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Interaction of HRC (Histidine-rich Ca2+-Binding Protein) and Triadin in the Lumen of Sarcoplasmic Reticulum

2001; Elsevier BV; Volume: 276; Issue: 43 Linguagem: Inglês

10.1074/jbc.m010664200

1083-351X

Han Gil Lee, Hara Kang, Do Han Kim, Woo Jin Park,

Molecular Biology Techniques and Applications

HRC (histidine-rich Ca2+binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca2+ 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 Ca2+-sensitive. Our data suggest that HRC may play a role in the regulation of Ca2+release from the sarcoplasmic reticulum by interaction with triadin. HRC (histidine-rich Ca2+binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca2+ 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 Ca2+-sensitive. Our data suggest that HRC may play a role in the regulation of Ca2+release from the sarcoplasmic reticulum by interaction with triadin. histidine-rich Ca2+-binding protein sarcoplasmic reticulum polyacrylamide gel electrophoresis glutathioneS-transferase polymerase chain reaction sarcoplasmic reticulum Ca2+-ATPase HRC1 (histidine rich Ca2+-binding protein) was first identified from rabbit skeletal muscle because of its ability to bind low density lipoprotein with high affinity. However, HRC did not appear to function as a lipoprotein receptor, as it was localized to the sarcoplasmic reticulum (SR) where it has no access to plasma lipoproteins (1Hofmann S.L. Goldstein J.L. Orth K. Moomaw C.R. Slaughter C.A. Brown M.S. J. Biol. Chem. 1989; 264: 18083-18090Abstract Full Text PDF PubMed Google Scholar). We have previously reported compelling evidence supporting that HRC resides in the lumen of the SR (2Suk J.Y. Kim Y.S. Park W.J. Biochem. Biophys. Res. Commun. 1999; 263: 667-671Crossref PubMed Scopus (29) Google Scholar), consistent with the results of the earlier immunofluorescence and immunoelectron microscopic studies (3Hofmann S.L. Brown M.S. Lee E. Pathak R.K. Anderson R.G. Goldstein J.L. J. Biol. Chem. 1989; 264: 8260-8270Abstract Full Text PDF PubMed Google Scholar). This suggests that HRC may play a role in Ca2+ homeostasis in the SR, although its physiological function is largely unknown. The deduced amino acid sequence of a full-length HRC cDNA reveals several unique features (1Hofmann S.L. Goldstein J.L. Orth K. Moomaw C.R. Slaughter C.A. Brown M.S. J. Biol. Chem. 1989; 264: 18083-18090Abstract Full Text PDF PubMed Google Scholar): 1) an amino-terminal signal sequence; 2) in the middle, nine nearly identical repeats of 29 residues containing a histidine-rich sequence HRHRGH and a stretch of 10 to 11 acidic amino acids; 3) a 13-residue stretch of polyglutamic acid near the carboxyl terminus; and 4) a cluster of 14 closely spaced cysteine residues in the carboxyl terminus. High contents of acidic amino acids may account for the high capacity Ca2+ binding of HRC, which is reminiscent of calsequestrin, a well characterized Ca2+-binding protein of the SR. Triadin was also first identified from rabbit skeletal muscle and found to be a single span membrane protein localized to skeletal muscle triads (4Caswell A.H. Brandt N.R. Brunschwig J.P. Purkerson S. Biochemistry. 1991; 30: 7507-7513Crossref PubMed Scopus (134) Google Scholar, 5Knudson C.M. Stang K.K. Moomaw C.R. Slaughter C.A. Campbell K.P. J. Biol. Chem. 1993; 268: 12646-23654Abstract Full Text PDF PubMed Google Scholar). Triadin was shown to be associated into a stable quaternary complex with the ryanodine receptor, calsequestrin, and junctin at the junctional membrane (6Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). Previous studies suggest that calsequestrin, the high capacity Ca2+-binding protein, actively participates in muscle contraction by regulating the amounts of Ca2+ released by the ryanodine receptor, the Ca2+ release channel (7Ikemoto N. Ronjat M. Meszaros L.G. Koshita M. Biochemistry. 1989; 28: 6764-6771Crossref PubMed Scopus (187) Google Scholar, 8Ikemoto N. Antoniu B. Kang J.J. Meszaros L.G. Ronjat M. Biochemistry. 1991; 30: 5230-5237Crossref PubMed Scopus (82) Google Scholar, 9Kawasaki T. Kasai M. Biochem. Biophys. Res. Commun. 