Limits...
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 batimastat on the amount of G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR or G805R-LDLR plasmids and the cells were cultured in the presence or absence of the metalloproteinase inhibitor batimastat (10 μM). Lysates and culture media were subjected to Western blot analysis. An antibody against the C-terminal HA tag was used to probe the lysates and an antibody against the ligand-binding domain was used to probe the media. In the media a 140 kDa ectdomain fragment and a 28 kDa fragment containing ligand-binding repeats 1–4 are shown. In the lysates the 160 kDa mature LDLR, the 120 kDa precursor LDLR and a C-terminal 17 kDa cleavage fragment are indicated. One representative of three separate experiments is shown.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048843&req=5

f0025: Effect of batimastat on the amount of G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR or G805R-LDLR plasmids and the cells were cultured in the presence or absence of the metalloproteinase inhibitor batimastat (10 μM). Lysates and culture media were subjected to Western blot analysis. An antibody against the C-terminal HA tag was used to probe the lysates and an antibody against the ligand-binding domain was used to probe the media. In the media a 140 kDa ectdomain fragment and a 28 kDa fragment containing ligand-binding repeats 1–4 are shown. In the lysates the 160 kDa mature LDLR, the 120 kDa precursor LDLR and a C-terminal 17 kDa cleavage fragment are indicated. One representative of three separate experiments is shown.

Mentions: In the culture medium of HepG2 cells transfected with the WT-LDLR plasmid, a 140 kDa N-terminal degradation product caused by ectodomain cleavage close to the cell membrane, can be observed (Fig. 5). When the cells were cultured in the presence of batimastat which inhibits a broad spectrum of metalloproteinases, no 140 kDa fragment was found in the culture medium (Fig. 5). These data are consistent with previous findings that metalloproteinase inhibitors prevent shedding of the 140 kDa ectodomain fragment at the cell surface [18]. Moreover, in the medium of cells transfected with the WT-LDLR plasmid, a 28 kDa fragment was observed (Fig. 5). This fragment represents the ligand-binding repeats 1–4 which is cleaved off from the 160 kDa mature LDLR at the cell surface [19]. The 140 kDa ectodomain fragment was also observed in the medium of cells transfected with the G805R-LDLR plasmid, but not when the cells were cultured in the presence of batimastat (Fig. 5). Thus, also G805R-LDLR is subjected to metalloproteinase cleavage.


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 batimastat on the amount of G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR or G805R-LDLR plasmids and the cells were cultured in the presence or absence of the metalloproteinase inhibitor batimastat (10 μM). Lysates and culture media were subjected to Western blot analysis. An antibody against the C-terminal HA tag was used to probe the lysates and an antibody against the ligand-binding domain was used to probe the media. In the media a 140 kDa ectdomain fragment and a 28 kDa fragment containing ligand-binding repeats 1–4 are shown. In the lysates the 160 kDa mature LDLR, the 120 kDa precursor LDLR and a C-terminal 17 kDa cleavage fragment are indicated. 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

f0025: Effect of batimastat on the amount of G805R-LDLR. HepG2 cells were transiently transfected with the WT-LDLR or G805R-LDLR plasmids and the cells were cultured in the presence or absence of the metalloproteinase inhibitor batimastat (10 μM). Lysates and culture media were subjected to Western blot analysis. An antibody against the C-terminal HA tag was used to probe the lysates and an antibody against the ligand-binding domain was used to probe the media. In the media a 140 kDa ectdomain fragment and a 28 kDa fragment containing ligand-binding repeats 1–4 are shown. In the lysates the 160 kDa mature LDLR, the 120 kDa precursor LDLR and a C-terminal 17 kDa cleavage fragment are indicated. One representative of three separate experiments is shown.
Mentions: In the culture medium of HepG2 cells transfected with the WT-LDLR plasmid, a 140 kDa N-terminal degradation product caused by ectodomain cleavage close to the cell membrane, can be observed (Fig. 5). When the cells were cultured in the presence of batimastat which inhibits a broad spectrum of metalloproteinases, no 140 kDa fragment was found in the culture medium (Fig. 5). These data are consistent with previous findings that metalloproteinase inhibitors prevent shedding of the 140 kDa ectodomain fragment at the cell surface [18]. Moreover, in the medium of cells transfected with the WT-LDLR plasmid, a 28 kDa fragment was observed (Fig. 5). This fragment represents the ligand-binding repeats 1–4 which is cleaved off from the 160 kDa mature LDLR at the cell surface [19]. The 140 kDa ectodomain fragment was also observed in the medium of cells transfected with the G805R-LDLR plasmid, but not when the cells were cultured in the presence of batimastat (Fig. 5). Thus, also G805R-LDLR is subjected to metalloproteinase cleavage.

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