Soluble LDL-R are formed by cell surface cleavage in response to phorbol esters.
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 4beta-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 [35S]methionine and chasing in the presence of PMA, we demonstrated that up to 20% of LDL-receptors 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 125I-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 hydoxamate-based inhibitor TAPI, indicating the resemblance of the LDL receptor cleavage mechanism to that of other surface released membrane proteins.
Begg
Michael J
MJ
Division of Medical Biochemistry, University of Cape Town, South Africa.
Sturrock
Edward D
ED
van der Westhuyzen
Deneys R
DR
D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D018076:DNA, Complementary; D004705:Endocytosis; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011973:Receptors, LDL / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D018076:DNA, Complementary; D011973:Receptors, LDL; D013755:Tetradecanoylphorbol Acetate