same cell type was verified by RT-PCR and immunocytochemistry of isolated vascular smooth muscle cells. The site of binding of S100A11 on annexin A6 was investigated by partial tryptic digestion and deletion mutagenesis. The unique NH₂ terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH₂- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca²⁺] leads to formation of a complex of S100A11 and annexin A6, which forms a physical connection between the plasma membrane and the cytoskeleton, or plays a role in the formation of signaling complexes at the level of the sarcolemma. smooth muscle; protein-protein interaction     

Isolation and characterization of recoverin-like Ca(2+)-binding protein from rat brain.

Rat brain was found, by immunoblot analysis, to have a protein of Mr 23,000 (P23k) that was clearly different from recoverin and was labeled with an antiserum raised against the NH2-terminus of recoverin. P23k could not be detected by an antiserum raised against the COOH-terminus of recoverin. Blots with 45Ca demonstrated that P23k bound Ca2+. This calciprotein was further purified by Ca(2+)-dependent hydrophobic interaction and ion-exchange chromatography. In SDS polyacrylamide gel electrophoresis, P23k had an apparent Mr of 21,000 in the presence of 10 microM Ca2+ and 23,000 in the absence of Ca2+ (0.1 mM EGTA). The isoelectric point of P23k was 5.6. Ca(2+)-binding analysis indicated that P23k bound 2 moles of Ca2+ per mole of protein and had two binding sites with dissociation constants of 13 microM and 0.2 microM. Purified P23k bound to the crude membrane fractions from the cerebellum, cerebrum and retina in a Ca(2+)-dependent manner. Partial amino acid sequence analysis of proteolytic fragments of P23k revealed the sequence homology between P23k and recoverin. These results suggested that P23k may act as a Ca(2+)-sensitive regulator by forming a complex with its target on the membrane.     

A Ca(2+)-dependent protein kinase phosphorylates phosphoenolpyruvate carboxylase in maize.

In C4 plants the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is regulated by phosphorylation/dephosphorylation which is mediated by light/dark signals. The study using protein kinase inhibitors showed that the inhibition pattern of maize PEPC-protein kinase (PEPC-PK) is similar to that of myosin light chain kinase, a Ca(2+)-calmodulin-dependent PK. The kinase activity was also inhibited by EGTA and the inhibition was relieved by Ca2+. These results suggest that PEPC-PK is Ca(2+)-dependent in contrast with previous observations by other research groups.     

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

Intralumenal sarcoplasmic reticulum Ca(2+)-binding proteins

The sarcoplasmic reticulum (SR) controls the level of intracellular Ca2+ in cardiac and skeletal muscle by storing and releasing Ca2+. A set of intralumenal SR Ca(2+)-binding proteins has been identified that may serve important roles in SR Ca2+ storage and mobilization. The most prominent of these SR proteins, calsequestrin, is discretely localized to junctional SR. Other intralumenal proteins are more widely distributed throughout the SR. All of these intralumenal SR Ca(2+)-binding proteins are acidic, stain blue with dye Stains-All, and appear to be substrates for casein kinase II. The biochemistry and cell biology of lumenal SR proteins may conform to a paradigm now emerging from the study of endoplasmic reticulum proteins.     

H-NMR study of rabbit skeletal muscle troponin C: Ca(2+)-dependent interaction with mastoparan.