1994; 199: 1120-1127Crossref PubMed Scopus (93) Google Scholar, 10Donoso P. Beltran M. Hidalgo C. Biochemistry. 1996; 35: 13419-13425Crossref PubMed Scopus (70) Google Scholar). This regulatory effect was suggested to be regulated by calsequestrin-anchoring proteins, triadin (11Guo W. Campbell K.P. J. Biol. Chem. 1995; 270: 9027-9030Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 12Guo W. Jorgensen A.O. Campbell K.P. Soc. Gen. Physiol. Ser. 1996; 51: 19-28PubMed Google Scholar) and junctin (13Mitchell R.D. Simmerman H.K. Jones L.R. J. Biol. Chem. 1988; 263: 1376-1381Abstract Full Text PDF PubMed Google Scholar, 14Jones L.R. Zhang L. Sanborn K. Jorgensen A.O. Kelley J. J. Biol. Chem. 1995; 270: 30787-30796Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Several triadin isoforms appear to arise from alternative splicing of the single triadin gene (5Knudson C.M. Stang K.K. Moomaw C.R. Slaughter C.A. Campbell K.P. J. Biol. Chem. 1993; 268: 12646-23654Abstract Full Text PDF PubMed Google Scholar, 15Guo W. Jorgensen A.O. Jones L.R. Campbell K.P. J. Biol. Chem. 1996; 271: 458-465Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 16Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1999; 274: 28660-28668Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). All triadin isoforms share identical sequences over the first 250–260 residues. This common region encompasses the short amino-terminal cytoplasmic region, the membrane-spanning region, and the highly charged lumenal domain. Particularly, the lumenal domain contains several clusters of charged amino acids, which are referred to as "KEKE" motifs (17Realini C. Rogers S.W. Rechsteiner M. FEBS Lett. 1994; 348: 109-113Crossref PubMed Scopus (158) Google Scholar) and have been proposed to facilitate protein-protein interactions by acting as polar zippers (18Realini C. Rechsteiner M. J. Biol. Chem. 1995; 270: 29664-29667Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 19Perutz M.F. Johnson T. Suzuki M. Finch J.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5355-5358Crossref PubMed Scopus (941) Google Scholar, 20West Jr., R.W. Kruger B. Thomas S. Ma J. Milgrom E. Gene. 2000; 243: 195-205Crossref PubMed Scopus (18) Google Scholar). A recent study has demonstrated that a single KEKE motif of the lumenal domain of triadin is indeed essential for the binding of triadin to calsequestrin (21Kobayashi Y.M. Alseikhan B.A. Jones L.R. J. Biol. Chem. 2000; 275: 17639-17646Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Since HRC shares many biochemical and structural features with calsequestrin, we reasoned that HRC might be involved in Ca2+ release from the SR by playing a similar role as calsequestrin. In addition, several lines of circumstantial evidence suggested that HRC might be associated with triadin (22Kim K.C. Caswell A.H. Talvenheimo J.A. Brandt N.R. Biochemistry. 1990; 29: 9281-9289Crossref PubMed Scopus (106) Google Scholar, 23Damiani E. Picello E. Saggin L. Margreth A. Biochem. Biophys. Res. Commun. 1995; 209: 457-465Crossref PubMed Scopus (44) Google Scholar, 24Sacchetto R. Turcato F. Damiani E. Margreth A. J. Muscle Res. Cell Motil. 1999; 20: 403-415Crossref PubMed Scopus (27) Google Scholar). In the present study, we have utilized immunoprecipitation and fusion protein binding assays to investigate the alleged interaction between HRC and triadin. Our data demonstrate that HRC indeed binds to triadin, and this binding occurs between the histidine-rich acidic region of HRC and the lumenal region of triadin containing the KEKE motif, which is also critical for binding to calsequestrin. Heavy SR vesicles were isolated from skeletal muscle of male New Zealand White rabbit according to the heavy SR preparation method described previously (25Kim D.H. Sreter F.A. Ohnishi S.T. Ryan J.F. Roberts J. Allen P.D. Meszaros L.G. Antoniu B. Ikemoto N. Biochim. Biophys. Acta. 1984; 775: 320-327Crossref PubMed Scopus (80) Google Scholar). Protein assays were performed using bovine serum albumin as a standard (26Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar). Rabbit skeletal heavy SR vesicles were solubilized at a protein concentration of 5 mg/ml in a buffer containing 2% Triton X-100, 1 m NaCl, 1 mmdithiothreitol, 20 mm Tris-Cl (pH 7.4), and protease inhibitor mixture (Roche Molecular Biochemicals) for 1 h at 4 °C. Solubilized proteins were obtained by centrifugation (a Beckman TLA-100.3 rotor) at 60,000 rpm for 45 min at 4 °C. Triton X-100 solubilized rabbit skeletal heavy SR proteins were diluted 10-fold in 20 mmTris-Cl (pH 7.4), 1 mm dithiothreitol, and protease inhibitor mixture (Roche Molecular Biochemicals). One ml of the diluted extracts was incubated with purified sheep polyclonal antibody raised against HRC (a gift from Dr. Sandra Hoffman, Southwestern Medical Center) or sheep polyclonal antibody raised against triadin (a gift from Dr. Kevin Campbell, University of Iowa College of Medicine) for 2 h at 4 °C, followed by further incubation with 50 µl of protein G-agarose beads (Roche Molecular Biochemicals) for 4 h at 4 °C. Immunoprecipitates were collected and washed several times with a buffer containing 20 mm Tris-Cl (pH 7.4), 0.15m NaCl, and 0.2% Triton X-100. Proteins were eluted from the affinity beads by boiling in SDS-PAGE sample buffer (60 mm Tris-Cl (pH 6.8), 2% SDS, 10% glycerol, 100 mm dithiothreitol, and 0.01% bromphenol blue) and subjected to SDS-PAGE. Purified sheep preimmune IgGs were used as a control. Fragments of HRC and triadin were amplified by PCR, digested with EcoRI andXhoI and subcloned into the EcoRI-XhoI sites of pGEX 4T-1 (Pharmacia). Primer sets used for PCR are as follows: HRC-N (27–199 residues), 5′-ATCCG AATTC ACCCA GCAGC TGAGA GG-3′, 5′-ATTCG TCGAC CTGTG CTCTG GGGAG ACC-3′; HRC-M (193–357 residues), 5′-TTCGG AATTC GAGGA GGTCT CCCCA GAGC-3′, 5′-AACGG TCGAC GACGT GATGG TGTTC ACC-3′; HRC-C (471–847 residues), 5′-ATGCG AATTC CACCA TCACG TCCCT CACC-3′, 5′-TTAGC TCGAG GGCGT CTCCA GCATG TCC-3′; Tri-a (1–66 residues), 5′ ATGAAT TCATGACTGAGATCACTGC-3′, 5′ATGTC GACTA ACAAC GGCAA CTGC-3′; Tri-b (79–160 residues), 5′-ATCCG AATTC TCTAT TGCCA AGATG GGC-3′, 5′-TTAGC TCGAG TGGTA TTTTC CTCTC AGG-3′; Tri-c(151–260 residues), 5′-AGTCG AATTC CAGGA AAAAC CTGAG AGG-3′, 5′-TTAGC TCGAG GTTTT GAAAC AGCAG CAG-3′; Tri-c1 (151–204 residues), 5′-AGTCG AATTC CAGGA AAAAC CTGAG AGG-3′, 5′-TCTTC TCGAG TTTTG TCACT GTCTT TGTTT CTGG-3′; Tri-c2 (204–260 residues), 5′-AGTCG AATTC GAGGA GAAGA AAGCT CG-3′, 5′-TTAGC TCGAG GTTTT GAAAC AGCAG CAG-3′. To produce more GST fusion proteins containing various numbers of histidine-rich acidic repeats of HRC, one forward and two backward primers were synthesized in addition to the HRC-M primer set. The primers were used in combinations. The additional forward primer is: 5′-TTCGG AATTC CACAG GCACC GGGGC CAC-3′. The additional backward primers are: 5′-AACGG TCGAC GGCCC TGGTG ACGGT CGCT-3′ and 5′-AACGG TCGAC TTCAC CTTCG TCATC ATC-3′. GST fusion constructs were induced in Escherichia coli JM109 with 0.5 mmisopropyl β-d-thiogalactopyranoside for 3 h at 30 °C. Cells were harvested, resuspended in phosphate buffered saline containing 1 mm phenylmethylsulfonyl fluoride, lysed by sonication on ice, and then incubated in 1% Triton X-100 for 1 h. The soluble fraction was obtained by centrifugation at 13,000 rpm for 10 min at 4 °C. The fusion proteins were immobilized by incubating 1 ml of the soluble E. coli fraction with 50 µl of bead volume of glutathione-Sepharose 4B (Pharmacia) for 3 h and washed three times with 1 ml phosphate-buffered saline. The immobilized fusion proteins were incubated with 1 ml of heavy SR proteins solubilized with Triton X-100 for 5 h at 4 °C. Beads were then washed with 20 mm Tris-Cl (pH 7.4), 0.15 mNaCl, and 0.2% Triton X-100. Bound proteins were eluted by boiling in SDS-PAGE sample buffer and subjected to SDS-PAGE. Protein samples were separated by 6 or 10% SDS-PAGE and then transferred to nitrocellulose filters by a semidry-transfer unit (Hoefer) by following the manufacturer's manual. The blots were rinsed briefly with TNT buffer (100 mm Tris-Cl (pH 8.0), 150 mm NaCl and 0.5% Tween 20), blocked