Inositol 1,4,5-trisphosphate receptor ubiquitination is mediated by mammalian Ubc7, a component of the endoplasmic reticulum-associated degradation pathway, and is inhibited by chelation of intracellular Zn2+

In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP(3)Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome. Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin- protein ligases (E3s). In the present study we have examined the enzymology of ubiquitination of endogenous InsP(3)Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum- associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p. Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP(3)R ubiquitination and down- regulation, but that MmUbc6 does not. These data indicate that InsP(3)Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP(3)Rs, that are subject to regulated ERAD. Additional studies showed that the Zn2+ chelator N,N,N',N'- tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP(3)R ubiquitination, suggesting that a RING finger domain- containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP(3)R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH- SY5Y cells reflect a physiological process.

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