Characterization and mapping of the 12 kDa FK506-binding protein (FKBP12)-binding site on different isoforms of the ryanodine receptor and of the inositol 1,4,5-trisphosphate receptor

We investigated the interaction of the 12 kDa FK506-binding protein (FKBP12 ) with two ryanodine-receptor isoforms (RyR1 and RyR3) and with two myo-ino sitol 1,4,5-trisphosphate (IP3) receptor isoforms (IP(3)R1 and IP(3)R3). Us ing glutathione S-transferase (GST)-FKBP12 affinity chromatography, we coul d efficiently extract RyR1 (42 +/- 7% of the solubilized RyR1) from termina l cisternae of skeletal muscle as well as RyR3 (32 +/- 4% of the solubilize d RyR3) from RyR3-overexpressing HEK-293 cells. These interactions were com pletely abolished by FK506 (20 muM) but were largely unaffected by RyR-chan nel modulators. In contrast, neither IP(3)R1 nor IP(3)R3 from various sourc es, including rabbit cerebellum, A7r5 smooth-muscle cells and IP3R-overexpr essing Sf9 insect cells from Spodoptera frugiperda, were retained on the GS T-FKBP12. matrix. Moreover, immunoprecipitation experiments indicated a hig h-affinity interaction of FKBP12 with RyR1 but not with IP(3)R1, In order t o determine the FKBP12-binding site, we fragmented both RyR1 and IP(3)R1 by limited proteolysis. We obtained a 45 kDa fragment of RyR1 that bound to t he GST-FKBP12 matrix, indicating that it retained all requirements for FKBP 12 binding. This fragment was identified by its interaction with antibody m 34C and must therefore contain its epitope (amino acids 2756-2803). However , no fragment of IP(3)R1 was retained on the column. These molecular data a re in agreement with the lack of correlation between FKBP12 and IP(3)R1 exp ression in various cell types. The observation that FKBP12 did not affect I P3-induced Ca2+ release but reduced caffeine-induced Ca2+ release also indi cated that mature IP(3)R1 and IP(3)R3, in contrast to RyR1 and RyR3, did no t display a specific, high-affinity interaction with FKBP12.