Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors

Abstract

The inositol 1,4,5-trisphosphate receptor (IP 3 R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP 3 Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP 3 at a distance. Defective allostery in IP 3 R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP 3 R type 1 (IP 3 R1) and negatively regulates IP 3 R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP 3 R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.L igand-gated ion channels function by allostery that is the regulation at a distance; the allosteric coupling of ligand binding with channel gating requires reversible changes in subunit configurations and conformations (1). Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) are ligand-gated ion channels that release calcium ions (Ca 2+ ) from the endoplasmic reticulum (ER) (2, 3). IP 3 Rs are allosteric proteins comprising four subunits that assemble a calcium channel with fourfold symmetry about an axis perpendicular to the ER membrane. The subunits of three IP 3 R isoforms (IP 3 R1, IP 3 R2, and IP 3 R3) are structurally divided into three domains: the IP 3 -binding domain (IBD), the regulatory domain, and the channel domain (3-6). Fitting of the IBD X-ray structures (7, 8) to a cryo-EM map (9) indicates that the IBD activates a remote Ca 2+ channel by allostery (8); however, the current X-ray structure only spans 5% of each tetramer, such that the mechanism underlying allosteric coupling of the IBD to channel gating remains unknown.The IP 3 R in the ER mediates intracellular calcium signaling that impinges on homeostatic control in various subsequent intracellular processes. Deletion of the genes encoding the type 1 IP 3 R (IP 3 R1) leads to perturbations in long-term potentiation/ depression (3, 10, 11) and spinogenesis (12), and the human genetic disease spinocerebellar ataxia 15 is caused by haploinsufficiency of the IP 3 R1 gene (13-15). Dysregulation of IP 3 R1 is also implicated in neurodegenerative diseases including Huntington disease (HD) (16-18) and Alzheimer's disease (AD) (19)(20)(21)(22). IP 3 Rs also control fundamental cellular processes-for example, mitochondrial energy production (23, 24), autophagy regulation (24-27), ER stress (28), hepatic gluconeogenesis (29), pancreatic exocytosis (30), and macrophag...

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

“…In that respect, it is noteworthy to mention that TGM2 can negatively affect autophagy by modifying ITPR1 (inositol 1,4,5trisphosphate receptor, type 1) and suppressing its Ca 2C -release activity. 966 It is also possible to analyze tissues ex vivo, and these studies can be particularly helpful in assessing autophagic flux as they avoid the risks of toxicity and bioavailability of compounds such as bafilomycin A 1 or other autophagy inhibitors. Along these lines, autophagic flux can be determined by western blot in retinas placed in culture for 4 h with protease inhibitors.…”

IP3 receptor mutations and brain diseases in human and rodents

“…In contrast to these physical but transient regulations, our group recently demonstrated the covalent posttranslational modifications of the COOH‐terminus of IP 3 R1 by transglutaminase (TG) (Hamada et al . ). TG is a Ca 2+ ‐dependent enzyme, which catalyzes a cross‐linking reaction in which a glutamine residue is linked with a lysine residue via an Nε‐(γ‐glutamyl) lysine isopeptide bond (Greenberg et al .…”

“…The cytosolic COOH-terminal region of IP 3 R1 is one of the critical regions for regulating channel activity by physically interacting with various proteins including Htt and HAP1 (Foskett et al 2007;Patterson et al 2004) and GIT1 (Zhang et al 2009). In contrast to these physical but transient regulations, our group recently demonstrated the covalent posttranslational modifications of the COOH-terminus of IP 3 R1 by transglutaminase (TG) (Hamada et al 2014). TG is a Ca 2+ -dependent enzyme, which catalyzes a cross-linking reaction in which a glutamine residue is linked with a lysine residue via an Ne-(c-glutamyl) lysine isopeptide bond (Greenberg et al 1991).…”

Modulation of Autophagy by Calcium Signalosome in Human Disease

“…The knockdown of IP3-R also leads to an accumulation of autophagosomes in HeLa cells. In this case, the effect is not due to the release of BECN1 from the complex with IP3-R since cells deficient in TGM2 (a regulator of IP3-R that inhibits IP3R-mediated Ca 21 release and IP3R-mediated autophagy) showed increased IP3-R-mediated Ca 21 signaling and increased autophagosome formation (Hamada et al, 2014). Finally, BECN1 is recruited by IP3-R during starvation and sensitizes IP3-R to low levels of IP3, allowing the release of Ca 21 from the ER (Decuypere et al, 2011).…”