Localization of the 12.6-kDa FK506-binding Protein (FKBP12.6) Binding Site to the NH2-terminal Domain of the Cardiac Ca2+ Release Channel (Ryanodine Receptor)

The 12.6-kDa FK506-binding protein (FKBP12.6) interacts with the cardiac ryanodine receptor (RyR2) and modulates its channel function. However, the molecular basis of FKBP12.6-RyR2 interaction is poorly understood. To investigate the significance of the isoleucine-proline (residues 2427–2428) dipeptide epitope, which is thought to form an essential part of the FKBP12.6 binding site in RyR2, we generated single and double mutants, P2428Q, I2427E/P2428A, and P2428A/L2429E, expressed them in HEK293 cells, and assessed their ability to bind GST-FKBP12.6. None of these mutations abolished GST-FKBP12.6 binding, indicating that this isoleucine-proline motif is unlikely to form the core of the FKBP12.6 binding site in RyR2. To systematically define the molecular determinants of FKBP12.6 binding, we constructed a series of internal and NH2- and COOH-terminal deletion mutants of RyR2 and examined the effect of these deletions on GST-FKBP12.6 binding. These deletion analyses revealed that the first 305 NH2-terminal residues and COOH-terminal residues 1937–4967 are not essential for GST-FKBP12.6 binding, whereas multiple sequences within a large region between residues 305 and 1937 are required for GST-FKBP12.6 interaction. Furthermore, an NH2-terminal fragment containing the first 1937 residues is sufficient for GST-FKBP12.6 binding. Co-expression of overlapping NH2 and COOH-terminal fragments covering the entire sequence of RyR2 produced functional channels but did not restore GST-FKBP12.6 binding. These data suggest that FKBP12.6 binding is likely to be conformationdependent. Binding of FKBP12.6 to the NH2-terminal domain may play a role in stabilizing the conformation of this region. The 12.6-kDa FK506-binding protein (FKBP12.6) interacts with the cardiac ryanodine receptor (RyR2) and modulates its channel function. However, the molecular basis of FKBP12.6-RyR2 interaction is poorly understood. To investigate the significance of the isoleucine-proline (residues 2427–2428) dipeptide epitope, which is thought to form an essential part of the FKBP12.6 binding site in RyR2, we generated single and double mutants, P2428Q, I2427E/P2428A, and P2428A/L2429E, expressed them in HEK293 cells, and assessed their ability to bind GST-FKBP12.6. None of these mutations abolished GST-FKBP12.6 binding, indicating that this isoleucine-proline motif is unlikely to form the core of the FKBP12.6 binding site in RyR2. To systematically define the molecular determinants of FKBP12.6 binding, we constructed a series of internal and NH2- and COOH-terminal deletion mutants of RyR2 and examined the effect of these deletions on GST-FKBP12.6 binding. These deletion analyses revealed that the first 305 NH2-terminal residues and COOH-terminal residues 1937–4967 are not essential for GST-FKBP12.6 binding, whereas multiple sequences within a large region between residues 305 and 1937 are required for GST-FKBP12.6 interaction. Furthermore, an NH2-terminal fragment containing the first 1937 residues is sufficient for GST-FKBP12.6 binding. Co-expression of overlapping NH2 and COOH-terminal fragments covering the entire sequence of RyR2 produced functional channels but did not restore GST-FKBP12.6 binding. These data suggest that FKBP12.6 binding is likely to be conformationdependent. Binding of FKBP12.6 to the NH2-terminal domain may play a role in stabilizing the conformation of this region. ryanodine receptor inositol 1,4,5-trisphosphate receptor glutathione S-transferase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Ryanodine receptors (RyRs)1 are intracellular Ca2+ channels located in the sarco(endo)plasmic reticulum of muscle and nonmuscle cells. They govern the release of Ca2+ from intracellular stores and play an essential role in various cellular processes