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Normal Sorting but Defective Endocytosis of the Low Density Lipoprotein Receptor in Mice with Autosomal Recessive Hypercholesterolemia*

  • Christopher Jones
    Affiliations
    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046
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  • Robert E. Hammer
    Affiliations
    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046

    Department of The Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046
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  • Wei-Ping Li
    Affiliations
    Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046
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  • Jonathan C. Cohen
    Affiliations
    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046

    Department of McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046

    Department of Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046
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  • Helen H. Hobbs
    Affiliations
    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046

    Department of The Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046
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  • Joachim Herz
    Correspondence
    To whom correspondence should be addressed: 5323 Harry Hines Blvd., Dallas, TX 75390-9046. Tel.: 214-648-5633; Fax: 214-648-8804
    Affiliations
    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046
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  • Author Footnotes
    * This study was supported by grants from the National Institutes of Health (Grants HL20948, HL63762, NS43408), the Alzheimer Association, the Howard Hughes Medical Institute, the Perot Family Foundation, the Donald W. Reynolds Foundation, and the Humboldt Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:May 13, 2003DOI:https://doi.org/10.1074/jbc.M304855200
      Autosomal recessive hypercholesterolemia (ARH) is a genetic form of hypercholesterolemia that clinically resembles familial hypercholesterolemia (FH). As in FH, the rate of clearance of circulating low density lipoprotein (LDL) by the LDL receptor (LDLR) in the liver is markedly reduced in ARH. Unlike FH, LDL uptake in cultured fibroblasts from ARH patients is normal or only slightly impaired. The gene defective in ARH encodes a putative adaptor protein that has been implicated in linking the LDLR to the endocytic machinery. To determine the role of ARH in the liver, ARH-deficient mice were developed. Plasma levels of LDL-cholesterol were elevated in the chow-fed Arh–/– mice (83 ± 8 mg/dl versus 68 ± 8 mg/dl) but were lower than those of mice expressing no LDLR (Ldlr–/–) (197 ± 8 mg/dl). Cholesterol feeding elevated plasma cholesterol levels in both strains. The fractional clearance rate of radiolabeled LDL was reduced to similar levels in the Arh–/– and Ldlr–/– mice, whereas the rate of removal of α2-macroglobulin by the LDLR-related protein, which also interacts with ARH, was unchanged. Immunolocalization studies revealed that a much greater proportion of immunodetectable LDLR, but not LDLR-related protein, was present on the sinusoidal surface of hepatocytes in the Arh–/– mice. Taken together, these results are consistent with ARH playing a critical and specific role in LDLR endocytosis in the liver.
      Low density lipoproteins (LDL)
      The abbreviations used are: LDL, low density lipoprotein; LDL-C, LDL cholesterol; LDLR, LDL receptor; LRP, LDLR-related protein; VLDL, very low density lipoprotein; α2M, α2-macroglobulin; ARH, autosomal recessive hypercholesterolemia; FH, familial hypercholesterolemia; PTB, phosphotyrosine binding.
      1The abbreviations used are: LDL, low density lipoprotein; LDL-C, LDL cholesterol; LDLR, LDL receptor; LRP, LDLR-related protein; VLDL, very low density lipoprotein; α2M, α2-macroglobulin; ARH, autosomal recessive hypercholesterolemia; FH, familial hypercholesterolemia; PTB, phosphotyrosine binding.
      particles are the major cholesterol transport vehicle in the circulation. Approximately 70% of cholesterol in human plasma is contained in the LDL fraction. The major fate of circulating LDL is uptake into the liver by LDLR-mediated endocytosis (
      • Osono Y.
      • Woollett L.A.
      • Herz J.
      • Dietschy J.M.
      ,
      • Goldstein J.
      • Hobbs H.
      • Brown M.
      ). The rate of removal of LDL from the plasma is a key determinant of plasma cholesterol levels. Reductions in hepatic LDLR number or activity result in elevated plasma levels of LDL-C. The most common monogenic cause of severe hypercholesterolemia is the autosomal dominant disorder, familial hypercholesterolemia (FH), caused by mutations in LDLR (
      • Goldstein J.
      • Hobbs H.
      • Brown M.
      ,
      • Goldstein J.L.
      • Brown M.S.
      ). FH homozygotes, who have mutations in both LDLR alleles, have a markedly decreased rate of removal of circulating LDL and a dramatic increase in plasma cholesterol levels (
      • Goldstein J.
      • Hobbs H.
      • Brown M.
      ). The hypercholesterolemia results in deposition of cholesterol in body tissues, including skin and tendons (xanthomas), and coronary arteries, causing premature coronary atherosclerosis (
      • Goldstein J.
      • Hobbs H.
      • Brown M.
      ). Recently, the molecular defect for a recessive form of hypercholesterolemia that clinically resembles FH but is not due to mutations in LDLR was identified (
      • Garcia C.K.
      • Wilund K.
      • Arca M.
      • Zuliani G.
      • Fellin R.
      • Maioli M.
      • Calandra S.
      • Bertolini S.
      • Cossu F.
      • Grishin N.
      • Barnes R.
      • Cohen J.C.
      • Hobbs H.H.
      ). This disorder, called autosomal recessive hypercholesterolemia, is caused by mutations in the putative adaptor protein ARH (
      • Wilund K.R.
      • Yi M.
      • Campagna F.
      • Arca M.
      • Zuliani G.
      • Fellin R.
      • Ho Y.K.
      • Garcia J.V.
      • Hobbs H.H.
      • Cohen J.C.
      ).
      Patients lacking ARH clear LDL from the circulation at rates as low as those observed in patients with no functioning LDLRs (
      • Zuliani G.
      • Arca M.
      • Signore A.
      • Bader G.
      • Fazio S.
      • Chianelli M.
      • Bellosta S.
      • Campagna F.
      • Montali A.
      • Maioli M.
      • Pacifico A.
      • Ricci G.
      • Fellin R.
      ). Immortalized lymphoblasts from ARH patients exhibit impaired LDL internalization, despite expressing 2-fold more LDLRs on the cell surface than lymphoblasts from unaffected individuals (
      • Wilund K.R.
      • Yi M.
      • Campagna F.
      • Arca M.
      • Zuliani G.
      • Fellin R.
      • Ho Y.K.
