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. 2013 Jun;54(6):1550-1559.
doi: 10.1194/jlr.M033167. Epub 2013 Apr 7.

S-nitrosylation of ARH is required for LDL uptake by the LDL receptor

Zhenze Zhao  1 , Shanica Pompey  1 , Hongyun Dong  1 , Jian Weng  1 , Rita Garuti  1 , Peter Michaely  2
Affiliations

S-nitrosylation of ARH is required for LDL uptake by the LDL receptor

Zhenze Zhao et al. J Lipid Res. 2013 Jun.

Abstract

The LDL receptor (LDLR) relies upon endocytic adaptor proteins for internalization of lipoproteins. The results of this study show that the LDLR adaptor autosomal recessive hypercholesterolemia protein (ARH) requires nitric oxide to support LDL uptake. Nitric oxide nitrosylates ARH at C199 and C286, and these posttranslational modifications are necessary for association of ARH with the adaptor protein 2 (AP-2) component of clathrin-coated pits. In the absence of nitrosylation, ARH is unable to target LDL-LDLR complexes to coated pits, resulting in poor LDL uptake. The role of nitric oxide on LDLR function is specific for ARH because inhibition of nitric oxide synthase activity impairs ARH-supported LDL uptake but has no effect on other LDLR-dependent lipoprotein uptake processes, including VLDL remnant uptake and dab2-supported LDL uptake. These findings suggest that cells that depend upon ARH for LDL uptake can control which lipoproteins are internalized by their LDLRs through changes in nitric oxide.

Keywords: autosomal recessive hypercholesterolemia protein; low density lipoprotein receptor; nitric oxide.