with 3% bovine serum albumin/TNT and then incubated with the primary antibody in 0.5% bovine serum albumin/TNT for 1 h at room temperature with agitation. The blots were washed with three changes of TNT for 15 min each and incubated with the horse radish peroxidase-conjugated secondary antibody (Zymed Laboratories Inc.) in 0.5% bovine serum albumin/TNT for 1 h at room temperature with gentle shaking. The blots were then washed again 3–4 times with TNT for 20 min each. Signals were detected using the ECL system (Pierce). Mouse monoclonal antibodies against rabbit skeletal triadin and SERCA1 were purchased from Affinity BioReagents. The interaction of HRC and triadin was first examined by co-immunoprecipitation experiments with specific antibodies. Solubilized heavy SR proteins were immunoprecipitated with either anti-HRC or anti-triadin antibodies (see "Experimental Procedures). The immunoprecipitated proteins were then analyzed by Western blot (Fig. 1) with anti-HRC, anti-triadin, or anti-SERCA antibodies. Immunoprecipitation of HRC (Fig. 1 A, left panel, lanes 2, 4) led to the co-precipitation of triadin (Fig.1 A, mid-panel, lanes 2, 4), whereas preimmune serum was unable to precipitate either of these proteins (Fig. 1 A, left andmid-panels, lane 3). Conversely, immunoprecipitation of triadin (Fig. 1 B, left panel, lanes 2, 4) led to the co-precipitation of HRC (Fig. 1 B, mid-panel,lanes 2, 4). Once again, preimmune serum precipitated none of these proteins (Fig. 1 B,left and mid-panels, lane 3). These results show that HRC indeed binds to triadin. SERCA, another SR protein, was co-precipitated by neither HRC nor triadin (Fig. 1,A and B, right panels), implying that the interaction between HRC and triadin is specific. Three segments of HRC were expressed and purified as GST fusion proteins to determine the triadin binding domain of HRC (Fig.2A). HRC-N does not contain any notable domains. HRC-M contains five of the nine nearly identical histidine-rich acidic repeats (see below), each consisting of a histidine-rich sequence HRHRGH, a stretch of 10 to 11 acidic amino acids and a sequence containing two serines and one threonine in a negatively charged context. HRC-C contains a 13-residue stretch of polyglutamic acids and a cluster of 14 closely spaced cysteine residues. The three GST fusion proteins and a GST control were incubated with solubilized heavy SR and precipitated using glutathione beads. The resulting precipitates were separated by SDS-PAGE and stained with Coomassie blue (Fig. 2 B, left panel), and the same samples were transferred to nitrocellulose and probed with anti-triadin antibody (right panel). The result indicates that only the HRC-M region binds to triadin. Thus, the histidine-rich acidic repeats may represent a novel domain that is involved in protein-protein interaction. The repeats are highly homologous in their amino acid sequences and the tenth repeat is an incomplete one (Fig. 3A). To analyze the triadin binding domain in the repeats with greater precision, we have generated more GST fusion proteins containing various sets of repeats by PCR (Fig. 3 B). Internal deletions of repeats in some fusion proteins might arise probably due to the highly repetitive nature of the template sequence. Protein binding assays were performed as described above (Fig. 3 C). GST fusion proteins a through i did not bind to triadin, suggesting that no particular repeat is uniquely essential for the triadin binding. This is consistent with the fact that all the repeats are nearly identical with a few conservative amino acid substitutions or deletions. GST fusion proteins, j, k, andl contained more than four and a half repeats and were shown to bind to triadin (HRC-M in Fig. 2 is identical to the fusion protein l). Based on these observations, we suggest that more than four and a half repeats are required for the triadin binding of HRC. Further structural studies should be necessary to reveal the exact mechanism of the protein binding activity of the repeats.Figure 3Analysis of the histidine-rich acidic repeats of HRC for triadin binding. Panel A, sequences of the histidine-rich acidic repeats of HRC are aligned. Panel B, structures of the analyzed GST-repeats fusion proteins are shown.Panel C, the GST-repeats fusion proteins were incubated with solubilized heavy SR. The bound proteins were separated by SDS-PAGE and stained with Coomassie Blue (left panel) or probed with anti-triadin antibody (right panel). GST and GST fusion proteins are denoted by dots.