A chimeric LDL receptor containing the cytoplasmic domain of the transferrin receptor is degraded by PCSK9

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the extracellular domain of the low density lipoprotein receptor (LDLR) at the cell surface, and disrupts the normal recycling of the LDLR. However, the exact mechanism by which the LDLR is re-routed for lysosomal degradation remains to be determined. To clarify the role of the cytoplasmic domain of the LDLR for re-routing to the lysosomes, 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. These studies were performed in CHO T-REx cells stably transfected with a plasmid encoding the chimeric receptor and a novel assay was developed to study the effect of PCSK9 on the LDLR in these cells. Localization, function and stability of the chimeric receptor were similar to that of the wild-type LDLR. The addition of purified gain-of-function mutant D374Y-PCSK9 to the culture medium of stably transfected CHO T-REx cells showed that the chimeric receptor was degraded, albeit to a lower extent than the wild-type LDLR. In addition, a mutant LDLR, which has the three lysines in the intracellular domain substituted with arginines, was also degraded by D374Y-PCSK9. Thus, the mechanism for the PCSK9-mediated degradation of the LDLR does not appear to involve an interaction between the endosomal sorting machinery and LDLR-specific motifs in the cytoplasmic domain. Moreover, ubiquitination of lysines in the cytoplasmic domain does not appear to play a critical role in the PCSK9-mediated degradation of the LDLR.

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].