SPFH1 and SPFH2 mediate the ubiquitination and degradation of inositol 1,4,5-trisphosphate receptors in muscarinic receptor-expressing HeLa cells

Yuan Wang, Margaret M.P. Pearce, Danielle A. Sliter, James A. Olzmann, John C. Christianson, Ron R. Kopito, Stephanie Boeckmann, Christine Gagen, Gil S. Leichner, Joseph Roitelman, Richard J.H. Wojcikiewicz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

Inositol 1,4,5-trisphosphate (IP3) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. While it is clear that IP3 receptors are polyubiquitinated and are transferred to the proteasome by a p97-based complex, currently very little is known about the proteins that initially select activated IP3 receptors for ERAD. Here, we have transfected HeLa cells to stably express m3 muscarinic receptors to allow for the study of IP3 receptor ERAD in this cell type, and show that IP3 receptors are polyubiquitinated and then degraded by the proteasome in response to carbachol, a muscarinic agonist. In seeking to identify proteins that mediate IP3 receptor ERAD we found that both SPFH1 and SPFH2 (also known as erlin 1 and erlin 2), which exist as a hetero-oligomeric complex, rapidly associate with IP3 receptors in a manner that precedes polyubiquitination and the association of p97. Suppression of SPFH1 and SPFH2 expression by RNA interference markedly inhibited carbachol-induced IP3 receptor polyubiquitination and degradation, but did not affect carbachol-induced calcium mobilization or IκBα processing, indicating that the SPFH1/2 complex is a key player in IP3 receptor ERAD, acting at a step after IP3 receptor activation, but prior to IP3 receptor polyubiquitination. Suppression of SPFH1 and SPFH2 expression had only slight effects on the turnover of some exogenous model ERAD substrates, and had no effect on sterol-induced ERAD of endogenous 3-hydroxy-3-methylglutaryl-CoA reductase. Overall, these studies show that m3 receptor-expressing HeLa cells are a valuable system for studying IP3 receptor ERAD, and suggest that the SPFH1/2 complex is a factor that selectively mediates the ERAD of activated IP3 receptors.

Original languageEnglish
Pages (from-to)1710-1718
Number of pages9
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1793
Issue number11
DOIs
StatePublished - Nov 2009
Externally publishedYes

Funding

FundersFunder number
National Institutes of HealthGM074874
National Institute of Diabetes and Digestive and Kidney DiseasesR01DK049194
Pharmaceutical Research and Manufacturers of America Foundation

    Keywords

    • Endoplasmic reticulum-associated degradation
    • Inositol 1,4,5-trisphosphate receptor
    • Proteasome
    • SPFH1
    • SPFH2
    • Ubiquitin

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