including muscle contraction, fertilization, secretion, and apoptosis (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4493) Google Scholar). These channels are regulated by a number of protein modulators, such as the 12- and 12.6-kDa FK506-binding proteins (FKBP12 and FKBP12.6) (2Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (843) Google Scholar, 3Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 4Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar, 5Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (596) Google Scholar, 6Fill M. Copello J.A. Physiol. Rev. 2002; 82: 893-922Crossref PubMed Scopus (889) Google Scholar). FKBP12 is tightly associated with the type 1 ryanodine receptor (RyR1) predominantly expressed in skeletal muscle (7Jayaraman T. Brillantes A.M. Timerman A.P. Fleischer S. Erdjument-Bromage H. Tempst P. Marks A.R. J. Biol. Chem. 1992; 267: 9474-9477Abstract Full Text PDF PubMed Google Scholar), whereas FKBP12.6 is selectively associated with the type 2 ryanodine receptor (RyR2) mainly expressed in cardiac muscle and in the brain (8Timerman A.P. Jayaraman T. Wiederrecht G. Onoue H. Marks A.R. Fleischer S. Biochem. Biophys. Res. Commun. 1994; 198: 701-706Crossref PubMed Scopus (114) Google Scholar, 9Timerman A.P. Onoue H. Xin H.B. Barg S. Copello J. Wiederrecht G. Fleischer S. J. Biol. Chem. 1996; 271: 20385-20391Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). The type 3 ryanodine receptor (RyR3), which is expressed at relatively low levels in a variety of tissues, has also been shown to be capable of interacting with both FKBP12 and FKBP12.6 (10Bultynck G. Rossi D. Callewaert G. Missiaen L. Sorrentino V. Parys J.B. De Smedt H. J. Biol. Chem. 2001; 276: 47715-47724Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The interactions between FKBP and RyR are believed to be involved in the stabilization of the full conductance state (11Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar), channel gating (12Gaburjakova M. Gaburjakova J. Reiken S. Huang F. Marx S.O. Rosemblit N. Marks A.R. J. Biol. Chem. 2001; 276: 16931-16935Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), and modulating the sensitivity to Ca2+ activation of RyR (13Mayrleitner M. Timerman A.P. Wiederrecht G. Fleischer S. Cell Calcium. 1994; 15: 99-108Crossref PubMed Scopus (104) Google Scholar, 14Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar). Alterations in these interactions have been implicated in cardiomyopathy (15Shou W. Aghdasi B. Armstrong D.L. Guo Q. Bao S. Charng M.J. Mathews L.M. Schneider M.D. Hamilton S.L. Matzuk M.M. Nature. 1998; 391: 489-492Crossref PubMed Scopus (353) Google Scholar), cardiac hypertrophy (16Xin H.B. Senbonmatsu T. Cheng D.S. Wang Y.X. Copello J.A., Ji, G.J. Collier M.L. Deng K.Y. Jeyakumar L.H. Magnuson M.A. Inagami T. Kotlikoff M.I. Fleischer S. Nature. 2002; 416: 334-338Crossref PubMed Scopus (259) Google Scholar), and heart failure (17Doi M. Yano M. Kobayashi S. Kohno M. Tokuhisa T. Okuda S. Suetsugu M. Hisamatsu Y. Ohkusa T. Matsuzaki M. Circulation. 2002; 105: 1374-1379Crossref PubMed Scopus (120) Google Scholar,18Marks A.R. Front. Biosci. 2002; 7: 970-977Crossref PubMed Google Scholar). Given the important roles of FKBPs in RyR regulation, a number of studies have focused on the structural basis of FKBP-RyR interactions, and some insights into the molecular determinants of FKBP binding have recently been revealed (10Bultynck G. Rossi D. Callewaert G. Missiaen L. Sorrentino V. Parys J.B. De Smedt H. J. Biol. Chem. 2001; 276: 47715-47724Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Gaburjakova M. Gaburjakova J. Reiken S. Huang F. Marx S.O. Rosemblit N. Marks A.R. J. Biol. Chem. 2001; 276: 16931-16935Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 14Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 19Cameron A.M. Nucifora F.C., Jr. