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 (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.
Webster
Jack M
JM
Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York 13210-2339, USA.
Tiwari
Swati
S
Weissman
Allan M
AM
Wojcikiewicz
Richard J H
RJ
D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002540:Cerebral Cortex / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005029:Ethylenediamines / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008025:Ligases / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism; D013347:Subcellular Fractions; D013438:Sulfhydryl Compounds; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D015032:Zinc / Q000378:metabolism*
D015220:Calcium Channels; D002614:Chelating Agents; D005029:Ethylenediamines; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008164:Luminescent Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011976:Receptors, Muscarinic; D013438:Sulfhydryl Compounds; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D002217:Carbachol; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D002118:Calcium