      • Garcia J.V.
      • Hobbs H.H.
      • Cohen J.C.
      ,
      • Norman D.
      • Sun X.-M.
      • Bourbon M.
      • Knight B.L.
      • Naoumova R.P.
      • Soutar A.K.
      ). In contrast to ARH-deficient lymphoblasts and hepatocytes, cultured fibroblasts from ARH patients have preserved LDLR function, with levels of LDLR activity ranging from 30 to 100% of normal (
      • Arca M.
      • Zuliani G.
      • Wilund K.
      • Campagna F.
      • Fellin R.
      • Bertolini S.
      • Calandra S.
      • Ricci G.
      • Glorioso N.
      • Maioli M.
      • Pintus P.
      • Carru C.
      • Cossu F.
      • Cohen J.
      • Hobbs H.H.
      ). Although ARH and FH homozygotes have similar rates of LDL clearance (
      • Zuliani G.
      • Arca M.
      • Signore A.
      • Bader G.
      • Fazio S.
      • Chianelli M.
      • Bellosta S.
      • Campagna F.
      • Montali A.
      • Maioli M.
      • Pacifico A.
      • Ricci G.
      • Fellin R.
      ), ARH patients generally have lower plasma cholesterol levels and later onset of cardiovascular disease than FH homozygotes (
      • Arca M.
      • Zuliani G.
      • Wilund K.
      • Campagna F.
      • Fellin R.
      • Bertolini S.
      • Calandra S.
      • Ricci G.
      • Glorioso N.
      • Maioli M.
      • Pintus P.
      • Carru C.
      • Cossu F.
      • Cohen J.
      • Hobbs H.H.
      ,
      • Zuliani G.
      • Vigna G.B.
      • Corsini A.
      • Maioli M.
      • Romagnoni F.
      • Fellin R.
      ).
      ARH contains a single phosphotyrosine-binding (PTB) domain, which is capable of binding the LDLR cytoplasmic tail in vitro (
      • He G.
      • Gupta S.
      • Yi M.
      • Michaely P.
      • Hobbs H.H.
      • Cohen J.C.
      ,
      • Mishra S.K.
      • Watkins S.C.
      • Traub L.M.
      ). Adaptor proteins containing PTB domains bind the conserved NPXY sequence motif located in the cytoplasmic domains of various cell surface receptors and mediate diverse cellular functions, including receptor trafficking and endocytosis (
      • Forman-Kay J.D.
      • Pawson T.
      ,
      • Yan K.S.
      • Kuti M.
      • Zhou M.-M.
      ). The LDLR cytoplasmic tail contains a single NPXY motif that is required for clustering and endocytosis of the receptors in fibroblasts (
      • Chen W.J.
      • Goldstein J.L.
      • Brown M.S.
      ,
      • Davis C.G.
      • Lehrman M.A.
      • Russell D.W.
      • Anderson R.G.
      • Brown M.S.
      • Goldstein J.L.
      ). Point mutations in this highly conserved sequence cause FH and eliminate binding of ARH to LDLR in vitro (
      • He G.
      • Gupta S.
      • Yi M.
      • Michaely P.
      • Hobbs H.H.
      • Cohen J.C.
      ,
      • Mishra S.K.
      • Watkins S.C.
      • Traub L.M.
      ,
      • Davis C.G.
      • Lehrman M.A.
      • Russell D.W.
      • Anderson R.G.
      • Brown M.S.
      • Goldstein J.L.
      ,
      • Garcia-Garcia A.B.
      • Real J.T.
      • Puig O.
      • Cebolla E.
      • Marin-Garcia P.
      • Martinez Ferrandis J.I.
      • Garcia-Sogo M.
      • Civera M.
      • Ascaso J.F.
      • Carmena R.
      • Armengod M.E.
      • Chaves F.J.
      ). In addition to binding the LDLR tail, ARH also binds the β2-adaptin subunit of AP-2 and the terminal domain of clathrin in a sequence-specific manner in vitro, so it has been proposed to link the LDLR to the endocytic machinery (
      • Garcia C.K.
      • Wilund K.
      • Arca M.
      • Zuliani G.
      • Fellin R.
      • Maioli M.
      • Calandra S.
      • Bertolini S.
      • Cossu F.
      • Grishin N.
      • Barnes R.
      • Cohen J.C.
      • Hobbs H.H.
      ,
      • He G.
      • Gupta S.
      • Yi M.
      • Michaely P.
      • Hobbs H.H.
      • Cohen J.C.
      ). In support of this scenario, immortalized lymphocytes from ARH subjects are defective in LDL internalization (
      • Norman D.
      • Sun X.-M.
      • Bourbon M.
      • Knight B.L.
      • Naoumova R.P.
      • Soutar A.K.
      ).
      Although human studies suggest that ARH is required specifically for normal LDLR function, it may also be involved in the internalization of other proteins containing an NPXY motif in the cytoplasmic tail, such as other members of the LDLR gene family. For instance, the LDLR-related protein (LRP) contains two NPXY motifs. One of them binds the PTB domain protein Dab1, an adaptor protein that is involved in the control of neuronal migration and signaling through LDLR family members and that also interacts with the LDLR (
      • Trommsdorff M.
      • Borg J.P.
      • Margolis B.
      • Herz J.
      ,
      • Trommsdorff M.
      • Gotthardt M.
      • Hiesberger T.
      • Shelton J.
      • Stockinger W.
      • Nimpf J.
      • Hammer R.E.
      • Richardson J.A.
      • Herz J.
      ). LRP is abundantly expressed in the liver, where it functions in concert with the LDLR in the clearance of chylomicron remnants (
      • Willnow T.E.
      • Sheng Z.
      • Ishibashi S.
      • Herz J.
      ,
      • Rohlmann A.
      • Gotthardt M.
      • Hammer R.E.
      • Herz J.
      ), but it is unknown whether LRP also functionally interacts with ARH. In the initial three ARH patients examined, an accumulation of chylomicron remnants was not reported (
      • Zuliani G.
      • Arca M.
      • Signore A.
      • Bader G.
      • Fazio S.
      • Chianelli M.
      • Bellosta S.
      • Campagna F.
      • Montali A.
      • Maioli M.
      • Pacifico A.
      • Ricci G.
      • Fellin R.