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 20Bultynck G., De Smet P. Rossi D. Callewaert G. Missiaen L. Sorrentino V., De Smedt H. Parys J.B. Biochem. J. 2001; 354: 413-422Crossref PubMed Scopus (81) Google Scholar, 21Zissimopoulos S. Lai F.A. Biophys. J. 2002; 82 (abstr.): 59Google Scholar). Using the yeast two-hybrid technique, it has been shown that a 114-amino acid region containing residues 2497–2520 of RyR1 interacts with FKBP12. An analogous region in the inositol 1,4,5-trisphosphate receptor (IP3R) has also been shown to bind FKBP12 (19Cameron A.M. Nucifora F.C., Jr. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). This 114-amino acid fragment contains a valine-proline (residues 2461 and 2462) dipeptide epitope thought to be the FKBP12 binding motif in RyR1 (19Cameron A.M. Nucifora F.C., Jr. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), and its significance in FKBP12 binding to the intact full-length RyR1 protein has been confirmed. Mutations of valine 2461 to glycine, glutamate, FKBP12 binding to RyR1 (12Gaburjakova M. Gaburjakova J. Reiken S. Huang F. Marx S.O. Rosemblit N. Marks A.R. J. Biol. Chem. 2001; 276: 16931-16935Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). This valine-proline dipeptide motif is in of the valine to also FKBP12 binding (10Bultynck G. Rossi D. Callewaert G. Missiaen L. Sorrentino V. Parys J.B. De Smedt H. J. Biol. Chem. 2001; 276: 47715-47724Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These that the valine-proline motif is essential for FKBP12 interaction with RyR1 and the of RyR2, the valine-proline motif is with The role of this motif in FKBP12.6 binding to RyR2 has not been Using the yeast two-hybrid Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google that a acid fragment of the RyR2 containing residues with FKBP12.6 and that this interaction by a to FKBP12.6 from RyR2. on these Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google that the FKBP12.6 binding site is by and analogous to the FKBP12 binding motif in and However, to this and the by Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar), studies by and Lai S. Lai F.A. Biophys. J. 2002; 82 (abstr.): 59Google the yeast two-hybrid and that of the overlapping fragments covering the entire sequence of RyR2, with Using the to interactions between the domain of the type 1 receptor and FKBP12.6 and FKBP12 S. Lai F.A. Biophys. J. 2002; 82 (abstr.): 59Google Scholar). and Lai S. Lai F.A. Biophys. J. 2002; 82 (abstr.): 59Google also that a large COOH-terminal fragment of RyR2 with to the that the FKBP12.6 binding site is located at the of RyR2 S. Lai F.A. Biophys. J. 2002; 82 (abstr.): 59Google Scholar). the of the FKBP12.6 binding site in RyR2 and has to be the we the significance of the isoleucine-proline motif in FKBP12.6 interaction with RyR2. Using a GST-FKBP12.6 we that mutations of this motif did not GST-FKBP12.6 from binding to RyR2, in to with RyR1 and This to studies to define the FKBP12.6 binding site in RyR2. and COOH-terminal deletion we that the first 1937 NH2-terminal residues are sufficient for GST-FKBP12.6 binding, whereas the COOH-terminal residues the FKBP12.6 binding motif are required sufficient for GST-FKBP12.6 binding. we that multiple within the NH2-terminal domain are required for GST-FKBP12.6 interaction. Furthermore, of overlapping COOH-terminal fragments to the of functional RyR channels but did not the GST-FKBP12.6 binding These suggest that FKBP12.6 binding may be and from The by the at the of the of from and from HEK293 in as K. L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). HEK293 on for with of type RyR Ca2+ J. T. Scholar). The RyR2 produced as B. H. D. Wang R. Y. L. T. Y. Lai F.A. T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). 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J. Physiol. 