title: Central domain of the human cardiac muscle ryanodine receptor does not mediate interaction with FKBP12.6.
abstract: The immunophilin, FK506-binding protein (FKBP12), is an essential component of the ryanodine receptor channel complex of skeletal muscle (RyR1) and modulates intracellular calcium signaling from the endoplasmic reticulum. The cardiac muscle RyR isoform (RyR2) specifically associates with a distinct FKBP isoform, FKBP12.6. Previous studies have led to the proposal that the central domain of RyR1 exclusively mediates the interaction with FKBP12. To characterize the topography of the FKBP12.6 binding site on the human cardiac RyR2, we have applied complementary protein-protein interaction methods using both in vivoyeast two-hybrid analysis and in vitroimmunoprecipitation experiments. Our results indicate an absence of interaction of FKBP12/12.6 with fragments containing the central domain of either RyR1, RyR2, or RyR3. Furthermore, no interaction was detected between FKBP12.6 with a series of overlapping fragments encompassing the entire RyR2, either individually or in multiple combination. We also found that a distinct, alternatively spliced variant of FKBP12.6 was unable to interact with RyR. In contrast, we successfully demonstrated a robust association between the cytoplasmic domain of transforming growth factor-beta receptor type I and both FKBP12 and FKBP12.6 in parallel positive control experiments, as well as between native RyR2 and FKBP12.6. These results suggest that the specific interaction of FKBP12.6 with RyR2, and generally of FKBPs with any RyR isoform, is not readily reconstituted by peptide fragments corresponding to central RyR domains. Further structural analysis will be necessary to unravel this intricate signaling system and the current model of FKBP12-RyR interaction via a single, central RyR epitope may therefore require revision.
authors.affiliation: Wales Heart Research Institute, Department of Cardiology, Wales College of Medicine, Cardiff University, UK. ZissimopoulouS@cardiff.ac.uk
authors.forename: Spyros
authors.initials: S
authors.lastname: Zissimopoulos
authors.forename: F Anthony
authors.initials: FA
authors.lastname: Lai
mesh_terms: D001665:Binding Sites; D002478:Cells, Cultured; D006801:Humans; D009206:Myocardium / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D022021:Tacrolimus Binding Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B