      ), suggesting that ARH may not be required for normal LRP endocytosis.
      The currently available limited data from human subjects suggest that ARH function may be tissue-specific, inasmuch as LDL internalization in ARH patients was found to be defective in the liver and in lymphoblasts, but is apparently largely normal in fibroblasts from the same patients. To facilitate the investigation of ARH function in the liver, the central organ that regulates LDL metabolism, we have now generated ARH-deficient mice by gene targeting. These animals replicate central features of the human disease phenotype, including abnormal sequestration of the LDLR at the cell surface and reduced internalization of LDLR but not LRP. ARH-deficient mice are currently the only physiological model system on which the role of this specialized adaptor protein in receptor-mediated endocytosis can be experimentally investigated.

      EXPERIMENTAL PROCEDURES

      General Methods—Unless otherwise indicated, DNA manipulations were performed by standard techniques (
      • Sambrook J.
      • Russel D.W.
      ). Cholesterol and triglycerides were determined enzymatically with assay kits obtained from Roche Applied Science and Sigma, respectively. Mouse LDL (d 1.019–1.063 g/ml) was isolated by sequential ultracentrifugation (
      • Goldstein J.L.
      • Basu S.K.
      • Brown M.S.
      ) from pooled plasma obtained from Ldlr–/– mice (
      • Ishibashi S.
      • Brown M.S.
      • Goldstein J.L.
      • Gerard R.D.
      • Hammer R.E.
      • Herz J.
      ) that had been fasted >6 h. Lipoproteins were radiolabeled with 125I by the iodine monochloride method (
      • Goldstein J.L.
      • Basu S.K.
      • Brown M.S.
      ). Methylamine-activated α2-macroglobulin was radiolabeled with 125I using the iodogen method as described previously (
      • Fraker P.J.
      • Speck Jr., J.C.
      ).
      Antibodies—Rabbit polyclonal antibodies against Rab5 (KAP-GP006), EEA1 (324610), and GRP78 (BiP) (SPA-826) were purchased from StressGen Biotechnologies (Victoria, BC, Canada) and Calbiochem. Rabbit polyclonal antibodies against Lamp1 (H-228) and Rab11 (H-87) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies were raised in rabbits against the PTB domain of mouse ARH and the C-terminal 13 residues of mouse LDLR. Rabbit polyclonal antibodies against LRP and purified LDLR from bovine adrenal have been described elsewhere (
      • Herz J.
      • Hamann U.
      • Rogne S.
      • Myklebost O.
      • Gausepohl H.
      • Stanley K.K.
      ,
      • Russell D.W.
      • Schneider W.J.
      • Yamamoto T.
      • Luskey K.L.
      • Brown M.S.
      • Goldstein J.L.
      ).
      Cloning of Mouse ARH cDNA—Murine ARH cDNA was amplified from a commercially available mouse liver cDNA library (Clontech) using the following primers: 5′-ATTCTAGACATGGACGCGCTCAAGTCGGCG-3′ and 5′-TTAAGCTTTCAGAAGGTGAAGACGTCATCATC-3′. Amplification products were TA-cloned into pCR2.1-TOPO (Invitrogen) and sequenced.
      Generation of ARH Knockout Mice—Two fragments were amplified from genomic DNA from mouse liver (129S6/SvEv strain) using long range PCR (Takara Biochemical, Inc., Berkeley, CA). An 8.5-kb fragment, corresponding to a region extending from intron 4 into the 3′-untranslated region, was derived using primers 5LA1 (5′-ACGGCGGCCGCCTGTGTGGCCTGAGTCCCTCCCTGG-3′) and 3LA1 (5′-CCTGCGGCCGCCGCCAGCATGAGCAAGC-3′), and a 1-kb fragment containing part of intron 3 was amplified using primers 5SA (5′-ACGCTCGAGGTGTGCCAGTGGGGAACCAGGAAGG-3′) and 3SA (5′-TGCCTCGAGGGGAGATGGAAATAAGAAATGAAGGAAGC-3′). Bold characters indicate inserted restriction sites. The two fragments were cloned into the targeting vector (
      • Gotthardt M.
      • Hammer R.E.
      • Hubner N.
      • Monti J.
      • Witt C.C.
      • McNabb M.
      • Richardson J.A.
      • Granzier H.
      • Labeit S.
      • Herz J.
      ) on either side of a PGKneobpA expression cassette (
      • Soriano P.
      • Montgomery C.
      • Geske R.
      • Bradley A.
      ) using NotI and XhoI sites, respectively. The vector also contained two copies of the herpes simplex virus thymidine kinase gene (
      • Mansour S.L.
      • Thomas K.R.
      • Capecchi M.R.
      ) in tandem at the 5′ end of the short arm. The linearized vector was electroporated into murine embryonic SM1 stem cells, and recombinant clones were selected using G418 and ganciclovir, as described (
      • Willnow T.E.
      • Herz J.
      ). Homologous recombination was identified by PCR (
      • Soriano P.
      • Montgomery C.
      • Geske R.
      • Bradley A.
      ) using primers ARH-T (5′-ACGCTCGAGCAGCCCAAATCCATGCTATCCATGGAC-3′) and Neo-36 (5′-CAGGACAGCAAGGGGGAGGATTGGGAAGAC-3′) and confirmed by Southern blot analysis after EcoRI and SacI digestion. Twelve independent stem cell clones containing a disrupted arh allele were injected into C57Bl/6 blastocysts, yielding a total of 18 chimeric males. Of these, 17 were fertile, and 6 gave offspring that carried the disrupted allele. Two separate lines were established for experimentation. No differences were detected between the two lines. Animals were genotyped by allele-specific PCR, using three primers. Primer ARH-T was used as the upstream oligonucleotide, whereas primers Neo-36 and ARH-GW (5′-CCTGTACTCCCAGACTACTTCATGATCCCCAC-3′) were used as the downstream primers for the disrupted and wild-type alleles, respectively.
      Animal Housing and Feeding—Mice were housed on a 12-h dark/12-h light cycle and given standard chow (number 7002; Harlan Teklad, Madison, WI) and water ad libitum. In cholesterol feeding experiments, animals were either fed the Paigen high cholesterol diet (1.25% cholesterol, 7.5% cocoa butter, 7.5% casein, and 0.5% cholic acid) or fed a Western-type diet containing 0.2% cholesterol (
      • Plump A.S.
