Wojcikiewicz, Richard; WRIGHT, FORREST (2017)
      Inositol 1,4,5-trisphosphate receptors (IP3Rs) are endoplasmic reticulum (ER) proteins that assemble into tetrameric IP3- and Ca2+-gated Ca2+ channels. Activation of IP3Rs begins with stimulation of cell surface receptors that elevate cytosolic IP3 levels. IP3, with its co-agonist Ca2+, binds to IP3Rs and causes a conformational change that results in the opening of the channel aperture, allowing for Ca2+ ions to flow from stores within the ER lumen to the cytosol and thereby promoting a number of Ca2+-dependent cellular events, including secretion, neurotransmitter release and cell division. Intriguingly, it appears that the same conformational change that IP3Rs undergo during activation makes them a target for degradation by the ubiquitin-proteasome pathway. This processing allows the cell to fine-tune its internal Ca2+ responses to extracellular stimuli. In the Wojcikiewicz lab, it was discovered that processing of activated IP3Rs is mediated by the Erlin1/2 complex, a large (~2MDa) complex composed of the proteins Erlin1 and Erlin2. Constitutively-associated with the Erlin1/2 complex is the E3 ubiquitin ligase RNF170. Thus, we employed TALEN and CRISPR/Cas9-mediated gene editing technologies to abrogate expression of these three proteins to define their roles in this process. Remarkably, analysis of cells lacking RNF170 showed that it is required for all ubiquitination of activated IP3Rs. Investigation into the roles of Erlin1 and Erlin2 uncovered that Erlin2 is the “dominant partner” in the Erlin1/2 complex, mediating complex interaction with activated IP3Rs and bringing RNF170 into place to allow for ubiquitination to proceed. Mutations to RNF170 (R199C) and Erlin2 (T65I) have been identified as causative for progressive neurodegenerative diseases. Investigation of the R199C mutation on IP3R processing by RNF170 uncovered that while the mutation did not affect normal RNF170 function, it destabilized the RNF170 protein, resulting in a significantly reduced cellular complement of RNF170 and inhibition of IP3R degradation. Analysis of the Erlin2 T65I mutation showed that the effect of the mutation on Erlin1/2 complex function was two-fold. First, Erlin2 T65I interaction with activated IP3Rs was completely blocked, thereby inhibiting recruitment of RNF170 and subsequent ubiquitination and processing. Second, normal binding of the Erlin1/2 complex to phosphoinositol-3-phosphate (PI(3)P) – the significance and function of which remains undefined – was drastically inhibited. Examination of complex assembly and stability by SDS-PAGE and Native PAGE showed no destabilization of individual Erlin2 proteins nor of overall Erlin1/2 complex assembly. These data demonstrate that proper and tight control of IP3R levels in the cell are critical to overall cellular homeostasis, as disruptive mutations to requisite mediators of IP3R processing - the Erlin1/2 complex and RNF170 – result in the development of progressive neurodegenerative disease.