2001; PubMed Scopus Google Scholar), with that with and and with of GST-FKBP12.6 protein that with and with of at for The and the protein with 1 2 2 2 and for The proteins to the by the of of Nature. PubMed Scopus Google and at for The proteins by The proteins with to at for at in the of to H. T. J. S. A. PubMed Scopus Google Scholar). The for with containing and The with the and for with containing The with the with for for the by the and as the Ca2+ in HEK293 with the Ca2+ as P. L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). has been shown that the at in into FKBP12 binding (19Cameron A.M. Nucifora F.C., Jr. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). To the significance of the in RyR2 to FKBP12.6 binding, we to shown in both the type RyR2 and the by GST-FKBP12.6 1 but not by indicating that the not FKBP12.6 binding to RyR2, in to with has also been shown that mutations of valine 2461 in RyR1 valine in FKBP12 binding and that the motif in the receptor is for FKBP12 binding (10Bultynck G. Rossi D. Callewaert G. Missiaen L. Sorrentino V. Parys J.B. De Smedt H. J. Biol. Chem. 2001; 276: 47715-47724Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Gaburjakova M. Gaburjakova J. Reiken S. Huang F. Marx S.O. Rosemblit N. Marks A.R. J. Biol. Chem. 2001; 276: 16931-16935Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, M. J. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). To the role of the RyR2 residues in FKBP12.6 we generated double and mutants by GST-FKBP12.6 3 and but not by the cells, HEK293 with mutants P2428Q, I2427E/P2428A, and Ca2+ release 1 These data that the isoleucine-proline motif is not essential for the interaction of RyR2 with the motif is for FKBP12 binding to and the To in RyR2 that are essential for FKBP12.6 binding, we a deletion shown in deletion of acid residues did not GST-FKBP12.6 binding, whereas deletion of residues that a region between is required for FKBP12.6 binding. with this deletion of this region also abolished GST-FKBP12.6 binding 2 The region between residues into of did not FKBP12.6 binding. be that the isoleucine-proline indicating that this motif and its are not required for FKBP12.6 binding, which is with the of single mutations the deletion of did FKBP12.6 binding. The of that are NH2-terminal to residues in FKBP12.6 binding assessed by deletion of FKBP12.6 binding. a large region between residues 305 and 1937 is required for FKBP12.6 binding to RyR2. The of these internal deletions on RyR2 channel assessed by the of HEK293 with deletion deletions abolished Ca2+ release in HEK293 and indicating that a large of the RyR2 sequence is required for activation by To their ability to form we deletion mutants with an RyR2 RyR2 in HEK293 and their have shown that HEK293 with the RyR2 Ca2+ release B. H. D. Wang R. Y. L. T. Y. Lai F.A. T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). that a deletion is capable of a channel with RyR2 of HEK293 with a deletion and RyR2 may their the mutants may the of channel shown in HEK293 with RyR2 and internal deletion mutants for 3 Ca2+ deletion mutants the ability to form functional that these deletions did not the channel To investigate the role of the NH2 in FKBP12.6 binding, we constructed a series of NH2-terminal deletion mutants and examined the effect of these deletions on GST-FKBP12.6 binding. shown in deletion of the first 305 NH2-terminal residues did not GST-FKBP12.6 binding indicating that the first 305 NH2-terminal residues are not essential for FKBP12.6 binding. the deletion of the first NH2-terminal residues abolished GST-FKBP12.6 binding This that a NH2-terminal region 305 is for FKBP12.6 binding, which is with the of internal deletion studies These data also that a acid COOH-terminal fragment of RyR2 the FKBP12.6 binding motif is for GST-FKBP12.6 binding. The effect of NH2-terminal deletions on channel is shown in HEK293 with deletion to indicating that the first 305 NH2-terminal acid residues are not essential for activation of RyR2. the deletion of the first NH2-terminal acid residues abolished Ca2+ release in HEK293 and Co-expression of these