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Calsequestrin, a Ca2+-binding protein of SR, binds to triadin and junctin in a Ca2+-sensitive manner. We therefore tested whether the interaction between HRC and triadin is also affected by Ca2+ concentration. HRC was immunoprecipitated from solubilized heavy SR in the presence of EGTA or various concentrations of Ca2+. The precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-HRC and anti-triadin antibodies (Fig.4A). The interaction between HRC and triadin was strongest when assayed in the absence of Ca2+ and inhibited by millimolar concentrations of Ca2+. This behavior is similar to the interaction between calsequestrin and triadin. Identical results were obtained when the HRC-M fusion protein was utilized to pull down the triadin protein (Fig. 4 B), showing once again that HRC-M is the domain responsible for triadin binding of HRC in a Ca2+ sensitive manner. All triadin isoforms in a given species have identical amino acid sequence over the first 250–260 residues. This common region contains a short amino-terminal cytoplasmic domain, a membrane-spanning region, and highly negatively charged luminal domains. The charged luminal domain has been shown to be responsible for binding to calsequestrin and the ryanodine receptor. We therefore assumed that this common region might also contain the HRC binding domain. Three GST fusion proteins were first generated: Tri-a contains the amino-terminal cytoplasmic region and part of the membrane-spanning region; Tri-b contains the proximal half of the common luminal domain; and Tri-c contains the distal half of the common luminal domain (Fig.5A). GST fusion proteins and GST alone were purified and incubated with solubilized heavy SR. The protein complexes were then recovered on glutathione beads, separated on SDS-PAGE, and then stained with Coomassie Blue (Fig. 5 B,left panel), or probed with anti-HRC antibody (mid-panel) or anti-triadin antibody (right panel). Only the Tri-c fusion protein was able to pull down HRC. We then generated two more GST fusion proteins, Tri-c1 and Tri-c2, which are the proximal and distal halves of the Tri-c region, respectively. The results of a similar binding assay showed that the Tri-c2 region contains the HRC binding activity (Fig. 5 B,mid-panel). Interestingly, the Tri-c2 region contains the KEKE motif that was previously shown to bind directly to calsequestrin (6Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). However, it is not yet certain that this particular KEKE motif also binds to HRC as well as to calsequestrin. Tri-c fusion protein also pulled down triadin itself (Fig. 5 B, right panel), implying that triadin can form a homo-oligomer. Unlike the HRC-triadin and calsequestrin-triadin interactions, the domains for triadin-triadin association appear to be dispersed throughout the Tri-c region. The Tri-c2 fusion protein was also used to test whether the interaction between HRC and triadin is Ca2+-sensitive, as shown in Fig.4. As expected, the interaction was strongest in the absence of Ca2+ and inhibited by increasing Ca2+concentration (Fig. 6,mid-panel). The triadin-triadin interaction, however, was rather Ca2+-resistant (right panel). It was shown that the ryanodine receptor, calsequestrin, triadin, and junctin form a stable quaternary structure at the inner surface of the junctional SR membrane (6Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). In this protein complex, triadin and junctin are suggested to serve as a scaffold structure that brings the Ca2+-binding protein (calsequestrin) into the proximity of the Ca2+ channel (ryanodine receptor). Because of this structural feature, it is postulated that a high concentration of Ca2+ can be stored at the inner surface of the junctional SR membrane, and thus Ca2+ ions can be rapidly released through the ryanodine receptor upon activation of Ca2+release in muscle cells. In the present study, we have shown that HRC, another SR Ca2+-binding protein, directly binds to triadin, suggesting that HRC could be an additional component of this protein complex. HRC is a high capacity and low affinity Ca2+-binding protein (27Picello E. Damiani E. Margreth A. Biochem. Biophys. Res. Commun. 