Soluble LDL‐R are formed by cell surface cleavage in response to phorbol esters

Abstract

A 140-kDa soluble form of the low density lipoprotein (LDL) receptor has been isolated from the culture medium of HepG2 cells and a number of other cell types. It is produced from the 160-kDa mature LDL receptor by a proteolytic cleavage, which is stimulated in the presence of 4b-phorbol 12-myristate 13-acetate (PMA), leading to the release of a soluble fragment that constitutes the bulk of the extracellular domain of the LDL receptor. By labeling HepG2 cells with [ 35 S]methionine and chasing in the presence of PMA, we demonstrated that up to 20% of LDLreceptors were released into the medium in a 2-h period. Simultaneously, the level of labeled cellular receptors was reduced by 30% in those cells treated with PMA compared to untreated cells, as was the total number of cell surface LDL-receptors assayed by the binding of 125 I-labeled antibody to whole cells. To determine if endocytosis was required for cleavage, internalization-defective LDL-receptors were created by mutagenesis or deletion of the NPXY internalization signal, transfected into Chinese hamster ovary cells, and assayed for cleavage in the presence and absence of PMA. Cleavage was significantly greater in the case of the mutant receptors than for wild-type receptors, both in the absence and presence of PMA. Similar results were seen in human skin fibroblasts homozygous for each of the internalization-defective LDL receptor phenotypes. LDL receptor cleavage was inhibited by the hydoxamatebased inhibitor TAPI, indicating the resemblance of the LDL receptor cleavage mechanism to that of other surface released membrane proteins.Keywords: internalization signal; LDL-R; low density lipoprotein; ectodomain shedding.The low density lipoprotein (LDL) receptor (LDL-R) is a cell surface protein that mediates the uptake and clearance of the cholesterol-rich lipoprotein LDL from the plasma [1]. The LDL-R plays an important role in regulating plasma LDL and cellular cholesterol levels and the activity of this receptor has a direct bearing on plasma cholesterol levels. The LDL receptor is regulated at the transcriptional level in response to intracellular sterol levels by means of sterol sensitive transcription factors SREBP1 and SREBP2 [2,3], however, it can also be upregulated by numerous cytokines [tumor necrosis factor (TNF)-a, interleukin (IL)-1, transforming growth factor (TGF)-b, oncostatin M, plateletderived growth factor and basic fibroblast growth factor (bFGF) [4][5][6][7][8], hormones (insulin and estradiol) [9,10] and second messenger systems [11,12]. Although much is known about the transcriptional mechanisms that control LDL-R expression, little is known about the mechanism(s) that control the turnover and degradation of this important protein. Earlier studies established that the LDL-R degradation mechanism(s) is a nonlysosomal process that is dependant on short lived mediator protein(s) and unaffected by the presence of ligand and/or sterol [13,14]. Some evidence suggests that degradation may also be able to modulate LDL-R number. Ness et al. [15...

Apolipoprotein E Isoform-Specific Effects on Lipoprotein Receptor Processing

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Soluble Megalin Is Accumulated in the Lumen of the Rat Endolymphatic Sac

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