deletion mutants with RyR2 their to activation indicating that these GST-FKBP12.6 NH2-terminal deletion mutants capable of functional The significance of the COOH-terminal region of RyR2 in FKBP12.6 interaction assessed by a series of COOH-terminal deletions and the effect of these deletions on GST-FKBP12.6 binding. of to COOH-terminal acid residues did not GST-FKBP12.6 binding, whereas deletion of the to to a of GST-FKBP12.6 binding These that the COOH-terminal region at 1937 is not required for GST-FKBP12.6 binding and that the first 1937 NH2-terminal residues are sufficient for GST-FKBP12.6 binding. These with the that multiple deletions within the NH2-terminal region GST-FKBP12.6 binding and channel 2 and the that the NH2-terminal region may a domain To this we this NH2-terminal fragment with the NH2-terminal deletion mutants, and in HEK293 and examined the of the cells. shown in Ca2+ release in HEK293 with the and fragments and with the and fragments the with and fragments did not Ca2+ release which is with the of internal deletion studies in which residues between are essential for activation 3 These that the NH2-terminal is capable of interacting with the COOH-terminal and examined a GST-FKBP12.6 NH2-terminal fragment to form functional channels with an overlapping COOH-terminal the NH2-terminal fragment containing residues with the COOH-terminal fragments and in HEK293 and examined Ca2+ release in Co-expression of fragments and which GST-FKBP12.6 binding, produced Ca2+ release channels in HEK293 the Ca2+ release in HEK293 with fragments and and with fragments and which have indicating that residues are for channel function. that of fragments and form functional it also be to restore the FKBP12.6 binding To this we and from HEK293 with fragments and shown in the to of the expressed whereas of these fragments by GST-FKBP12.6. These data that overlapping fragments covering the entire sequence of RyR2 are not sufficient to form a FKBP12.6 binding is believed that the isoleucine-proline (residues and motif an essential part of the FKBP12.6 binding site in RyR2 S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, A.R. Front. Biosci. 2002; 7: 970-977Crossref PubMed Google Scholar, A.R. Circulation. 2002; PubMed Scopus Google Scholar, C. N. M. G. R. Sorrentino Circulation. 2001; PubMed Scopus Google Scholar, N. A. A. F. G. B. K. B. M. C. G. A. Mol. 2001; PubMed Scopus Google Scholar). This mainly from the that a fragment of RyR2 this motif interacts with FKBP12.6 in the yeast two-hybrid and that the motif in and is for FKBP12 and FKBP12.6 binding (10Bultynck G. Rossi D. Callewaert G. Missiaen L. Sorrentino V. Parys J.B. De Smedt H. J. Biol. Chem. 2001; 276: 47715-47724Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Gaburjakova M. Gaburjakova J. Reiken S. Huang F. Marx S.O. Rosemblit N. Marks A.R. J. Biol. Chem. 2001; 276: 16931-16935Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 14Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 19Cameron A.M. Nucifora F.C., Jr. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 20Bultynck G., De Smet P. Rossi D. Callewaert G. Missiaen L. Sorrentino V., De Smedt H. Parys J.B. Biochem. J. 2001; 354: 413-422Crossref PubMed Scopus (81) Google Scholar). However, the significance of the isoleucine-proline motif this motif in FKBP12.6-RyR2 interaction has not been and it has recently been S. Lai F.A. Biophys. J. 2002; 82 (abstr.): 59Google Scholar). the we examined the role of this isoleucine-proline motif and containing this motif in the interaction of FKBP12.6 with the full-length RyR2. and deletion revealed that mutations of this motif not GST-FKBP12.6 binding to RyR2. the of large fragments containing the motif not the ability of the RyR2 to bind GST-FKBP12.6. These the first that the isoleucine-proline motif and the motif are unlikely to form the core of the FKBP12.6 binding site in RyR2. These from with RyR1 and in which mutations in