Intramembrane glycine mediates multimerization of Insig-2, a requirement for sterol regulation in Chinese hamster ovary cells

Peter C W Lee, Russell A. DeBose-Boyd

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Sterol-induced binding of endoplasmic reticulum (ER) membrane proteins Insig-1 and Insig-2 to SREBP cleavage-activating protein (Scap) and HMG-CoA reductase triggers regulatory events that limit cholesterol synthesis in animal cells. Binding of Insigs to Scap prevents proteolytic activation of sterol-regulatory element binding proteins (SREBPs), membrane-bound transcription factors that enhance cholesterol synthesis, by trapping Scap-SREBP complexes in the ER. Insig binding to reductase causes ubiquitination and subsequent proteasome-mediated degradation of the enzyme from ER membranes, slowing a ratelimiting step in cholesterol synthesis. Here, we report the characterization of mutant Chinese hamster ovary cells, designated SRD-20, that are resistant to 25-hydroxycholesterol, which potently inhibits SREBP activation and stimulates degradation of reductase. SRD-20 cells were produced by mutagenesis of Insig-1-deficient SRD-14 cells, followed by selection in 25-hydroxycholesterol. DNA sequencing reveals that SRD-20 cells harbor a point mutation in one Insig-2 allele that results in production of a truncated, nonfunctional protein, whereas the other allele contains a point mutation that results in substitution of glutamic acid for glycine-39. This glycine residue localizes to the first membrane-spanning segment of Insig-2 and is also present in the corresponding region of Insig-1. Mutant forms of Insig-1 and Insig-2 containing the Glu-to-Gly substitution fail to confer sterol regulation upon overexpressed Scap and reductase. These studies identify the intramembrane glycine as a key residue for normal sterol regulation in animal cells.

Original languageEnglish (US)
Pages (from-to)192-201
Number of pages10
JournalJournal of lipid research
Volume51
Issue number1
DOIs
StatePublished - Jan 6 2010

Keywords

  • Cholesterol
  • HMG CoA reductase
  • SREBP cleavage-activating protein
  • Sterol-regulatory element binding protein

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

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