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

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 rate-limiting 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.

Pubmed ID: 19617589

Authors

  • Lee PC
  • DeBose-Boyd RA

Journal

Journal of lipid research

Publication Data

January 16, 2010

Associated Grants

  • Agency: NHLBI NIH HHS, Id: HL-20948
  • Agency: Howard Hughes Medical Institute, Id:

Mesh Terms

  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Endoplasmic Reticulum
  • Glycine
  • Hydroxycholesterols
  • Hydroxymethylglutaryl CoA Reductases
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Molecular Sequence Data
  • Mutation
  • Protein Multimerization
  • Sequence Alignment