Abstract
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.
Highlights
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 the present study we have examined the enzymology of ubiquitination of endogenous InsP3 receptors (InsP3Rs) in muscarinic agonist-stimulated SHSY5Y 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
In the present study we have examined whether the ERADlinked Ubcs, mamUbc6 and mamUbc7, mediate G protein-coupled receptors (GPCRs)-initiated ubiquitination and down-regulation of endogenous InsP3Rs in SH-SY5Y human neuroblastoma cells
Summary
InsP3, inositol 1,4,5-trisphosphate; ER, endoplasmic reticulum; InsP3R, inositol 1,4,5-trisphosphate receptor; [Ca2ϩ]i, cytoplasmic free Ca2ϩ concentration; GPCR, G protein-coupled receptor; Ubc or E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; HECT-E3, ubiquitin-protein ligase containing a domain homologous to the E6-associated protein carboxyl terminus; RING-E3, RING finger domain-containing ubiquitin-protein ligase; U-box-E3, U-. The HECT ligases box domain-containing ubiquitin-protein ligase; ERAD, endoplasmic reticulum-associated degradation; HMGR, 3-hydroxy 3-methylglutarylcoenzyme A reductase; mamUbc, mammalian homologue of yeast Ubc6p; mamUbc, mammalian homologue of yeast Ubc7p; MmUbc, murine homologue of yeast Ubc6p; MmUbc, murine homologue of yeast Ubc7p; DN, dominant negative; HA, hemagglutinin; DTT, dithiothreitol; PBS, phosphate-buffered saline; TPEN, N,N,NЈ,NЈ-tetrakis(2pyridylmethyl)ethylenediamine; ALLN, N-acetyl-Leu-Leu-norleucinal; RERAD, regulated endoplasmic reticulum-associated degradation; GFP, green fluorescent protein In the present study we have examined whether the ERADlinked Ubcs, mamUbc and mamUbc, mediate GPCR-initiated ubiquitination and down-regulation of endogenous InsP3Rs in SH-SY5Y human neuroblastoma cells We accomplished this by creating SH-SY5Y cell lines stably expressing murine homologues of Ubc6p and Ubc7p (MmUbc and MmUbc7), and their respective “dominant-negative” (DN) mutants, in which the active site cysteine residues have been mutated to serine [22, 38]. We provide evidence for the involvement of a RING-E3 in InsP3R ubiquitination and down-regulation, and demonstrate that muscarinic receptor activation can induce InsP3R ubiquitination in rat cerebral cortex
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