the FKBP12 binding motif FKBP12 and FKBP12.6 interactions (10Bultynck G. Rossi D. Callewaert G. Missiaen L. Sorrentino V. Parys J.B. De Smedt H. J. Biol. Chem. 2001; 276: 47715-47724Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Gaburjakova M. Gaburjakova J. Reiken S. Huang F. Marx S.O. Rosemblit N. Marks A.R. J. Biol. Chem. 2001; 276: 16931-16935Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), a as to the FKBP12.6 binding site in RyR2 is from the binding site in RyR1 and has been shown that a 114-amino acid fragment containing residues 2497–2520 of RyR1 is sufficient for FKBP12 binding (19Cameron A.M. Nucifora F.C., Jr. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). However, we that RyR2 fragments containing the region not bind FKBP12.6 whereas an NH2-terminal fragment of RyR2 containing the first 1937 residues is sufficient for FKBP12.6 binding These suggest that the FKBP12.6 binding site in RyR2 and the FKBP12 binding site in RyR1 are located in of However, this is with the of a which that the of FKBP12.6 in RyR2 is to that of FKBP12 in RyR1 Jeyakumar L.H. Fleischer S. T. Biophys. J. 2002; 82 (abstr.): Scholar). it is that both the NH2-terminal and are involved in binding with FKBP12 and but to FKBP12.6 binding may on the NH2-terminal whereas FKBP12 binding may mainly on the region that the valine-proline FKBP12.6 may bind tightly to RyR2 to RyR1 and a the region may have on FKBP12.6 binding to RyR2 to RyR1 and This may mutations in the region FKBP12.6 binding to RyR1 and but not to RyR2. To the roles of these in FKBP12 and FKBP12.6 interactions, it be of to the NH2-terminal fragment of RyR1 and is capable of interacting with FKBP12.6 and FKBP12. also be important to RyR1 and proteins that the NH2-terminal region but the valine-proline motif are sufficient to bind FKBP12 and that a RyR2 the first NH2-terminal acid residues to with GST-FKBP12.6 also to be with the of studies the yeast two-hybrid a acid fragment including residues of RyR2 to be sufficient to with FKBP12.6 S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar), in FKBP12.6 to bind to a region in RyR2, likely at the S. Lai F.A. Biophys. J. 2002; 82 (abstr.): 59Google Scholar). The for these are not is that are multiple FKBP12.6 binding in RyR2 with binding The GST-FKBP12.6 in the may not be to low FKBP12.6 binding to the and COOH-terminal as by the yeast two-hybrid the the yeast two-hybrid may to FKBP12.6 binding to the NH2-terminal the RyR2 fragments in this may not of the sequences to form a FKBP12.6 binding site as on it is likely that the NH2-terminal region of RyR2 the binding site for FKBP12.6 multiple binding An important of is that multiple between residues 305 and 1937 of RyR2 are required for GST-FKBP12.6 binding. is unlikely that of these are involved in with of these are likely to be involved in the of the FKBP12.6 binding the binding of FKBP12.6 to RyR2 is likely to be have that of large overlapping fragments covering the full sequence of RyR2 channel but not GST-FKBP12.6 binding and Lai S. Lai F.A. Biophys. J. 2002; 82 (abstr.): 59Google have shown that overlapping fragments that the entire RyR2 sequence to bind to FKBP12.6 in multiple overlapping of the sequence for binding, are to a FKBP12.6 binding the that FKBP12.6 binding is on the of the binding This in that binding of FKBP12.6 to the binding site its have shown that an NH2-terminal fragment is to form functional channels with overlapping COOH-terminal fragments that the NH2-terminal region contains functional capable of interacting with the COOH-terminal of the channel is that binding of FKBP12.6 to the NH2-terminal region may interactions within the NH2-terminal region between the NH2-terminal and COOH-terminal of the residues involved in interaction with FKBP12.6 important into its roles in RyR and the at the of the of for the R. for for the of the and for and for of the

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