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Journal of Lipid Research, Vol. 51, 192-201, January 2010
Copyright © 2010 by American Society for Biochemistry and Molecular Biology

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

Peter C. W. Lee^* and Russell A. DeBose-Boyd^1,

Howard Hughes Medical Institute, University of Texas Southwestern Medical Center Dallas TX 75390-9046
^* Department of Molecular Genetics, University of Texas Southwestern Medical Center Dallas TX 75390-9046

¹ To whom correspondence should be addressed. e-mail: russell.debose-boyd{at}utsouthwestern.edu

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.

Supplementary key words cholesterol • sterol-regulatory element binding protein • SREBP cleavage-activating protein • HMG CoA reductase

Abbreviations: 25-HC, 25-hydroxycholesterol; CHO, Chinese hamster ovary; CMV, cytomegalovirus; EMS, ethylmethane sulfonate; ER, endoplasmic reticulum; LPDS, lipoprotein-deficient serum; Scap, SREBP cleavage-activating protein; SRD, sterol regulatory deficient; SREBP, sterol-regulatory element binding protein

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