      • Smith J.D.
      • Hayek T.
      • Aalto-Setala K.
      • Walsh A.
      • Verstuyft J.G.
      • Rubin E.M.
      • Breslow J.L.
      ). All experiments were performed with F2 or F3 generation descendents, which were hybrids between C57Bl/6 and 129Sv strains. No differences in the results were observed when littermates were used in the experiments or when animals from vertically inbred lines were compared.
      Clearance of LDL and α2-Macroglobulin from Plasma—Clearance of lipoprotein and α2-macroglobulin (α2M) was determined as described previously (
      • Ishibashi S.
      • Brown M.S.
      • Goldstein J.L.
      • Gerard R.D.
      • Hammer R.E.
      • Herz J.
      ,
      • Choi S.Y.
      • Cooper A.D.
      ). Briefly, mice were anesthetized with sodium pentobarbital (80 mg/kg) and injected via the external jugular vein with a 0.2-ml intravenous bolus of either 15 μg of 125I-LDL or 5 μg of 125I-α2M in 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.2% (w/v) bovine serum albumin. At various time points, blood samples were drawn from the retro-orbital plexus into EDTA-coated tubes (Microvette 500 KE, Sarstedt, Newton, NC). The plasma content of 125I-labeled protein was measured by trichloroacetic acid precipitation followed by γ counting. The amount of LDL or α2M remaining in the blood was expressed as a percent of the initial blood concentration, measured as the average 125I-radioactivity in the plasma 2 min after injection.
      Interaction of ARH PTB Domain with Lipoprotein Receptor Cytoplasmic Tails—The LDLR family cytoplasmic tails/LexA fusion protein constructs and the Dab1 prey construct have been described previously (
      • Gotthardt M.
      • Trommsdorff M.
      • Nevitt M.F.
      • Shelton J.
      • Richardson J.A.
      • Stockinger W.
      • Nimpf J.
      • Herz J.
      ). The ARH-PTB domain was amplified from the cloned cDNA using the primers 5APY (5′-ATGGAATTCAGCCTCAAGTACCTTGGTATGACG-3′) and 3APY (5′-TTGTCGACTCAGGACACCTGCCAAAACTCAAAGGC-3′). Bold characters indicate inserted restriction sites. The resulting PCR product was digested with EcoRI and SalI, inserted into the EcoRI-XhoI-digested prey vector pB42AD (MATCHMAKER system, Clontech), and sequenced. Yeast transformations and matings were performed following the manufacturer's instructions in the MATCHMAKER manual, and interactions were assessed as described previously (
      • Gotthardt M.
      • Trommsdorff M.
      • Nevitt M.F.
      • Shelton J.
      • Richardson J.A.
      • Stockinger W.
      • Nimpf J.
      • Herz J.
      ).
      Immunohistochemistry—Anesthetized mice were perfused by cardiac puncture with warm Hank's balanced salt solution followed by 4% (w/v) paraformaldehyde in phosphate-buffered saline. The livers were removed and divided into 0.5-cm2 sections. The tissue was fixed for an additional hour at 25 °C in 4% (w/v) paraformaldehyde in phosphate-buffered saline followed by an overnight incubation in 30% (w/v) sucrose solution. The tissue was frozen in OCT compound 4583 (Miles Laboratories, Elkhart, IN) over dry ice and stored at –70 °C until cutting. Sections of 7 μm were cut on a Leitz Cryostat (E. Leitz, Inc., Rockleigh, NJ) at –20 °C and mounted onto poly-l-lysine-coated slides. Samples were blocked by incubation for 1 h with 10 mm Tris-HCl, pH 9.0, 150 mm NaCl (Tris-buffered saline) containing 20% (v/v) normal goat serum and 1% (w/v) bovine serum albumin. Sections were then incubated overnight with rabbit antiserum raised against the PTB domain of ARH (1:800 dilution), rabbit antiserum against LRP (
      • Herz J.
      • Hamann U.
      • Rogne S.
      • Myklebost O.
      • Gausepohl H.
      • Stanley K.K.
      ) (1:200), or polyclonal rabbit IgG directed against the LDLR (
      • Russell D.W.
      • Schneider W.J.
      • Yamamoto T.
      • Luskey K.L.
      • Brown M.S.
      • Goldstein J.L.
      ) (1:400). Slides were washed three times in Tris-buffered saline/0.1% bovine serum albumin, and bound primary antibody was detected by incubation for 2 h with 20 μg/ml Alexa-Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Slides were washed three times in Tris-buffered saline/0.1% bovine serum albumin, rinsed once with water, and mounted under a coverslip with Immu-mount (Shandon, Pittsburgh, PA). Images were taken using the 63 × 0.70 objective on a Leica confocal microscope.
      Sucrose Gradients—Male wild-type, Arh–/–, and Ldlr–/– mice aged 15 weeks were fasted for 6 h and sacrificed. The livers were removed, rinsed briefly in cold phosphate-buffered saline, and minced in a weigh boat over ice. The minced liver was homogenized at 20% (w/v) in cold homogenization buffer (50 mm Tris-HCl, pH 7.4, 250 mm sucrose, 25 mm KCl, 5 mm MgCl2, 3 mm imidazole, Roche protease inhibitor mixture) with 20 strokes in a Dounce homogenizer. Nuclei and other debris were removed by low speed centrifugation at 1000 × g for 10 min at 4 °C. 300 μl of the supernatant was loaded on a 4-ml continuous 10–40% sucrose gradient and centrifuged using a Beckman SW60 Ti rotor for 16 h at 40,000 rpm, as described previously (
      • Stockinger W.
      • Sailler B.
      • Strasser V.
      • Recheis B.
      • Fasching D.
      • Kahr L.
      • Schneider W.J.
      • Nimpf J.
      ). A 20-gauge needle was used to puncture the bottom of each tube, and ∼200-μl fractions were collected. Protein was precipitated with trichloroacetic acid, neutralized with NaOH, and dissolved in 100 μl of sample buffer. A 10-μl aliquot was used for Western blotting.

