Interaction of FKBP12.6 with the Cardiac Ryanodine Receptor C-terminal Domain

Abstract

The ryanodine receptor-calcium release channel complex (RyR) plays a pivotal role in excitation-contraction coupling in skeletal and cardiac muscle. RyR channel activity is modulated by interaction with FK506-binding protein (FKBP), and disruption of the RyR-FKBP association has been implicated in cardiomyopathy, cardiac hypertrophy, and heart failure. Evidence for an interaction between RyR and FKBP is well documented, both in skeletal muscle (RyR1-FKBP12) and in cardiac muscle (RyR2-FKBP12.6), however definition of the FKBP-binding site remains elusive. Early reports proposed interaction of a short RyR central domain with FKBP12/12.6, however this site has been questioned, and recently an alternative FKBP12.6 interaction site has been identified within the N-terminal half of RyR2. In this study, we report evidence for the human RyR2 C-terminal domain as a novel FKBP12.6-binding site. Using competition binding assays, we find that short C-terminal RyR2 fragments can displace bound FKBP12.6 from the native RyR2, although they are unable to exclusively support interaction with FKBP12.6. However, expression of a large RyR2 C-terminal construct in mammalian cells encompassing the pore-forming transmembrane domains exhibits rapamycin-sensitive binding specifically to FKBP12.6 but not to FKBP12. We also obtained some evidence for involvement of the RyR2 N-terminal, but not the central domain, in FKBP12.6 interaction. Our studies suggest that a novel interaction site for FKBP12.6 may be present at the RyR2 C terminus, proximal to the channel pore, a sterically appropriate location that would enable this protein to play a central role in the modulation of this critical ion channel.

N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins

“…FKBP Localization-The location of the binding site for FKBP12 within the RyR1 protein sequence remains controversial. Although initial reports indicated a binding site proximal to the VP dipeptide at position 2461 of RyR1 (17,33), subsequent reports examining in vitro binding of FKBP12.6 to RyR2 fragments (18,19,34) failed to support this finding. However, FKBP binding data to RyR fragments or deletion mutants is difficult to interpret because this technique is predicated on the unlikely assumption that all RyR fragments fold and behave exactly as they would within the full-length RyR.…”

“…Instead, an N-terminal FKBP binding site has been proposed based on sequence deletions that abolish FKBP12.6 binding to RyR2 (18). Finally, findings from a third group suggest the presence of an FKBP binding site in the C-terminal transmembrane assembly of RyR2, as fragments derived from this region can bind FKBP12.6 in vitro (19). However, a C-terminal FKBP binding site appears unlikely given the cytoplasmic localization of FKBP observed in cryo-EM reconstructions, which is Ͼ100 Å from the transmembrane assembly of RyR1 (13,14).…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…Mutation of the amino acid Val2461 abolishes the FKBP12 binding Avila et al 2003). The amino-terminal and the carboxy-terminal regions of RyR2 have also been suggested to interact with FKBP12.6 Xiao et al 2004;Zissimopoulos and Lai 2005). Difference mapping of threedimensional reconstructions of RyR with and without FKBP12 or 12.6 places the FKBPs binding site between subdomains 3, 5, and 9 (Wagenknecht et al 1996;Wagenknecht et al 1997;Samsó et al 2006;Sharma et al 2006).…”

Genetic and Biochemical Approaches for <em>In Vivo</em> and <em>In Vitro</em> Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

“…We routinely carry out co-IP experiments (Figure 3) following transient expression in a mammalian cell line (HEK293), as an independent biochemical assay to reinforce the Y2H findings [2][3][4][12][13][14][21][22][23][24] . To verify RyR2 N-terminus self-interaction, two separate plasmids encoding for RyR2 residues 1 -906 tagged with either the cMyc or HA peptide epitope (BT4L and AD4L, respectively), were co-transfected in HEK293 cells using the calcium phosphate precipitation method 3 .…”

“…However, since co-IP and cross-linking are in vitro assays making use of homogenized cells, it is necessary to confirm whether the two protein partners are co-localized in the intact cell 11 . We routinely use transfection of mammalian HEK293 cells to transiently express mammalian integral membrane and cytosolic proteins using the calcium phosphate precipitation method [2][3][4][12][13][14] , described here in detail. This is an inexpensive way to efficiently deliver the plasmid DNA inside cells but it is dependent on the particular cell line used and cell confluence, as well as the purity of the plasmid DNA 11,15 .…”