1992; 186: 659-667Crossref PubMed Scopus (44) Google Scholar) like calsequestrin. We do not yet understand the role of HRC, as the more abundant Ca2+-binding protein calsequestrin appears to serve its role as a Ca2+ store perfectly well. It was shown that calsequestrin plays a role in the regulation of the ryanodine receptor (28Ohkura M. Furukawa K. Fujimori H. Kuruma A. Kawano S. Hiraoka M. Kuniyasu A. Nakayama H. Ohizumi Y. Biochemistry. 1998; 37: 12987-12993Crossref PubMed Scopus (84) Google Scholar, 29Groh S. Marty I. Ottolia M. Prestipino G. Chapel A. Villaz M. Ronjat M. J. Biol. Chem. 1999; 274: 12278-12283Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) in addition to the Ca2+ store function. Thus, HRC may perhaps also have a regulatory role. It remains to be seen whether this hypothesis is correct or whether HRC functions as a mere auxiliary Ca2+ store. The first evidence of an interaction between HRC and triadin was presented in an earlier study (22Kim K.C. Caswell A.H. Talvenheimo J.A. Brandt N.R. Biochemistry. 1990; 29: 9281-9289Crossref PubMed Scopus (106) Google Scholar). In this study, the authors observed by using affinity chromatography that a 95-kDa protein, now known as triadin, bound to the α1 subunit of the dihydropyridine receptor and to a novel 170-kDa protein. This 170-kDa protein was strongly stained with Stains-all, which specifically stains Ca2+-binding proteins (30Campbell K.P. MacLennan D.H. Jorgensen A.O. J. Biol. Chem. 1983; 258: 11267-11273Abstract Full Text PDF PubMed Google Scholar). Because HRC shows similar electrophoretic mobility on SDS-PAGE, and it is the only Stains-all stained SR protein in this molecular range, we reasoned that this 170-kDa protein is most likely HRC. Recently, Sacchetto et al. (24Sacchetto R. Turcato F. Damiani E. Margreth A. J. Muscle Res. Cell Motil. 1999; 20: 403-415Crossref PubMed Scopus (27) Google Scholar) performed filter binding assays with digoxigenin-labeled HRC and drew the conclusion that HRC binds to the short amino-terminal cytoplasmic region of triadin. This is inconsistent with our data and also with the earlier immunofluorescent and electron microscopic studies (3Hofmann S.L. Brown M.S. Lee E. Pathak R.K. Anderson R.G. Goldstein J.L. J. Biol. Chem. 1989; 264: 8260-8270Abstract Full Text PDF PubMed Google Scholar) and tryptic digestion and biotinylation experiments (2Suk J.Y. Kim Y.S. Park W.J. Biochem. Biophys. Res. Commun. 1999; 263: 667-671Crossref PubMed Scopus (29) Google Scholar), which suggested that HRC resides in the lumen of the SR. It is possible that denaturation of the protein samples during the filter binding assay may alter the specificity of protein-protein interactions and even generate false fortuitous interactions. Therefore, the results of filter binding assays should probably be interpreted with much caution. Our data show that HRC binds to the lumenal domain of triadin, which is highly enriched in charged amino acids. We further narrowed the interaction domain to the Tri-c2 region (205–260 residues) of triadin (Fig. 5). Notably, this region contains the KEKE motif that was shown to directly bind to calsequestrin (21Kobayashi Y.M. Alseikhan B.A. Jones L.R. J. Biol. Chem. 2000; 275: 17639-17646Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). KEKE motifs are defined as regions of sequences greater than 12 residues in length, devoid of tryptophan, tyrosine, phenylalanine, and proline, with more than 60% alternating lysine and glutamate/aspartate residues and lacking five positively or negatively charged residues in a row (17Realini C. Rogers S.W. Rechsteiner M. FEBS Lett. 1994; 348: 109-113Crossref PubMed Scopus (158) Google Scholar). This motif was suggested to promote association between proteins (18Realini C. Rechsteiner M. J. Biol. Chem. 1995; 270: 29664-29667Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 19Perutz M.F. Johnson T. Suzuki M. Finch J.