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Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum.

Strøm TB, Tveten K, Laerdahl JK, Leren TP - FEBS Open Bio (2014)

Bottom Line: Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum.This led to reduced amounts of the mature 160 kDa LDLR at the cell surface.It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

View Article: PubMed Central - PubMed

Affiliation: Unit for Cardiac and Cardiovascular Genetics, Department of Medical Genetics, Oslo University Hospital, Oslo, Norway.

ABSTRACT
More than 1700 mutations in the low density lipoprotein receptor (LDLR) gene have been found to cause familial hypercholesterolemia (FH). These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

No MeSH data available.


Related in: MedlinePlus

Internalization of DiD–LDL by the cell-surface G805R-LDLR. CHO T-REx cells stably transfected with the WT-LDLR plasmid or the G805R-LDLR plasmid were incubated with tetracycline (1 μg/ml) to induce the expression of the transgenes, and incubated with increasing concentrations of DiD–LDL for 2 h at 37 °C. The amount of internalized DiD–LDL was determined by flow cytometry. The amount of fluorescence detected after incubation with 150 μg/ml of DiD–LDL was given an arbitrary value of 1.0 for cells transfected with each of the plasmids. Mean (±SD) values from three separate experiments are shown. SD for the WT-LDLR is shown as upward symbols and SD for the G805R-LDLR is shown as downward symbols.
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f0030: Internalization of DiD–LDL by the cell-surface G805R-LDLR. CHO T-REx cells stably transfected with the WT-LDLR plasmid or the G805R-LDLR plasmid were incubated with tetracycline (1 μg/ml) to induce the expression of the transgenes, and incubated with increasing concentrations of DiD–LDL for 2 h at 37 °C. The amount of internalized DiD–LDL was determined by flow cytometry. The amount of fluorescence detected after incubation with 150 μg/ml of DiD–LDL was given an arbitrary value of 1.0 for cells transfected with each of the plasmids. Mean (±SD) values from three separate experiments are shown. SD for the WT-LDLR is shown as upward symbols and SD for the G805R-LDLR is shown as downward symbols.

Mentions: As determined by Western blot analysis, a proportion of the 120 kDa G805R-LDLR escaped metalloproteinase cleavage to generate a mature 160 kDa G805R-LDLR located at the cell surface (Fig. 2 and Supplementary Fig. S2). Studies were therefore undertaken to determine the functionality of the 160 kDa G805R-LDLR at the cell surface. CHO T-REx cells stably transfected with the G805R-LDLR plasmid were used to determine the kinetics of internalization of DiD-labelled LDL. As can be seen from Fig. 6, cells expressing G805R-LDLR internalized DiD–LDL with high affinity and saturation kinetics similarly to that of cells expressing the WT-LDLR. This finding indicates that the proportion of G805R-LDLR which escapes metalloproteinase cleavage, functions normally.


Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum.

Strøm TB, Tveten K, Laerdahl JK, Leren TP - FEBS Open Bio (2014)

Internalization of DiD–LDL by the cell-surface G805R-LDLR. CHO T-REx cells stably transfected with the WT-LDLR plasmid or the G805R-LDLR plasmid were incubated with tetracycline (1 μg/ml) to induce the expression of the transgenes, and incubated with increasing concentrations of DiD–LDL for 2 h at 37 °C. The amount of internalized DiD–LDL was determined by flow cytometry. The amount of fluorescence detected after incubation with 150 μg/ml of DiD–LDL was given an arbitrary value of 1.0 for cells transfected with each of the plasmids. Mean (±SD) values from three separate experiments are shown. SD for the WT-LDLR is shown as upward symbols and SD for the G805R-LDLR is shown as downward symbols.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048843&req=5

f0030: Internalization of DiD–LDL by the cell-surface G805R-LDLR. CHO T-REx cells stably transfected with the WT-LDLR plasmid or the G805R-LDLR plasmid were incubated with tetracycline (1 μg/ml) to induce the expression of the transgenes, and incubated with increasing concentrations of DiD–LDL for 2 h at 37 °C. The amount of internalized DiD–LDL was determined by flow cytometry. The amount of fluorescence detected after incubation with 150 μg/ml of DiD–LDL was given an arbitrary value of 1.0 for cells transfected with each of the plasmids. Mean (±SD) values from three separate experiments are shown. SD for the WT-LDLR is shown as upward symbols and SD for the G805R-LDLR is shown as downward symbols.
Mentions: As determined by Western blot analysis, a proportion of the 120 kDa G805R-LDLR escaped metalloproteinase cleavage to generate a mature 160 kDa G805R-LDLR located at the cell surface (Fig. 2 and Supplementary Fig. S2). Studies were therefore undertaken to determine the functionality of the 160 kDa G805R-LDLR at the cell surface. CHO T-REx cells stably transfected with the G805R-LDLR plasmid were used to determine the kinetics of internalization of DiD-labelled LDL. As can be seen from Fig. 6, cells expressing G805R-LDLR internalized DiD–LDL with high affinity and saturation kinetics similarly to that of cells expressing the WT-LDLR. This finding indicates that the proportion of G805R-LDLR which escapes metalloproteinase cleavage, functions normally.

Bottom Line: Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum.This led to reduced amounts of the mature 160 kDa LDLR at the cell surface.It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

View Article: PubMed Central - PubMed

Affiliation: Unit for Cardiac and Cardiovascular Genetics, Department of Medical Genetics, Oslo University Hospital, Oslo, Norway.

ABSTRACT
More than 1700 mutations in the low density lipoprotein receptor (LDLR) gene have been found to cause familial hypercholesterolemia (FH). These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

No MeSH data available.


Related in: MedlinePlus