      RESULTS

      Generation of ARH-deficient Mice—To disrupt the Arh gene in the mouse, targeted homologous recombination was used to replace exon 4 of Arh with a neomycin resistance cassette (Fig. 1A). Gene disruption was confirmed by Southern blotting (Fig. 1B). Deletion of exon 4 disrupts the PTB domain and is predicted to render the protein unable to bind the NPXY motifs of LDLR family members. If splicing occurs between exons 3 and 5, a frameshift and nonsense mutation would be introduced upstream of the putative binding sites for clathrin and AP-2 (
      • He G.
      • Gupta S.
      • Yi M.
      • Michaely P.
      • Hobbs H.H.
      • Cohen J.C.
      ). No immunoreactive ARH protein was detected in liver lysates of mice homozygous for the disruption using an antibody directed against the PTB domain (Fig. 1C). Levels of both LDLR and LRP protein were unchanged in the Arh–/– mice. Arh–/– mice were fertile and had normal litter sizes.
      Figure thumbnail gr1
      Fig. 1Targeted disruption of the arh gene. A, schematic structure of the mouse Arh gene locus and the knockout construct. The ARH PTB domain is encoded by exons 2–5, indicated by the gray shading. Homologous recombination resulted in the replacement of exon 4 with a PGKneobpA selection cassette (
      • Gotthardt M.
      • Hammer R.E.
      • Hübner N.
      • Monti J.
      • Witt C.C.
      • McNabb M.
      • Richardson J.A.
      • Granzier H.
      • Labeit S.
      • Herz J.
      ), introducing a diagnostic SacI (S) restriction site. B, Southern blot of genomic DNA isolated from mouse tails of the indicated genotypes and digested with EcoRI (E) and SacI (S) and hybridized with the an ARH cDNA probe. An 8.2-kb band is generated from the wild-type allele, and a 2.4- and a 6.4-kb band are present in the disrupted allele due to the presence of a SacI site in neo R. C, immunoblot analysis of LDLR and LRP in ARH-deficient mice. Whole liver cell lysates were prepared as described (
      • Rohlmann A.
      • Gotthardt M.
      • Willnow T.E.
      • Hammer R.E.
      • Herz J.
      ), and 30 μg of protein was fractionated using SDS-PAGE. Samples were analyzed by immunoblotting using antibodies against ARH, LDLR (
      • Russell D.W.
      • Schneider W.J.
      • Yamamoto T.
      • Luskey K.L.
      • Brown M.S.
      • Goldstein J.L.
      ), and LRP (
      • Herz J.
      • Hamann U.
      • Rogne S.
      • Myklebost O.
      • Gausepohl H.
      • Stanley K.K.
      ). Blots were stripped and incubated with antibodies to BiP (GRP78), which was used as a loading control.
      Arh–/– Mice Are Hypercholesterolemic and Sensitive to Dietary Cholesterol—Plasma cholesterol levels were only mildly elevated in the chow-fed Arh–/– mice and not significantly different in heterozygous mice when compared with wild-type littermate controls (Table I and Fig. 2A). Female Arh+/–, Arh–/–, Ldlr–/–, and wild-type mice were fed chow alone, chow supplemented with 0.2% cholesterol-containing Western-type diet, or chow supplemented with 1.25% cholesterol (Paigen diet) for 4 weeks. On the chow diet, the mean plasma level of cholesterol in the ARH–/– mice was intermediate (83 mg/dl) to the wild-type (68 mg/dl) and Ldlr–/– mice (196 mg/dl). On the Western diet, plasma cholesterol levels were increased to a similar level in the Arh–/– and Ldlr–/– mice (Table I and Fig. 2A). Feeding of the Paigen diet resulted in dramatic elevations of plasma cholesterol levels to an average of 1270 mg/dl for the Arh–/– mice and 1442 mg/dl for the Ldlr–/– mice. No significant differences were seen in plasma lipid levels between the Arh+/– mice and wild-type mice on any of the diets, which is consistent with the autosomal recessive inheritance pattern of ARH in humans. Liver cholesterol and triglyceride levels were not elevated in the Arh–/– mice over their wild-type littermates (Table I).
      Table ICholesterol and triglyceride levels in response to cholesterol feeding
      0.02% cholesterol0.2% cholesterol1.25% cholesterol/0.5% cholic acid
      Plasma cholesterol (mg/dl)
      Wild type67.7 ± 8.1125.9 ± 38.5184.9 ± 35.8
      ARH+/-94.6 ± 18.5157.1 ± 22.3155.7 ± 25.2
      ARH-/-83.1 ± 7.9307.9 ± 62.91270.0 ± 7.9
      LDLR-/-196.4 ± 7.6239.1 ± 80.51442.4 ± 42.0
      Plasma triglycerides (mg/dl)
      Wild type58.8 ± 4.556.6 ± 9.647.2 ± 5.0
      ARH+/-56.2 ± 9.361.4 ± 6.546.2 ± 1.8
      ARH-/-58.7 ± 7.8100.0 ± 12.549.0 ± 10.4
      LDLR-/-97.4 ± 12.194.8 ± 22.7129.2 ± 1.4
      Liver tissue cholesterol (mg/g)
      Wild type2.64 ± 0.056.80 ± 1.7319.58 ± 0.75
      ARH+/-2.69 ± 0.1010.44 ± 2.6415.97 ± 2.00
      ARH-/-2.67 ± 0.156.76 ± 0.7523.23 ± 0.21
      LDLR-/-3.27 ± 0.334.46 ± 0.4020.96 ± 0.76
      Liver tissue triglycerides (mg/g)
      Wild type26.0 ± 3.127.3 ± 6.729.0 ± 1.2
      ARH+/-22.3 ± 7.533.5 ± 2.419.9 ± 7.0
      ARH-/-22.9 ± 8.338.6 ± 10.821.5 ± 7.7
      LDLR-/-26.4 ± 6.136.3 ± 11.111.3 ± 0.7
      Figure thumbnail gr2
      Fig. 2Mean plasma cholesterol levels (A), and lipoprotein profiles (B–D) of Arh–/–, Ldlr–/–, and wild-type mice on chow and cholesterol-enriched diets. Female, 10–12-week-old Arh–/–, Arh+/–, and wild-type littermates and age-matched Ldlr–/– mice (four of each genotype) were fed a chow or cholesterol-enriched diet for 2 weeks. Plasma cholesterol levels were individually measured as described under “Experimental Procedures.” Error bars represent S.E. B–D, fast protein liquid chromatography profiles of plasma lipoproteins. Wild-type (□), Arh–/– (♦), and Ldlr–/– (▵) mice fed regular mouse chow containing 0.02% (w/w) cholesterol (B); chow supplemented with 0.2% (w/w) cholesterol (Western diet) (C); or chow supplemented with 1.25% (w/w) cholesterol (Paigen diet) (D). Mice were sacrificed after 2 weeks, and blood was collected from the inferior vena cava. Aliquots of plasma from the three animals in each group was pooled and subjected to fast protein liquid chromatography gel filtration (
      • Yokode M.
