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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 lactacystine on the amount of G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR or the G805R-LDLR plasmids and the cells were cultured in the presence or absence of proteasome inhibitor lactacystine (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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f0020: Effect of lactacystine on the amount of G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR or the G805R-LDLR plasmids and the cells were cultured in the presence or absence of proteasome inhibitor lactacystine (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: One mechanism for degradation of mutant proteins in the ER, is ER-associated degradation which involves ubiquitination of the cytoplasmic domain and subsequent proteasomal degradation [16]. To study whether the low amount of the precursor G805R-LDLR was due to proteasomal degradation, transfected HepG2 cells were cultured in the presence or absence of the proteasome inhibitor lactacystine. Whereas, lactacystine increased the amounts of the precursor and mature forms of the WT-LDLR by 61% (±10) and 18% (±16), which is consistent with previous reports [12,17], lactacystine increased the amount of the precursor G805R-LDLR by 13% (±23) and reduced the amount of the mature G805R-LDLR by 21% (±19) (Fig. 4). Thus, the low amounts of G805R-LDLR in cell lysates were apparently not due to proteasomal degradation of the precursor form of G805R-LDLR.


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 lactacystine on the amount of G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR or the G805R-LDLR plasmids and the cells were cultured in the presence or absence of proteasome inhibitor lactacystine (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

f0020: Effect of lactacystine on the amount of G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR or the G805R-LDLR plasmids and the cells were cultured in the presence or absence of proteasome inhibitor lactacystine (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: One mechanism for degradation of mutant proteins in the ER, is ER-associated degradation which involves ubiquitination of the cytoplasmic domain and subsequent proteasomal degradation [16]. To study whether the low amount of the precursor G805R-LDLR was due to proteasomal degradation, transfected HepG2 cells were cultured in the presence or absence of the proteasome inhibitor lactacystine. Whereas, lactacystine increased the amounts of the precursor and mature forms of the WT-LDLR by 61% (±10) and 18% (±16), which is consistent with previous reports [12,17], lactacystine increased the amount of the precursor G805R-LDLR by 13% (±23) and reduced the amount of the mature G805R-LDLR by 21% (±19) (Fig. 4). Thus, the low amounts of G805R-LDLR in cell lysates were apparently not due to proteasomal degradation of the precursor form of G805R-LDLR.

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