Abstract
Oligomerization of all three mammalian ryanodine receptor isoforms, a structural requirement for normal intracellular Ca2+ release channel function, is displayed by the discrete N-terminal domain which assembles into homo- and hetero-tetramers. This is demonstrated in yeast, mammalian cells and native tissue by complementary yeast two-hybrid, chemical cross-linking and co-immunoprecipitation assays. The IP3 (inositol 1,4,5-trisphosphate) receptor N-terminus (residues 1–667) similarly exhibits tetrameric association as indicated by chemical cross-linking and co-immunoprecipitation assays. The presence of either a 15-residue splice insertion or of the cognate ligand IP3 did not affect tetramerization of the IP3 receptor N-terminus. Thus N-terminus tetramerization appears to be an essential intrinsic property that is conserved in both the ryanodine receptor and IP3 receptor families of mammalian intracellular Ca2+ release channels.
Highlights
ryanodine receptor (RyR) and IP3 receptor (IP3R) [inositol 1 (IP3) receptors] are homologous membrane protein channel families that mediate the acute Ca2+ release from internal stores essential for the activation of numerous physiological processes [1,2]
We provide evidence that N-terminus tetramerization is a structural feature that is conserved across the three mammalian RyR isoforms and, significantly, this innate ability to oligomerize into tetramers is observed in the N-terminus of the related IP3R1 intracellular Ca2+ release channel
We have recently used a complementary series of yeast two-hybrid (Y2H), chemical cross-linking and co-immunoprecipitation assays to demonstrate that an RyR2 N-terminal fragment is capable of self-association, leading to tetramer assembly [16]
Summary
RyRs (ryanodine receptors) and IP3Rs [IP3 (inositol 1,4,5trisphosphate) receptors] are homologous membrane protein channel families that mediate the acute Ca2+ release from internal stores essential for the activation of numerous physiological processes [1,2]. To examine the precise stoichiometry of the RyR1/3 N-terminus oligomer, we expressed RyR1/3 N-terminal fragments tagged with the c-Myc peptide epitope (BT4LR1/3) in mammalian HEK-293 cells and carried out chemical cross-linking using glutaraldehyde.
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