      • Hammer R.E.
      • Ishibashi S.
      • Brown M.S.
      • Goldstein J.L.
      ).
      To determine the distribution of cholesterol in the lipoprotein fractions, pooled plasma from mice of the same genotype on the different diets was subjected to fast protein liquid chromatography. On the regular chow diet, there was very little accumulation of LDL in the Arh–/– animals (Fig. 2B). On the cholesterol-enriched diets, most of the cholesterol was in the LDL fraction in Arh–/– and in Ldlr–/– mice (Fig. 2, C and D). The relative increase in LDL-C in the mice of different genotypes was estimated by taking the sum of the cholesterol content of each column fraction in the LDL peak. On the chow diet, LDL-C was increased 1.4-fold in the Arh–/– mice relative to wild-type littermates, and the difference between the plasma cholesterol levels of these two strains increased further after cholesterol feeding. On a Western diet, the mean LDL-C level was 4.7-fold higher in the Arh–/– mice than in the littermate controls and 7.3-fold higher than the Arh–/– mice on a chow diet. In contrast to these results, plasma LDL-C levels increased only 2-fold in Ldlr–/– mice fed with the Western-type diet.
      Plasma LDL-C levels were increased 11-fold in the Arh–/– mice on the Paigen diet when compared with wild-type littermates and 42-fold when compared with chow-fed Arh–/– mice. Ldlr–/– mice on the Paigen diet had LDL-C levels that were similar to those of Arh–/– mice on the same diet. LDL-C levels of Ldlr–/– animals were 12-fold increased over wild-type mice fed the same diet and 11-fold increased over Ldlr–/– mice that had been fed a 0.2% cholesterol-containing Western-diet. Thus, cholesterol feeding was associated with a more dramatic elevation of cholesterol in the Arh–/– mice.
      Clearance of 125I-labeled LDL Is Reduced in Arh–/– Mice—To determine the effect of ARH deficiency on hepatic LDLR function, we compared the rates of removal of 125I-labeled LDL from the circulation of Arh–/– mice to wild-type and Ldlr–/– mice. The half-time for disappearance of 125I-LDL was over twice as long in the Arh–/– mice as in wild-type animals (5.5 versus 2 h) (Fig. 3A). The rate of clearance of LDL from the circulation was as slow in the Arh–/– mice as in mice expressing no LDLR. To test whether the absence of ARH also impacted on the activity of other endocytic receptors in the liver, we examined the clearance of radiolabeled α2M, which is removed from the circulation by LRP. No differences were found between the rates of removal of 125I-α2M in the Arh–/– and wild-type mice (Fig. 3B).
      Figure thumbnail gr3
      Fig. 3Turnover of 125I-labeled LDL and 125I-α2M in Arh–/–, Ldlr–/–, and wild-type mice. A, four wild-type (□), Arh–/– (♦), and Ldlr–/– (▵) 12–14-week-old male mice were injected in the external jugular vein with a bolus of 15 μg of 125I-LDL (550 cpm/ng of protein). B, three wild-type (□) and Arh–/– (♦) 12–14-week-old female mice were injected with 5 μg of 125I-α2M (1250 cpm/ng). Blood samples were collected by retro-orbital puncture at the indicated times, and the plasma content of trichloroacetic acid-precipitable 125I-radioactivity was measured. Radioactivity remaining in the plasma was plotted as a percentage of the activity present 2 min after injection of the labeled ligand. Before the experiment, the mice were fasted for 6 h and anesthetized with sodium pentobarbital (80 mg/kg of intraperitoneal).
      ARH Interacts with the Cytoplasmic Tails of Several LDLR Family Members—The cytoplasmic tail of each LDLR family member contains one or more characteristic NPXY motifs, to which the PTB domain of ARH is likely to bind. To identify other receptors with which ARH might interact, we performed a yeast two-hybrid assay with a panel of LDLR family members. Surprisingly, in this assay ARH bound to the LDLR tail only relatively weakly but bound strongly to both splice variants of ApoER2 and also to the VLDL receptor. Since the NPXY motif in the cytoplasmic tails of both of these receptors is identical to that in the LDLR (Fig. 4A), this finding suggests that the ARH PTB domain requires additional flanking amino acid residues for optimal binding. In addition, ARH was also capable of interacting with the first NPXY motif of megalin and the second NPXY motif of LRP. A paradigm for a receptor-specific role of PTB domain-containing adaptor proteins in endocytosis is also supported by the finding that Dab2, another PTB domain-containing protein that is most closely related to Dab1, binds to the cytoplasmic tail of megalin and is required for its endocytosis in the proximal tubule of the kidney (
      • Morris S.M.
      • Tallquist M.D.
      • Rock C.O.
      • Cooper J.A.
      ).
      Figure thumbnail gr4
      Fig. 4Interaction of ARH with LDLR family members (A) and redistribution of LDLR, but not LRP, to the sinusoidal membrane in mice lacking ARH (B). As shown in A, cytoplasmic tails of LDLR family members interact with the ARH PTB domain. During the yeast mating assays, clones containing bait, prey, and the reporter plasmid p8op-lacZ were selected on Trp-, Ura-, His-deficient plates. Four individual clones were transferred to patches on 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal) plates deficient in Trp, Ura, His, and Leu. Growth and blue staining was evaluated during 3 days at 30 °C (no growth, –; weak to strong growth and staining +, ++, or +++). LRP and LRP-B tails were self-activating, but lacZ activity was nevertheless enhanced by the ARH PTB domain. The + and – after the ApoER2 tail designate the presence of an alternatively spliced insert in the tail. The empty vectors (pLexA and pB42AD) were used as negative controls. Dab1 has been previously shown to interact with several LDLR family member tails and was used as a positive control. VLDLR, VLDL receptor. Meg, megalin. B, immunohistochemical staining of liver sections from wild-type, Arh–/–, Ldlr–/– mice. Frozen sections of liver from wild-type, Arh–/–, and Ldlr–/– mice were incubated with rabbit polyclonal antibodies against ARH, LDLR, and LRP. Bound IgG was detected with 20 μg/ml Alexa-Fluor 488-labeled goat anti-rabbit IgG as described under “Experimental Procedures.”
