Three Different Rearrangements in a Single Intron Truncate Sterol Regulatory Element Binding Protein-2 and Produce Sterol-resistant Phenotype in Three Cell Lines

Abstract

The cholesterol analogue 25-hydroxycholesterol kills animal cells by blocking the proteolytic activation of two sterol-regulated transcription factors designated sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2). These proteins, each approximately 1150 amino acids in length, are embedded in the membranes of the nucleus and endoplasmic reticulum by virtue of hydrophobic COOH-terminal segments. In cholesterol-depleted cells the proteins are cleaved to release soluble NH2-terminal fragments of approximately 480 amino acids that enter the nucleus and activate genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. 25-Hydroxycholesterol blocks this cleavage, and cells die of cholesterol deprivation. We previously described a mutant 25-hydroxycholesterol-resistant hamster cell line (SRD-1 cells) in which the SREBP-2 gene had undergone a recombination between the intron following codon 460 and an intron in an unrelated gene. The SREBP-2 sequence terminated at residue 460, eliminating the membrane attachment domain and producing a constitutively active factor that no longer required proteolysis and thus was not inhibited by 25-hydroxycholesterol. Here, we report that two additional sterol-resistant cell lines (SRD-2 and SRD-3) have also undergone genomic rearrangements in the intron following codon 460 of the SREBP-2 gene. Although the molecular rearrangements differ in the three mutant lines, each leads to the production of a constitutively active transcription factor whose SREBP-2 sequence terminates at residue 460. These findings provide a dramatic illustration of the advantage that introns provide in allowing proteins to gain new functions in response to new environmental challenges.

Isolation of Mutant Cells Lacking Insig-1 through Selection with SR-12813, an Agent That Stimulates Degradation of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase

“…In mutant cell lines identified previously, the persistence of nuclear SREBP-2 upon 25-hydroxycholesterol treatment was indicative of the ability of the cells to survive chronic treatment with the oxysterol (9,12,(27)(28)(29). However, in the experiment of Fig.…”

“…Mutant cells survive chronic 25-hydroxycholesterol treatment because they do not turn off cholesterol synthesis in the presence of oxysterols, forming the genetic basis for their isolation. However, the sterol-resistant mutants isolated to date either harbor point mutations in the sterol-sensing domain of SCAP or produce abnormal truncated versions of SREBP-2 that enter the nucleus without requiring proteolysis (9,12,(27)(28)(29). Thus, to facilitate the isolation of mutant cells incapable of accelerating reductase degradation, selection of mutagenized cells must be carried out with a reagent that replaces sterols to promote reductase degradation but not ER retention of SCAP.…”

Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells.

“…The terminator codon occurs at the same position as the terminator codon in the SREBP-1a460 construct that was described previously (2). Both of these proteins terminate before the first transmembrane segment and enter the nucleus directly without a requirement for proteolysis, thereby making them immune to the normal process of downregulation (11,22).…”

“…The net effect is to reduce the synthesis of cholesterol and fatty acids and their uptake from lipoproteins such as LDL. This feedback mechanism is designed to maintain a constant level of cholesterol in cell membranes, and its disruption by mutation in cultured cells leads to either overaccumulation or deficiency of cholesterol (11,14,22).…”

Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis

“…Monoclonal antibody IgG-7D4 against hamster SREBP-2 (amino acids 32-350) (20), monoclonal antibody IgG-9D5 against hamster Scap (amino acids 540 -707) (5), and monoclonal antibody IgG-7G5 against hamster Scap (amino acids 46 -269) (10) are described in the indicated references.…”