A chimeric LDL receptor containing the cytoplasmic domain of the transferrin receptor is degraded by PCSK9
Introduction
The plasma level of low density lipoprotein (LDL)1 cholesterol is regulated both by environmental and genetic factors and the LDL receptor (LDLR) plays a key role in determining plasma LDL cholesterol levels [1]. The importance of the LDLR is illustrated by the severe hypercholesterolemia in patients with familial hypercholesterolemia, who have defective LDLR due to mutations in the LDLR gene [1].
The number of LDLR is regulated both at the transcriptional level and at the post-transcriptional level. Regulation at the transcriptional level is mediated by transcription factors which bind to regulatory elements of the promoter region. Whereas, transcription factor Sp1 is constitutively expressed [2], the sterol regulatory element binding protein is activated when intracellular levels of cholesterol are low [3]. Regulation of the LDLR at the post-transcriptional level is mediated partly by factors affecting mRNA stability [4] and translation efficiency [5] and partly by post-translational degradation mediated by proprotein convertase subtilisin/kexin type 9 (PCSK9) [6], [7], [8]. Very recently myosin regulatory light chain interacting protein [9] has also been found to mediate degradation of the LDLR [10].
PCSK9 is a zymogen which undergoes autocatalytic cleavage in the endoplasmic reticulum and is secreted as a complex consisting of the cleaved prodomain non-covalently bound to the mature PCSK9 [11]. However, unlike other proprotein convertases, PCSK9 does not appear to undergo a second autocatalytic event to release an active convertase [12]. Thus, PCSK9 is found as an enzymatically inactive protein in the media of cultured cells and in human plasma.
Secreted PCSK9 is able to bind to the epidermal growth factor (EGF)-like repeat A of the LDLR at the cell surface [13], and the PCSK9:LDLR complex is internalized by endocytosis through clathrin-coated pits [7], [13]. At the acidic pH of endosomes, the affinity of PCSK9 to bind to the LDLR increases approximately 150-fold [14], [15] and bound PCSK9 somehow disrupts the normal recycling of the LDLR [6], [7], [8]. As a consequence, the LDLR with bound PCSK9 is directed to the lysosomes for degradation [13]. Thus, by binding to the LDLR at the cell surface, PCSK9 post-translationally regulates the number of cell-surface LDLR. However, the exact mechanism by which bound PCSK9 reshuttles the LDLRs to the lysosomes, remains to be determined. Mutations in the PCSK9 gene may increase or decrease the LDLR-degrading activity of the mutant PCSK9 [16], [17]. Mutations which cause autosomal dominant hypercholesterolemia or autosomal dominant hypocholesterolemia are referred to as gain-of-function or loss-of-function mutations, respectively.
Whereas, the structural requirements of the extracellular part of the LDLR for PCSK9-mediated degradation have been elegantly depicted [13], [18], the mechanism by which the LDLR is reshutteled to lysosomal degradation, is unknown. A possible mechanism of action could be that PCSK9 disrupts the normal recycling of the LDLR by affecting the interaction between the cytoplasmic domain of the LDLR and the endosomal sorting machinery. To determine the role of the cytoplasmic domain of the LDLR for PCSK9-mediated degradation, we have studied the ability of PCSK9 to degrade a chimeric receptor which contains the extracellular and transmembrane domains of the LDLR and the cytoplasmic domain of the transferrin receptor (TFRC). Thus, in the chimeric LDLR-TFRC the 50 residues of the cytoplasmic domain of the LDLR have been replaced by the 61 residues of the cytoplasmic domain of the TFRC. There are no common motifs in the cytoplasmic domains of the two receptors (Fig. 1). Moreover, the TFRC itself is not degraded by PCSK9 [6], [19].
Section snippets
Cell cultures
CHO T-REx cells (Invitrogen, Carlsbad, CA) are stably transfected with a tetracycline repressor which enables tetracycline-induced expression of genes cloned into plasmids containing the tetracycline operator 2 element. Stably transfected CHO T-REx cells were maintained in Ham’s F-12 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml blasticidine and 100 μg/ml zeocin in a humidified atmosphere (37 °C, 5% CO2). Stably transfected
Establishing an assay to study the effect of PCSK9 on LDLR in stably transfected CHO T-REx cells
The wild-type (WT)-LDLR synthesized from the pcDNA4-LDLR plasmid in stably transfected CHO T-REx cells was found to be relatively insensitive to degradation by PCSK9 due to high expression of the pcDNA4-LDLR plasmid by the strong cytomegalovirus immediate-early promoter of the pcDNA4 vector. This is shown in Fig. 2 where CHO T-REx cells stably transfected with pcDNA4-LDLR plasmid were incubated with 0–20 μg/ml of purified D374Y-PCSK9. The gain-of-function mutant D374Y-PCSK9 was used for these
Discussion
In this report we have performed studies to determine whether the PCSK9-mediated degradation of the LDLR depends on specific residues in the cytoplasmic domain of the LDLR. A chimeric LDLR-TFRC containing the membrane-spanning and extracellular domains of the LDLR and the cytoplasmic domain of the TFRC was used for these studies. In order to be able to study PCSK9-mediated degradation of highly expressed LDLR in stably transfected cells, a novel assay was developed. We found that the chimeric
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2013, Journal of Lipid ResearchCitation Excerpt :These cells are stably transfected with a tetracycline repressor that enables tetracycline-induced expression of transgenes containing the tetracycline operator 2 element. Before PCSK9 was added to stably transfected CHO T-REx cells, the cells had been cultured in the presence of tetracycline followed by 8 h in the absence of tetracycline to reduce the LDLR synthesis, as previously described (19). HepG2 cells (European Collection of Cell Cultures, Wiltshire, UK) were maintained in modified Eagle's medium (Gibco, Carlsbad, CA) containing 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin and grown in collagen-coated flasks (BD Biosciences, San Jose, CA).