Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

• Published in: The Journal of biological chemistry Vol. 290; no. 38; pp. 23385 - 23400
• Main Authors: Le, Quoc-Tuan, Blanchet, Matthieu, Seidah, Nabil G., Labonté, Patrick
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.09.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Animals; Cell Biology; cell surface protein; CHO Cells; Cricetinae; Cricetulus; Gene Expression Regulation - physiology; Humans; Life Sciences; lipoprotein receptor; Multiprotein Complexes - genetics; Multiprotein Complexes - metabolism; Mutation, Missense; Proprotein Convertase 9; proprotein convertase subtilisin/kexin type 9 (PCSK9); Proprotein Convertases - genetics; Proprotein Convertases - metabolism; protein sorting; protein-protein interaction; Proteolysis; Receptors, LDL - biosynthesis; Receptors, LDL - genetics; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Tetraspanin 28 - genetics; Tetraspanin 28 - metabolism; protein-protein interaction; cell biology; cell surface protein; lipoprotein receptor; protein sorting; proprotein convertase subtilisin/kexin type 9 (PCSK9)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.642991

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in plasma cholesterol regulation through modulation of low density lipoprotein receptor (LDLR) levels. Naturally occurring mutations can lead to hyper- or hypocholesterolemia in human. Recently, we reported that PCSK9 was also able to modulate CD81 in Huh7 cells. In the present study, several gain-of-function and loss-of-function mutants as well as engineered mutants of PCSK9 were compared for their ability to modulate the cell surface expression of LDLR and CD81. Although PCSK9 gain-of-function D374Y enhanced the degradation both receptors, D374H and D129N seemed to only reduce LDLR levels. In contrast, mutations in the C-terminal hinge-cysteine-histidine-rich domain segment primarily affected the PCSK9-induced CD81 degradation. Furthermore, when C-terminally fused to an ACE2 transmembrane anchor, the secretory N-terminal catalytic or hinge-cysteine-histidine-rich domain domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway as reported for LDLR. Using immunocytochemistry, a proximity ligation assay, and co-immunoprecipitation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81 and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect. Background: PCSK9 is a modulator of LDLR and the tetraspanin CD81. Results: Although CD81 is targeted for degradation by PCSK9 in an LDLR-independent manner, it can associate with the LDLR. Conclusion: CD81 and LDLR are two independent targets of PCSK9 that bind to each other. Significance: Structure-based mutagenesis of PCSK9 reveals functional interactions of CD81 with LDLR and PCSK9.