      LDLR Distribution Is Altered in the Liver of Arh–/– Mice—To determine the role of ARH on the subcellular distribution of the LDLR, we performed immunolocalization studies on liver sections from Arh–/–, Ldlr–/–, and wild-type mice using indirect immunofluorescence confocal microscopy. A diffuse cytoplasmic staining pattern was seen in sections from wild-type mice using a polyclonal anti-ARH antibody. Punctate ARH-specific staining was most intense at and immediately beneath the sinusoidal membrane. No differences in the pattern of ARH expression were seen in liver sections from the Ldlr–/– mice (Fig. 4B). Specific staining was absent from liver sections from Arh–/– mice and from sections to which the preimmune rabbit control serum had been applied (data not shown).
      LDLR distribution in the same liver sections was assessed using a rabbit polyclonal anti-mouse LDLR antibody. Cell surface staining as well as intracellular punctate staining in the cytoplasm was seen in the liver sections from wild-type mice. A very different distribution of immunodetectable LDLR was seen in the Arh–/– mice. In the absence of ARH, LDLR was sequestered virtually exclusively to the sinusoidal membrane. To determine whether the absence of ARH affected the cellular location of other endocytic receptors, the distribution of LRP was examined. Surprisingly, despite the strong binding of ARH to the LRP tail in the yeast two-hybrid assay, the subcellular distribution of LRP was not affected by the ARH disruption and indistinguishable among the liver sections from the Arh–/–, Ldlr–/–, and wild-type mice. Thus, absence of ARH had a specific effect on the distribution of the LDLR and was associated with a relative increase in cell surface staining.
      Isopycnic Centrifugation of Liver Lysates on a Continuous Sucrose Gradient—To determine the relative distribution of ARH and the LDLR within the various endocytic compartments in cells, analytical centrifugation was performed to fractionate vesicles from livers of Arh–/–, Ldlr–/–, and wild-type mice on a continuous sucrose gradient. The majority of ARH co-sedimented with vesicles containing early endosomal antigen 1 (EEA1) and rab5. Both proteins are associated with early endocytic compartments. Only a small percentage of ARH was present in the same fractions as the LDLR (Fig. 5). The distribution of ARH did not differ in the absence or presence of the LDLR. These results are consistent with a subset of ARH being recruited to LDLR-containing early endocytic compartments independently of LDLR.
      Figure thumbnail gr5
      Fig. 5Subcellular fractionation of ARH by isopycnic centrifugation on a continuous density gradient. Livers from 15-week-old male wild-type and Ldlr–/– mice were homogenized, and a postnuclear supernatant was loaded on top of a 10–40% continuous sucrose gradient. After centrifugation, fractions were collected, precipitated with trichloroacetic acid, and redisolved in 100 μl of loading buffer. Proteins were separated by SDS-PAGE and analyzed by immunoblotting using antibodies against the respective proteins as described under “Experimental Procedures.”

      DISCUSSION

      To explore the physiological function of ARH in hepatocytes and in the intact mammalian organism, we have developed an ARH-deficient mouse using a gene knockout approach. This animal model accurately reflects many hallmarks of the human disease phenotype of autosomal recessive hypercholesterolemia. When fed a normal mouse chow, which contains virtually no cholesterol, Arh–/– mice are only mildly hypercholesterolemic. However, the same animals are extremely sensitive to dietary cholesterol intake. Plasma cholesterol levels rise many-fold when the animals are fed cholesterol-enriched Paigen or Western-type diets, approaching those of Ldlr–/– mice on the same diets. Immunohistochemical analysis of the subcellular localization of LDLR in Arh–/– livers indicate that the LDLR is expressed at normal levels in the Arh knockout but that it is sequestered at the hepatocyte surface. This finding suggests that ARH mediates a specific step that is necessary for the internalization of LDLR in hepatocytes.
      Interestingly, plasma cholesterol levels in the Arh–/– mice are only mildly elevated on a chow diet, although the fractional clearance rate of LDL is greatly reduced in these animals and indeed the same as in Ldlr–/– mice. These data suggest that the apparent rate of LDL production must be reduced in the Arh–/– mice. A possible explanation for this could be that in the Arh–/– mice LDLRs, which are predominantly sequestered at the cell surface, may trap nascent VLDL particles, making them available for enrichment with ApoE and internalization by LRP (
      • Kowal R.C.
      • Herz J.
      • Weisgraber K.H.
      • Mahley R.W.
      • Brown M.S.
      • Goldstein J.L.
      ). Such a mechanism would effectively reduce the circulating precursor pool of lipoproteins from which LDL is formed. Alternatively, the rate of secretion of VLDL may be reduced due to LDLRs in the secretory pathway binding apolipoprotein B and targeting it for degradation (
      • Twisk J.
      • Gillian-Daniel D.L.
      • Tebon A.
      • Wang L.
      • Barrett P.H.R.
      • Attie A.D.
      ).
      ARH-deficient patients have LDL half-lives similar to those of FH homozygotes (
      • Zuliani G.
      • Arca M.
      • Signore A.
      • Bader G.
      • Fazio S.
      • Chianelli M.
      • Bellosta S.
      • Campagna F.
      • Montali A.
      • Maioli M.
      • Pacifico A.
      • Ricci G.
      • Fellin R.
      ). The clearance of 125I-LDL from the plasma of Arh–/– mice is also reduced to a rate comparable with that of Ldlr–/– mice. Two mutations in the cytoplasmic tail of the LDLR, J.D. (Y807C) and FH-Turku (G823D), have been shown to cause FH through distinctly different mechanisms. The J.D. mutation of the tyrosine in the NPXY motif of the LDLR disrupts the interaction of ARH with the LDLR tail in vitro and leads to the production of a receptor that is defective in endocytosis in fibroblasts (
      • Davis C.G.
