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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

Effect of mutation G805R on the amount of precursor and mature G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR plasmid or LDLR plasmids containing mutations G805R, G805A or G805L. The cells were cultured in the presence or absence of the γ-secretase inhibitor DAPT (10 μM). Cell lysates were subjected to Western blot analysis using an antibody against the C-terminal HA tag. The 160 kDa mature LDLR, the 120 kDa precursor LDLR and a C-terminal 17 kDa cleavage fragment are indicated. Beta-tubulin was used as a loading control. One representative of three separate experiments is shown.
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f0010: Effect of mutation G805R on the amount of precursor and mature G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR plasmid or LDLR plasmids containing mutations G805R, G805A or G805L. The cells were cultured in the presence or absence of the γ-secretase inhibitor DAPT (10 μM). Cell lysates were subjected to Western blot analysis using an antibody against the C-terminal HA tag. The 160 kDa mature LDLR, the 120 kDa precursor LDLR and a C-terminal 17 kDa cleavage fragment are indicated. Beta-tubulin was used as a loading control. One representative of three separate experiments is shown.

Mentions: Cell lysates of transfected HepG2 cells were subjected to Western blot analysis using an antibody against the C-terminal HA-tag. This antibody detects the 120 kDa precursor LDLR, the 160 kDa mature LDLR and a 17 kDa C-terminal cleavage fragment (Fig. 2). HepG2 cells transfected with the G805R-LDLR plasmid had markedly lower amounts of both the 120 kDa and the 160 kDa LDLR forms as compared to cells transfected with the WT-LDLR plasmid, of 21% (±5) and 25% (±6), respectively. However, the relative intensities of the bands representing the two LDLR forms, were similar to that of the WT-LDLR (Fig. 2). Because mutation G805R caused reduced amounts of both the precursor 120 kDa and mature 160 kDa G805R-LDLR and not a selective reduction of the mature 160 kDa form, it appears that this mutation primarily reduced the amount of the precursor form which led to reduced amounts of the mature form at the cell surface. Reduced amounts of the cell-surface G805R-LDLR were also demonstrated by confocal laser-scanning microscopy (Supplementary Fig. S2).


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)

Effect of mutation G805R on the amount of precursor and mature G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR plasmid or LDLR plasmids containing mutations G805R, G805A or G805L. The cells were cultured in the presence or absence of the γ-secretase inhibitor DAPT (10 μM). Cell lysates were subjected to Western blot analysis using an antibody against the C-terminal HA tag. The 160 kDa mature LDLR, the 120 kDa precursor LDLR and a C-terminal 17 kDa cleavage fragment are indicated. Beta-tubulin was used as a loading control. One representative of three separate experiments is shown.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0010: Effect of mutation G805R on the amount of precursor and mature G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR plasmid or LDLR plasmids containing mutations G805R, G805A or G805L. The cells were cultured in the presence or absence of the γ-secretase inhibitor DAPT (10 μM). Cell lysates were subjected to Western blot analysis using an antibody against the C-terminal HA tag. The 160 kDa mature LDLR, the 120 kDa precursor LDLR and a C-terminal 17 kDa cleavage fragment are indicated. Beta-tubulin was used as a loading control. One representative of three separate experiments is shown.
Mentions: Cell lysates of transfected HepG2 cells were subjected to Western blot analysis using an antibody against the C-terminal HA-tag. This antibody detects the 120 kDa precursor LDLR, the 160 kDa mature LDLR and a 17 kDa C-terminal cleavage fragment (Fig. 2). HepG2 cells transfected with the G805R-LDLR plasmid had markedly lower amounts of both the 120 kDa and the 160 kDa LDLR forms as compared to cells transfected with the WT-LDLR plasmid, of 21% (±5) and 25% (±6), respectively. However, the relative intensities of the bands representing the two LDLR forms, were similar to that of the WT-LDLR (Fig. 2). Because mutation G805R caused reduced amounts of both the precursor 120 kDa and mature 160 kDa G805R-LDLR and not a selective reduction of the mature 160 kDa form, it appears that this mutation primarily reduced the amount of the precursor form which led to reduced amounts of the mature form at the cell surface. Reduced amounts of the cell-surface G805R-LDLR were also demonstrated by confocal laser-scanning microscopy (Supplementary Fig. S2).

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