The autosomal recessive hypercholesterolemia (ARH) protein interfaces directly with the clathrin-coat machinery

Abstract

The low density lipoprotein (LDL) receptor plays a pivotal role in cholesterol metabolism. Inherited mutations that disturb the activity of the receptor lead to elevations in plasma cholesterol levels and early-onset coronary atherosclerosis. Defects in either the LDL receptor or apolipoprotein B, the proteinaceous component of LDL particles that binds the LDL receptor, elevate circulating LDL-cholesterol levels in an autosomal-dominant fashion, with heterozygotes displaying values between homozygous and normal individuals. Rarely, similar clinical phenotypes occur with a recessive pattern of inheritance, and several genetic lesions in the autosomal recessive hypercholesterolemia (ARH) gene on chromosome 1 have been mapped in this class of patients. ARH has an N-terminal phosphotyrosine-binding (PTB) domain evolutionarily related to that found in Disabled-2 and numb, two endocytic proteins. PTB domains bind to the consensus sequence FXNPXY, corresponding to the internalization motif of the LDL receptor. We show here that in addition to the FXNPXY sequence, ARH binds directly to soluble clathrin trimers and to clathrin adaptors by a mode involving the independently folded appendage domain of the beta subunit. At steady state, ARH colocalizes with endocytic proteins in HeLa cells, and the LDL receptor fluxes through peripheral ARH-positive sites before delivery to early endosomes. Because ARH also binds directly to phosphoinositides, which regulate clathrin bud assembly at the cell surface, our data suggest that in ARH patients, defective sorting adaptor function in hepatocytes leads to faulty LDL receptor traffic and hypercholesterolemia.

Fig 1.

ARH binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] and FXNPXY motifs synchronously. (A) Schematic illustration of the overall domain organization of ARH and the various GST-ARH constructs used. (B) Thrombin-cleaved ARH-(1–308) was added to 3 μM together with carrier BSA and 0.4 mg/ml control (asterisk, lanes a and b) or PtdIns(4,5)P₂-containing liposomes alone (lanes c, d, g, and h) or liposomes plus either 15 μM NWRLKNINSIFDNPVYQKTT (lanes e, f, i–l) or NWRLKNINSIFDAPVAQKTT (lanes m and n) peptide as indicated and incubated on ice for 60 min. After centrifugation, aliquots of 1/25th of each supernatant (S) or one-fourth of each pellet (P) were analyzed by SDS/PAGE and stained with Coomassie blue. The bound peptide migrates at the dye front and the position of the molecular mass standards (in kDa) is indicated on the left.

Fig 2.

ARH interacts with clathrin, AP-1, and AP-2. Approximately 150 μg of either GST (lanes a, b, e, and f) or GST-ARH-(180–308) (lanes c, d, g, and h) immobilized on GSH-Sepharose was incubated with either rat brain (lanes a–d) or liver (lanes e–h) cytosol. After centrifugation, aliquots corresponding to 1/60th of each supernatant (S) and one-fifth of each washed pellet (P) were resolved by SDS/PAGE and either stained with Coomassie blue (Left) or transferred to nitrocellulose (Right). Portions of the blots were probed with mAb TD.1 [clathrin heavy chain (HC)], mAb 100/2 (α subunits), anti-μ2 serum, affinity-purified AE/1 (γ subunit), RY/1 serum (μ1 subunit), affinity-purified DE/1 (σ1 subunit) or KQ/1 (δ subunit), or anti-σ3 serum. The position of the molecular mass standards (in kDa) is indicated on the left, and only the relevant portion of each blot is shown.

Fig 3.

(A) A type-I clathrin box. Approximately 150 μg of GST (lanes a and b), GST-ARH-(180–308) (lanes c and d), or GST-ARH-(180–308) LLD → AAA (lanes e and f) immobilized on GSH-Sepharose was incubated with rat brain cytosol. After centrifugation, aliquots corresponding to 1/60th of each supernatant (S) and one-fifth of each washed pellet (P) were resolved by SDS/PAGE and either stained with Coomassie blue (Left) or transferred to nitrocellulose (Right). Portions of the blots were probed with the anti-clathrin HC mAb TD.1 or anti-AP-2 α-subunit mAb 100/2. (B) Approximately 100 μg of GST (lanes a and b), GST-ARHC3-(180–209) (lanes c and d), GST-ARHC2-(180–263) (lanes e and f), or GST-ARHC1-(180–308) (lanes g and h) immobilized on GSH-Sepharose was incubated with rat liver cytosol. After centrifugation, aliquots corresponding to 1/60th of each supernatant (S) and one-fifth of each washed pellet (P) were resolved by SDS/PAGE and either stained with Coomassie blue (Left) or transferred to nitrocellulose (Right). Portions of the blots were probed with the anti-clathrin HC mAb TD.1, anti-AP-2 α-subunit mAb 100/2, or anti-AP-2 μ2-subunit antiserum.

Fig 4.

ARH expression. Equal aliquots of total cell lysate from father (GM00965A) or affected brother (GM00697) were fractionated by SDS/PAGE and either stained with Coomassie blue (lanes a and b) or transferred to nitrocellulose (lanes c–f). Blots were probed with anti-ARH (c and d) or anti-clathrin HC mAb TD.1 and anti-β-subunit mAb 100.1 or anti-Dab2 antibodies (e and f).

Fig 5.

Intracellular localization. Cultured fibroblasts from either the asymptomatic heterozygous father (A–C) or ARH-affected (homozygous) brother (D–F) were permeabilized, fixed, and probed with affinity-purified anti-ARH antibodies (green) and anti-clathrin HC mAb X22 (red), followed by Alexa 488-labeled anti-rabbit IgG and Cy3-labeled anti-mouse IgG secondary antibodies. A single optical section at the base of the cells is shown. (Bar = 20 μm.)

Fig 6.

Subcellular colocalization. Fixed and permeabilized HeLa cells were incubated with anti-ARH antibodies (A, D, G, and J) (green) and anti-clathrin HC mAb X22 (B), anti-AP2 α-subunit mAb AP.6 (E), an anti-AP-1 γ-subunit mAb (H), or an anti-EEA1 mAb (K) (red). Single optical sections near the base of the adherent cells are shown with merged images in C, F, I, and L. (Bar = 20 μm.)

Fig 7.

LDL receptor internalization. HeLa cells precultured in LPDS were incubated with anti-LDL receptor mAb IgG-C7 (red) on ice for 60 min before warming to 37°C for 30 sec (A), 1 min (B–E), 2 min (F), or 10 min (G and H). After fixation, the cells were incubated with anti-ARH antibodies (green). A single optical section at the base of the cells is shown except in H, which is a medial section. (Bar = 20 μm.)

Fig 8.

ARH in liver clathrin-coated vesicles. Aliquots of 20 μg of liver homogenate (lanes a), cytosol (lanes b), microsomes (lanes c), and 10 μg of crude vesicles (lanes d) or purified clathrin-coated vesicles (lanes e) were fractionated by SDS/PAGE and either stained (Left) or transferred to nitrocellulose (Center and Right). Portions of the blots were probed with mAb 100.1 (anti-β1/2-subunits), RY/1 serum (μ1 subunit), anti-AP-2 μ2 serum, mAb anti-CALM, affinity-purified anti-Dab2, anti-epsin, anti-ARH antibodies, or mAb C2 (anti-actin).