Abstract
ARH is an adaptor protein required for efficient endocytosis of low density lipoprotein (LDL) receptors (LDLRs) in selected tissues. Individuals lacking ARH (ARH-/-) have severe hypercholesterolemia due to impaired hepatic clearance of LDL. Immortalized lymphocytes, but not fibroblasts, from ARH-deficient subjects fail to internalize LDL. To further define the role of ARH in LDLR function, we compared the subcellular distribution of the LDLR in lymphocytes from normal and ARH-/- subjects. In normal lymphocytes LDLRs were predominantly located in intracellular compartments, whereas in ARH-/- cells the receptors were almost exclusively on the plasma membrane. Biochemical assays and quantification of LDLR by electron microscopy indicated that ARH-/- lymphocytes had >20-fold more LDLR on the cell surface and a approximately 27-fold excess of LDLR outside of coated pits. The accumulation of LDLR on the cell surface was not due to failure of receptors to localize in coated pits since the number of LDLRs in coated pits was similar in ARH-/- and normal cells. Despite the dramatic increase in cell surface receptors, LDL binding was only 2-fold higher in the ARH-/- lymphocytes. These findings indicate that ARH is required not only for internalization of the LDL.LDLR complex but also for efficient binding of LDL to the receptor and suggest that ARH stabilizes the associations of the receptor with LDL and with the invaginating portion of the budding pit, thereby increasing the efficiency of LDL internalization.
Highlights
autosomal recessive hypercholesterolemia (ARH) is an adaptor protein required for efficient endocytosis of low density lipoprotein (LDL) receptors (LDLRs) in selected tissues
The degradation of 125I-LDL is severely compromised in ARHϪ/Ϫ lymphocytes despite an increased amount of LDL binding to the surfaces of these cells [16, 17]
To determine whether the decreased LDL degradation in ARHϪ/Ϫ cells is due to a failure of low density lipoprotein receptor (LDLR) internalization, ARHϪ/Ϫ and normal lymphocytes were treated with monensin to block LDLR recycling, and the rates of disappearance of LDLR from the cell surface were compared in the two cell lines (Fig. 1)
Summary
Materials—All cell culture reagents were from Invitrogen. Rabbit anti-LDLR IgG used for immunofluorescence and immunoelectron microscopy was from Maine Biotechnology Services, Inc. (Portland, ME). The lymphocytes were formaldehyde-fixed as described above, rinsed in Buffer B, and incubated with rabbit anti-LDLR IgG antibody (1 g/ml) in Buffer B for 17 h. Quantification of Cell Surface LDLR—A total of 5 ϫ 106 lymphocytes were washed with ice-cold Medium C (1ϫ Eagle’s modified minimal essential medium supplemented with 20 mM HEPES, pH 7.4) and incubated on a rotator with 125I-labeled monoclonal antibody to human LDLR (C7) or a rabbit polyclonal antibody to the human LDLR (4548) (7 g/ml) for 90 min at 4 °C. Cells were washed twice with cold PBS and lysed with 60 l of lysis buffer (1% Triton X-100, 4 mM EGTA, 10 mM Tris-HCl, pH 8) at room temperature for 30 min with end-over-end mixing. Ligand blot assays were performed as described previously [27]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