1H-NMR spectroscopy is employed to study the interaction between rabbit skeletal muscle troponin (C (TnC) and wasp venom tetradecapeptide mastoparan. We monitored the spectral change of the following species of TnC as a function of mastoparan concentration: apoTnC, Ca(2+)-saturated TnC (Ca4TnC) and Ca(2+)-half loaded TnC (Ca2TnC). When apo-TnC is titrated with mastoparan, line-broadening is observed for the ring-current shifted resonance of Phe-23, Ile-34, Val-62 and Phe-72 and the downfield-shifted CH alpha-resonances of Asp-33, Thr-69 and Asp-71; these residues are located in the N-domain. When Ca4TnC is titrated with mastoparan, chemical shift change is observed for the ring-current shifted resonances of Phe-99, Ile-110 and Phe-148 and the downfield-shifted CH alpha-resonances of Asn-105, Ala-106, Ile-110 and Ile-146 and aromatic resonance of Tyr-109 and His-125; these residues are located in the C-domain. The resonance of Phe-23, Asp-33, Asp-71, Phe-72, Phe-99, Tyr-109, Ile-146, His-125 and Phe-148 in both N- and C-domains changes when Ca2TnC is titrated with mastoparan. These results suggest that mastoparan binds to the N-domain of apo-TnC, the C-domain of Ca4TnC and the N- and C-domains of Ca2TnC; the hydrophobic cluster in each domain is involved in binding. As mastoparan binds to TnC, the above resonances shift to their normal chemical shift positions. The stability of the cluster and the beta-sheet is reduced by mastoparan-binding. These results suggest that the conformation of the hydrophobic cluster and the neighboring beta-sheet change to a loose form. The stability of the N-domain of Ca2TnC and Ca4TnC increases when these species bind 1 mol of mastoparan at the C-domain. These results suggest a mastoparan-induced interaction between the N- and C-domains of TnC.     

Na(+)/Ca(2+) exchange facilitates Ca(2+)-dependent activation of endothelial nitric-oxide synthase.

Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na(+) gradients and after loading of endothelial cells with Na(+) ions using the ionophore monensin. Monensin-induced Na(+) loading markedly reduced Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-dependent activation of eNOS was strictly dependent on the presence of Na(+) ions during treatment of the cells with monensin. Na(+)-induced facilitation of eNOS activation was not due to a direct effect of Na(+) ions on the Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) mimicked the effects of Na(+) loading. The effects of monensin were completely blocked by 3', 4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na(+) gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.     

The EF-hand Ca(2+)-binding protein p22 associates with microtubules in an N-myristoylation-dependent manner.

Proteins containing the EF-hand Ca(2+)-binding motif, such as calmodulin and calcineurin B, function as regulators of various cellular processes. Here we focus on p22, an N-myristoylated, widely expressed EF-hand Ca(2+)-binding protein conserved throughout evolution, which was shown previously to be required for membrane traffic. Immunofluorescence studies show that p22 distributes along microtubules during interphase and mitosis in various cell lines. Moreover, we report that p22 associates with the microtubule cytoskeleton indirectly via a cytosolic microtubule-binding factor. Gel filtration studies indicate that the p22-microtubule-binding activity behaves as a 70- to 30-kDa globular protein. Our results indicate that p22 associates with microtubules via a novel N-myristoylation-dependent mechanism that does not involve classic microtubule-associated proteins and motor proteins. The association of p22 with microtubules requires the N-myristoylation of p22 but does not involve p22's Ca(2+)-binding activity, suggesting that the p22-microtubule association and the role of p22 in membrane traffic are functionally related, because N-myristoylation is required for both events. Therefore, p22 is an excellent candidate for a protein that can mediate interactions between the microtubule cytoskeleton and membrane traffic.     

Crystal structure of the Ca(2+)-form and Ca(2+)-binding kinetics of metastasis-associated protein, S100A4

S100A4 takes part in control of tumour cell migration and contributes to metastatic spread in in vivo models. In the active dimeric Ca(2+)-bound state it interacts with multiple intracellular targets. Conversely, oligomeric forms of S100A4 are linked with the extracellular function of this protein. We report the 1.5A X-ray crystal structure of Ca(2+)-bound S100A4 and use it to identify the residues involved in target recognition and to derive a model of the oligomeric state. We applied stopped-flow analysis of tyrosine fluorescence to derive kinetics of S100A4 activation by Ca(2+) (k(on)=3.5 microM(-1)s(-1), k(off)=20s(-1)).     