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5355-5358Crossref PubMed Scopus (941) Google Scholar, 20West Jr., R.W. Kruger B. Thomas S. Ma J. Milgrom E. Gene. 2000; 243: 195-205Crossref PubMed Scopus (18) Google Scholar). The subdomains Tri-b, Tri-c1, and Tri-c2 of triadin contain one, two, and two KEKE motifs, respectively (Fig. 5). Of these, the proximal KEKE motif of Tri-c2 is responsible for interaction with calsequestrin (indicated by an asterisk in Fig. 5). Our data are not able to distinguish whether this proximal KEKE motif alone, the distal one, or both KEKE motifs are involved in the HRC-triadin interaction. HRC contains nine nearly identical tandem repeats of 29 residues, each consisting of a histidine-rich sequence HRHRGH, a stretch of 10 to 11 acidic amino acids, and a sequence containing two serines and one threonine in a negatively charged context (1Hofmann S.L. Goldstein J.L. Orth K. Moomaw C.R. Slaughter C.A. Brown M.S. J. Biol. Chem. 1989; 264: 18083-18090Abstract Full Text PDF PubMed Google Scholar). Our data show that this domain directly binds to triadin and thus seems to represent a novel motif for protein-protein interactions. However, the mechanism underlying the interaction between HRC and triadin is unclear. It was suggested that KEKE motifs often stabilize protein-protein interactions by acting as polar zippers. It appears that the histidine-rich acidic repeats are unable to form such a zipper, therefore the HRC-triadin interaction may be facilitated by other mechanisms. Both the HRC-triadin interaction (this study) and the calsequestrin-triadin interaction (6Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 13Mitchell R.D. Simmerman H.K. Jones L.R. J. Biol. Chem. 1988; 263: 1376-1381Abstract Full Text PDF PubMed Google Scholar, 14Jones L.R. Zhang L. Sanborn K. Jorgensen A.O. Kelley J. J. Biol. Chem. 1995; 270: 30787-30796Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) are Ca2+-sensitive, implying that a common mechanism underlies both of these interactions. Intriguingly, the triadin binding region of calsequestrin was localized to the carboxyl-terminal stretch of glutamate residues (31Shin D. Ma J. Kim D.H. FEBS Lett. 2000; 486: 178-182Crossref PubMed Scopus (97) Google Scholar). In a similar fashion, the stretch of acidic amino acids in the histidine-rich acidic repeats of HRC may be essential for triadin binding of HRC. However, it should be noted that the other stretch of glutamate residues (721–733 residues) in HRC does not bind to triadin, thus implying that the mode of HRC-triadin and calsequestrin-triadin interactions seems more complex. We report here that triadin-triadin association occurs through the Tri-c (151–260 residues) region of triadin and this interaction is not affected by high Ca2+ concentration. Similarly, triadin was shown to associate with junctin in a Ca2+-resistant manner (6Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). We propose that triadin and junctin constitute a possibly huge scaffold structure by associating with each other and themselves and that the integrity of this scaffold is not affected by changes in intralumenal Ca2+ concentrations. It seems possible that triadin-triadin or triadin-junctin interactions are facilitated by forming polar zippers that are in this case Ca2+-resistant. In conclusion, we have shown that HRC binds to triadin in a Ca2+-sensitive manner in SR lumen. This and other biochemical characteristics of HRC suggest that HRC may play a similar role to calsequestrin during excitation-contraction coupling. Further investigation will be required to address this issue including production of transgenic mice that overexpress HRC, which is currently in progress.