Unique Regulatory Properties of Heterotetrameric Inositol 1,4,5-trisphosphate Receptors Revealed by Studying Concatenated Receptor Constructs.

摘要

Inositol 1,4,5-trisphosphate receptors (IP3R) are a family of ubiquitous, endoplasmic reticulum localized, tetrameric Ca2+ release channels. There are three main subtypes of the IP3Rs (namely R1, R2 and R3), each encoded by a distinct gene (ITPR1, ITPR2 and ITPR3, respectively), that share ∼ 60-70% primary sequence identity. The diversity of Ca 2+ signals generated by IP3Rs is thought to be largely the result of differential tissue expression, intracellular localization and subtype-specific regulation of the three subtypes by various cellular factors. These factors include IP3 (the agonist), Ca2+ (co-agonist), ATP and other adenine nucleotides, phosphorylation by various kinases and interactions with various binding proteins. The regulation of the individual subtypes by these different modulators has been extensively studied.;Another largely unexplored, level of IP3R mediated Ca 2+ signaling diversity is thought to be mediated by the assembly of both homo and heterotetrameric IP3R channels. Although the three subtypes are ubiquitously and differentially expressed in all tissues and do associate in complexes, the biochemical evidence for hetero-oligomerization fail to exclude the possibility that the observed interactions are the result of larger inter-molecular complexes of homotetrameric channels. To address this, a combination of gel filtration chromatography and co-immunoprecipitation assays was used to provide strong biochemical evidence for native heterotetramer formation. Additionally, we provide biochemical evidence for homotetramer formation through the sequential immunodepletion of IP3R isoforms from mouse pancreatic lysates.;Typically, when multiple subtypes of IP3Rs are co-expressed, as is the case cultured cells or isolated tissues, the subunit composition of individual channels cannot be specifically defined. Therefore, understanding the contribution of heterotetrameric IP3R channels with differing subunit composition to shaping the spatio-temporal properties of IP3 -mediated Ca2+ signals has proven to be troublesome. To address this, our approach was to engineer concatenated IP3Rs constructs of defined subunit composition, whereby the CT of one IP3 R monomer is connected to the NT of a subsequent monomer by a short flexible amino acid linker. In this thesis, we establish our ability to successfully engineer and stably express concatenated homo and heteromeric IP3Rs of defined subunit composition in a null background cell line (DT40-3KO cells). These concatenated proteins oligomerize into tetrameric channels, localize to internal membranes and are functionally indistinguishable from IP 3Rs assembled from monomeric constructs. Accordingly, we use this approach to the contribution of individual subtypes within defined concatenated heterotetramers to the shaping of Ca2+ signals.;Under conditions where key regulators of IP3R function are optimal for Ca2+ release (200 nM Ca2+ and 5 mM ATP), we demonstrate that individual monomers within heteromeric IP 3Rs contributed equally towards generating a distinct, 'blended' sensitivity to IP3. Homologous competitive IP3 binding assays indicate that this intermediate IP3 sensitivity is likely dictated by the unique, 'blended' apparent IP3 binding affinity of heteromers, which is determined by the constituent subtypes. However, under sub-optimal conditions where [ATP] were varied, we found that one subtype dictated the ATP regulatory properties of heteromers. Specifically focusing on R2 containing concatemers, we show that two R2 monomers within a heterotetramer were both necessary and sufficient for R2 to dictate the ATP regulatory properties. This stoichiometry of R2 dominance was also observed with the patterns of Ca2+ signals generated when intact cells were stimulated with receptor tyrosine kinase / PLC-gamma coupled cell surface agonists.;Lastly, we sought to address questions of regulatory stoichiometry of IP3Rs using a mutagenic approach. Specifically, the critical ATPB site in R2 was mutagenically rendered non-functional. We found that two functional R2 monomers were sufficient to maintain ATP regulation in R2 homotetramers, although it is unclear whether one ATPB site is enough. Interestingly, R2 DeltaR1 and R3R2Delta heterotetramers, rather than switching to an R1 or R3 mode of regulation, lose the ability to be ATP regulated, suggesting that R2 still dictates the ATP regulation in the heterotetramer.;In summary, this thesis demonstrates that the concatenated approach to studying the properties of heterotetrameric IP3Rs is useful in answering fundamental questions regarding IP3R function.