Ca(2+)-dependent ubiquitination of calmodulin in yeast.

Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.     

Annexins--a new family of Ca(2+)-binding proteins

Annexins, the Ca(2+)- and phospholipid-binding proteins, are able to induce Ca(2+)-dependent aggregation of biomembranes. All the representatives of this family contain four or eight tandem repeats, 60-80 amino acids each. All these repeats include a highly conservative 17-member amino acid consensus sequence (an endonexin fold). The central domain comprises all these repeats and contains, in addition, the site(s) with a binding affinity for Ca2+ and phospholipids. Annexins are devoid of the classical "EF-hand" Ca(2+)-binding domain and can therefore be assigned to a new family of Ca(2+)-binding proteins.     

Regulation of the erythrocyte Ca(2+)-ATPase by mutant calmodulins with Glu----Ala substitutions in the Ca(2+)-binding domains.

We have used four mutant calmodulins to study the regulation of human erythrocyte Ca(2+)-ATPase by the calmodulin-dependent pathway; the conserved Glu at position 12 in each of the four Ca(2+)-binding domains of calmodulin (Glu31, Glu67, Glu104, or Glu140) was replaced by Ala. At pCa 7, where unmodified calmodulin maximally activates the erythrocyte Ca(2+)-ATPase, all four mutants stimulated Ca(2+)-ATPase activity to the same maximal velocity. However, the concentrations of mutant calmodulins required for half-maximal activation (KCaM) were significantly higher than that for unmodified calmodulin and were strongly dependent on the domain in which the mutated Glu was located; substitution in either the first or second Ca(2+)-binding domain had little effect (2-3-fold increase in KCaM), whereas substitution in either the third or fourth domain resulted in a dramatic, 25-71-fold increase in KCaM. The same order of sensitivity was observed when the Ca2+ dependence of enzyme activation was measured at a constant 100 nM concentration of mutant calmodulin. These data point to dramatic differences in the functional significance of the replacement of the Glu at position 12 in each of the four Ca(2+)-binding domains for activation of the Ca(2+)-ATPase. The 2 Glu residues located in the carboxyl-terminal half of calmodulin (particularly Glu140) are crucial for activation of the Ca(2+)-ATPase at physiologically significant Ca2+ concentrations.     

Ca(2+)-dependent interaction between FKBP12 and calcineurin regulates activity of the Ca(2+) release channel in skeletal muscle

Calcineurin is a Ca(2+) and calmodulin-dependent protein phosphatase with diverse cellular functions. Here we examined the physical and functional interactions between calcineurin and ryanodine receptor (RyR) in a C2C12 cell line derived from mouse skeletal muscle. Coimmunoprecipitation experiments revealed that the association between RyR and calcineurin exhibits a strong Ca(2+) dependence. This association involves a Ca(2+) dependent interaction between calcineurin and FK506-binding protein (FKBP12), an accessory subunit of RyR. Pretreatment with cyclosporin A, an inhibitor of calcineurin, enhanced the caffeine-induced Ca(2+) release (CICR) in C2C12 cells. This effect was similar to those of FK506 and rapamycin, two drugs known to cause dissociation of FKBP12 from RyR. Overexpression of a constitutively active form of calcineurin in C2C12 cells, DeltaCnA(391-521) (deletion of the last 131 amino acids from calcineurin), resulted in a decrease in CICR. This decrease in CICR activity was partially recovered by pretreatment with cyclosporin A. Furthermore, overexpression of an endogenous calcineurin inhibitor (cain) or an inactive form of calcineurin (DeltaCnA(H101Q)) in C2C12 cells resulted in up-regulation of CICR. Taken together, our data suggest that a trimeric-interaction among calcineurin, FKBP12, and RyR is important for the regulation of the RyR channel activity and may play an important role in the Ca(2+) signaling of muscle contraction and relaxation.