      • Lehrman M.A.
      • Russell D.W.
      • Anderson R.G.
      • Brown M.S.
      • Goldstein J.L.
      ). In contrast, the FH-Turku mutation, which resides downstream of the NPXY sequence, does not impair LDLR internalization in fibroblasts. However, LDL internalization in the liver is prevented because the receptors are inappropriately sorted to the bile canalicular membrane in hepatocytes, thereby effectively sequestering them from the circulation and making them inaccessible to circulating LDL (
      • Koivisto U.M.
      • Hubbard A.L.
      • Mellman I.
      ).
      The available data from human patients did not exclude such abnormal sorting of the LDLR in the liver as a possible cause for the LDL clearance defect in ARH patients. Our present results now show, however, that LDLRs are sorted normally to the sinusoidal surface in Arh–/– mouse livers and that the LDL internalization defect in these mice is instead caused by the inability of the receptors to enter the endocytic cycle. Although LDLRs are present not only on the surface, but also in intracellular vesicles in the cytoplasm of wild-type hepatocytes, no intracellular receptors are detectable by immunohistochemistry in Arh–/– hepatocytes. Similarly, a 2-fold enrichment of LDLR on the cell surface was detected in ARH–/– human lymphoblasts (
      • Wilund K.R.
      • Yi M.
      • Campagna F.
      • Arca M.
      • Zuliani G.
      • Fellin R.
      • Ho Y.K.
      • Garcia J.V.
      • Hobbs H.H.
      • Cohen J.C.
      ,
      • Norman D.
      • Sun X.-M.
      • Bourbon M.
      • Knight B.L.
      • Naoumova R.P.
      • Soutar A.K.
      ,
      • Eden E.R.
      • Patel D.D.
      • Sun X.M.
      • Burden J.J.
      • Themis M.
      • Edwards M.
      • Lee P.
      • Neuwirth C.
      • Naoumova R.P.
      • Soutar A.K.
      ). After reintroduction of ARH by lenti-viral gene transfer, LDLRs were again readily detectable in intracellular compartments of ARH–/– lymphoblasts (
      • Eden E.R.
      • Patel D.D.
      • Sun X.M.
      • Burden J.J.
      • Themis M.
      • Edwards M.
      • Lee P.
      • Neuwirth C.
      • Naoumova R.P.
      • Soutar A.K.
      ). Taken together, all these observations are consistent with a model in which ARH functions as an adaptor protein that is required for LDLR internalization in polarized cells, such as hepatocytes and lymphoblasts. The virtually normal LDLR activity in fibroblasts from ARH patients suggests that another adaptor protein that is not present in the liver may be capable of substituting for ARH in certain non-polarized cell types. Alternatively, this hypothetical alternative protein may be present but incapable of supporting LDLR endocytosis in polarized cells.
      Similarly to the LDLR, LRP is highly expressed in the liver and participates in the clearance of apoE-containing lipoproteins. The LRP cytoplasmic domain contains two NPXY motifs, and in our yeast two-hybrid experiment, the interaction of ARH with LRP was even stronger than that seen between ARH and LDLR. This interaction with ARH and also the similarities in function and endocytosis rate between LRP and LDLR (
      • Li Y.
      • Lu W.
      • Marzolo M.P.
      • Bu G.
      ) suggested that LRP endocytosis might also be dependent upon ARH. Yet the clearance of the LRP-specific ligand α2M is normal in ARH-deficient mice, whereas mice lacking hepatic LRP have a profound deficiency in α2M uptake (
      • Rohlmann A.
      • Gotthardt M.
      • Willnow T.E.
      • Hammer R.E.
      • Herz J.
      ). However, in contrast to the striking difference in LDLR distribution between Arh–/– and wild-type hepatocytes, no alteration in the subcellular localization of LRP was observed. That a receptor that is functionally and structurally so closely related to the LDLR operates independently of ARH suggests that the ARH phenotype is not caused by a global defect in receptor-mediated endocytosis but is restricted to the LDLR. How this specificity is conferred remains unclear. Conceivably, interaction with another scaffolding protein that may stabilize the complex through interaction with ARH and the LDLR tail may be involved.
      Since ARH profoundly affects the internalization of the LDLR, we tested whether the LDLR was required for recruitment of ARH to specific vesicular compartments. ARH associates with plasma membrane sheets independently of the LDLR, possibly by binding to phosphatidylinositol 4,5-bisphosphate on the inner leaflet of the plasma membrane (
      • Mishra S.K.
      • Watkins S.C.
      • Traub L.M.
      ). ARH has also been shown to interact in vitro and colocalize in vivo with AP-2 and clathrin (
      • He G.
      • Gupta S.
      • Yi M.
      • Michaely P.
      • Hobbs H.H.
      • Cohen J.C.
      ,
      • Mishra S.K.
      • Watkins S.C.
      • Traub L.M.
      ). These proteins may also be involved in recruitment of ARH to the plasma membrane in vivo. The majority of ARH, however, appears in fractions from which the LDLR is absent, co-sedimenting with rab5 and overlapping with EEA1-containing fractions. Rab5 is involved in endocytic internalization and early endosome fusion (
      • Somsel Rodman J.
      • Wandinger-Ness A.
      ). Previously, ARH has been shown to associate with the LDLR during the early stages of endocytosis prior to entry to early endosomes (
      • Mishra S.K.
      • Watkins S.C.
      • Traub L.M.
      ), consistent with the co-sedementation of ARH with components of the early endocytic pathway.
      Although the genetic and metabolic studies in humans have revealed the clinical importance of ARH and have also provided initial glimpses into the biochemical mechanisms in which ARH is involved, direct experimental cell biological studies have been restricted to peripheral tissues, such as fibroblasts and lymphoblasts. Although ARH is clearly required for LDLR internalization, it is not yet clear whether ARH operates in the recruitment of the receptor to clathrin-coated pits or whether it is required to maintain the LDLR in the pits during invagination and endocytosis. The Arh–/– mouse now makes it possible to investigate the molecular basis for this defect, which is responsible for the clinical ARH phenotype in humans, in a model organism that is physiologically comparable with humans.

      Acknowledgments

      We thank J. Hayes, Richard Gibson, and Wen Ling Niu for excellent technical assistance.

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