The conserved sites for the FK506-binding proteins in ryanodine receptors and inositol 1,4,5-trisphosphate receptors are structurally and functionally different.
We compared the interaction of the FK506-binding protein (FKBP) with the type 3 ryanodine receptor (RyR3) and with the type 1 and type 3 inositol 1,4,5-trisphosphate receptor (IP(3)R1 and IP(3)R3), using a quantitative GST-FKBP12 and GST-FKBP12.6 affinity assay. We first characterized and mapped the interaction of the FKBPs with the RyR3. GST-FKBP12 as well as GST-FKBP12.6 were able to bind approximately 30% of the solubilized RyR3. The interaction was completely abolished by FK506, strengthened by the addition of Mg(2+), and weakened in the absence of Ca(2+) but was not affected by the addition of cyclic ADP-ribose. By using proteolytic mapping and site-directed mutagenesis, we pinpointed Val(2322), located in the central modulatory domain of the RyR3, as a critical residue for the interaction of RyR3 with FKBPs. Substitution of Val(2322) for leucine (as in IP(3)R1) or isoleucine (as in RyR2) decreased the binding efficiency and shifted the selectivity to FKBP12.6; substitution of Val(2322) for aspartate completely abolished the FKBP interaction. Importantly, the occurrence of the valylprolyl residue as alpha-helix breaker was an important determinant of FKBP binding. This secondary structure is conserved among the different RyR isoforms but not in the IP(3)R isoforms. A chimeric RyR3/IP(3)R1, containing the core of the FKBP12-binding site of IP(3)R1 in the RyR3 context, retained this secondary structure and was able to interact with FKBPs. In contrast, IP(3)Rs did not interact with the FKBP isoforms. This indicates that the primary sequence in combination with the local structural environment plays an important role in targeting the FKBPs to the intracellular Ca(2+)-release channels. Structural differences in the FKBP-binding site of RyRs and IP(3)Rs may contribute to the occurrence of a stable interaction between RyR isoforms and FKBPs and to the absence of such interaction with IP(3)Rs.
Bultynck
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Laboratorium voor Fysiologie, K.U.Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium.
Rossi
D
D
Callewaert
G
G
Missiaen
L
L
Sorrentino
V
V
Parys
J B
JB
De Smedt
H
H
D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism; D036563:Cyclic ADP-Ribose; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D008274:Magnesium / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D016559:Tacrolimus / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection; D014633:Valine / Q000737:chemistry
D015220:Calcium Channels; D018076:DNA, Complementary; C506926:ITPR1 protein, human; D007166:Immunosuppressive Agents; D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D007532:Isoleucine; D036563:Cyclic ADP-Ribose; D000246:Adenosine Diphosphate Ribose; D005982:Glutathione Transferase; D022021:Tacrolimus Binding Proteins; D007930:Leucine; D014633:Valine; D008274:Magnesium; D002118:Calcium; D016559:Tacrolimus