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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

Stability of G805R-LDLR. CHO T-REx cells stably transfected with the WT-LDLR plasmid or the G805R-LDLR plasmid were incubated with tetracycline (tet) (1 μg/ml) to induce the expression of the transgenes. The media were removed and replaced with media without tetracycline and cultured for the indicated time intervals (h: hours), before Western blot analysis of cell lysates was performed using an antibody against the ligand-binding domain of the LDLR. Western blot of cells cultured in the absence of tetracycline to induce gene expression (No tet) was used as a control. One representative of three separate experiments is shown.
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f0035: Stability of G805R-LDLR. CHO T-REx cells stably transfected with the WT-LDLR plasmid or the G805R-LDLR plasmid were incubated with tetracycline (tet) (1 μg/ml) to induce the expression of the transgenes. The media were removed and replaced with media without tetracycline and cultured for the indicated time intervals (h: hours), before Western blot analysis of cell lysates was performed using an antibody against the ligand-binding domain of the LDLR. Western blot of cells cultured in the absence of tetracycline to induce gene expression (No tet) was used as a control. One representative of three separate experiments is shown.

Mentions: Studies were also performed to determine the stability of the mature 160 kDa G805R-LDLR at the cell surface. This was done by measuring the decay of the 160 kDa G805R-LDLR after removal of the transcription inducer tetracycline. These analyses revealed that the 160 kDa G805R-LDLR was slightly less stable than the WT-LDLR with half-lives of 13 and 16 h, respectively (Fig. 7, Quantified in Supplementary Fig. S3). A tendency for reduced stability of the G805R-LDLR at the cell surface, could possibly result from an increased propensity of the 160 kDa G805R-LDLR to undergo metalloproteinase cleavage also at the cell surface.


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)

Stability of G805R-LDLR. CHO T-REx cells stably transfected with the WT-LDLR plasmid or the G805R-LDLR plasmid were incubated with tetracycline (tet) (1 μg/ml) to induce the expression of the transgenes. The media were removed and replaced with media without tetracycline and cultured for the indicated time intervals (h: hours), before Western blot analysis of cell lysates was performed using an antibody against the ligand-binding domain of the LDLR. Western blot of cells cultured in the absence of tetracycline to induce gene expression (No tet) was used as a 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

f0035: Stability of G805R-LDLR. CHO T-REx cells stably transfected with the WT-LDLR plasmid or the G805R-LDLR plasmid were incubated with tetracycline (tet) (1 μg/ml) to induce the expression of the transgenes. The media were removed and replaced with media without tetracycline and cultured for the indicated time intervals (h: hours), before Western blot analysis of cell lysates was performed using an antibody against the ligand-binding domain of the LDLR. Western blot of cells cultured in the absence of tetracycline to induce gene expression (No tet) was used as a control. One representative of three separate experiments is shown.
Mentions: Studies were also performed to determine the stability of the mature 160 kDa G805R-LDLR at the cell surface. This was done by measuring the decay of the 160 kDa G805R-LDLR after removal of the transcription inducer tetracycline. These analyses revealed that the 160 kDa G805R-LDLR was slightly less stable than the WT-LDLR with half-lives of 13 and 16 h, respectively (Fig. 7, Quantified in Supplementary Fig. S3). A tendency for reduced stability of the G805R-LDLR at the cell surface, could possibly result from an increased propensity of the 160 kDa G805R-LDLR to undergo metalloproteinase cleavage also at the cell surface.

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