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Figures

Fig. 1.
Fig. 1.
ARH is nitrosylated at C199 and C286. (A) Domain structure of ARH. ARH has a PTB domain (PTB), Clathrin Box module (C), and an AP-2-binding module (AP2). Below the domain structure is the amino acid sequence of the C-terminal 44 amino acids. (B) Identification of a posttranslational modification in the C-terminal half of ARH. HEK293 cells were transfected with C-terminal V5-tagged human ARH constructs encompassing the entire ARH protein (WT-V5), residues 1–187 (NT-V5), or residues 188–308 (CT-V5), and then lysed. Lysates were run on SDS-PAGE and immunoblotted for V5. (C) Posttranslational modification resides between residues 284 and 292. HEK293 cells were transfected with untagged human ARH variants bearing premature stop codons at the indicated residue positions and lysed. Lysates were run on SDS-PAGE and immunoblotted for ARH. (D) C286 is posttranslationally modified. HEK293 cells were transfected with human ARH variants bearing alanine mutations at the indicated positions and lysed. Lysates were run on SDS-PAGE and immunoblotted for ARH. (E) C286 participates in an intramolecular disulfide bond. Lysates of normal human fibroblasts were treated with the indicated concentrations of DTT for 30 min at room temperature, run on SDS-PAGE, and immunoblotted for ARH. (F) C286 forms a disulfide bridge with C199, and both C199 and C286 are nitrosylated. The C-terminal 121 residues of human ARH have two cysteines, C199 and C286. HEK293 cells were transfected with the indicated ARH variants and lysed. Lysates were divided into two portions. The first portion was processed by biotin switch to label nitrosylated cysteines with biotin. Biotinylated proteins were precipitated with neutravidin agarose and run with the second, untreated portion of the lysate on SDS-PAGE and immunoblotted for ARH. CC>AA indicates the ARH variant with both the C199A and C286A mutations.
Fig. 2.
Fig. 2.
Generation of Arh−/−;Dab2−/−;hLDLR+/+ mouse embryonic fibroblasts and stable cell lines. (A) Isolated embryonic fibroblasts from Arh−/−;Dab2flox/flox;hLDLR+/+ mice were infected with adenoviruses encoding the cre-recombinase and clonally selected for cells that lack Dab2 expression. Cells lacking Dab2 were infected with retroviruses encoding no ARH (Vector), WT ARH (WT), ARH-C199A (C199A), ARH-C286A (C286A), or ARH-C199A/C286A (CC>AA). All introduced ARH cDNAs used the human ARH sequence. The mRNA produced by the viral vector is bicistronic and encodes both ARH and GFP with an internal ribosome entry site separating the two. Thus, ARH-expressing cells also express GFP, allowing transgenic cells to be FACS sorted by GFP fluorescence. (B) Immunoblots showing Dab2, ARH, LDLR, and α-tubulin expression by the parental Arh−/−;Dab2flox/flox;hLDLR+/+ fibroblasts (Dab2) and by the Vector, WT, C199A, C286A, and CC>AA transgenic cells.
Fig. 3.
Fig. 3.
ARH nitrosylation is necessary for LDL uptake but not β-VLDL uptake. Vector, WT, C199A, C286A, CC>AA, and Dab2 cells were pretreated or not with 4 mM L-NAME for 30 min and then assayed by FACS for lipoprotein uptake at 1, 2, 3, and 4 h using 10 μg/ml Alexa546-labeled LDL or 5 μg/ml Alexa546-labeled β-VLDL. Data for each time point are shown in supplementary Fig. I. Linear regression of uptake data was used to determine rate constants for LDL uptake and β-VLDL uptake. Rate data are plotted as mean rates ± SD (n = 3 trials; 10,000 cells per time point per trial). Significance determined by one-way ANOVA. ‡P < 0.05, ‡‡P < 0.005 for untreated cells versus L-NAME-treated cells; *P < 0.05, **P < 0.005 for untreated cells compared with untreated WT cells.
Fig. 4.
Fig. 4.
Loss of ARH function increases surface LDLRs. (A) Vector and CC>AA cells have twice as much surface LDL-binding capacity as WT, C199A, and C286A cells. Surface binding of 125I-labeled LDL was performed as described in Materials and Methods and is reported as nanograms of LDL bound per milligram of cellular protein. Experiments were performed in quadruplicate and data is shown as means ± SD. (B) Vector and CC>AA cells have more surface LDLRs than WT, C199A, and C286A cells. Relative surface number of LDLRs was determined by flow cytometry using the C7 antibody against the LDLR. Cellular fluorescence data are reported as a fraction of the mean fluorescence of WT cells. Experiments were performed in triplicate, and data is shown as means ± SD. *P < 0.05 compared with WT.
Fig. 5.
Fig. 5.
ARH nitrosylation promotes association with AP-2 and is necessary for efficient targeting of LDL to coated pits. (A) Representative images of coated pits with and without LDL-gold labeling. The indicated cells were incubated with 10 μg/ml colloidal gold-labeled LDL for 2 h on ice, and then washed, fixed and processed for thin-section EM. Quantification of enrichment is shown below representative examples of coated pits. (B) CC>AA cells have poor ability to target LDL-LDLR complexes to coated pits. Coated pit enrichment is reported using the summation of 10 random micrographs ± SEM of enrichments calculated from each micrograph separately. The P-value relative to WT is indicated above the error bar. (C) CC>AA cells have poor ability to support LDL internalization. Surface receptors were saturated with 10 μg/ml 125I-LDL at 4°C and then shifted to 37°C in the presence of 10 μg/ml 125I-LDL. At the indicated times, internalization was stopped and surface-bound and internalized pools of LDL were assayed as described in Materials and Methods. Data is shown as the mean ratio of internal/surface ± SEM, n = 16.
Fig. 6.
Fig. 6.
Nitrosylation is required for normal interaction of ARH with AP-2. Cells were treated or not with 4 mM L-NAME for 30 min, and then lysed and immunoprecipitated with antibodies to ARH or AP-2. Immunoprecipitants were run on SDS-PAGE and immunoblotted for the presence of ARH, AP-2, and clathrin heavy chain (CHC).
Fig. 7.
Fig. 7.
Model for regulation of ARH activity. We propose that ARH exists in three states: a free sulfhydryl state, a nitrosylated state, and a disulfide-bonded state. The free sulfhydryl form can react with nitric oxide produced by NOS enzymes to generate the active, nitrosylated form of ARH. Nitrosylation catalyzes disulfide bond formation, leading to inactivation of ARH. Cytosolic reductases (e−) reduce disulfide-bonded ARH to the free sulfhydryl form. Active ARH is presented as a hexagon, and inactive ARH is presented as a square.
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