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J. Biol. Chem., Vol. 278, Issue 40, 38238-38246, October 3, 2003

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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 Zn^2+*

Jack M. Webster , Swati Tiwari , Allan M. Weissman  and Richard J. H. Wojcikiewicz  ¶

From the Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York 13210-2339 and the Regulation of Protein Function Laboratory, Center for Cancer Research, NCI-Frederick, Frederick, Maryland 21702

In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP₃Rs), 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 InsP₃Rs 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 InsP₃R ubiquitination and down-regulation, but that MmUbc6 does not. These data indicate that InsP₃Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP₃Rs, that are subject to regulated ERAD. Additional studies showed that the Zn²⁺ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP₃R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP₃R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process.

Received for publication, May 28, 2003 , and in revised form, June 27, 2003.

^* This work was supported by National Institutes of Health Grant 5RO1DK49194 and by an advanced predoctoral fellowship in pharmacology/toxicology from the Pharmaceutical Research and Manufacturers of America Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, State University of New York Upstate Medical University, 750 E. Adams St., Syracuse, NY 13210-2339. Tel.: 315-464-7956; Fax: 315-464-8014; E-mail: wojcikir{at}upstate.edu.

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