ARH Is a Modular Adaptor Protein That Interacts with the LDL Receptor, Clathrin, and AP-2

Mutations in the phosphotyrosine binding domain protein ARH cause autosomal recessive hypercholesterolemia, a disorder caused by defective internalization of low density lipoprotein receptors (LDLR) in the liver. To examine the function of ARH, we used pull-down experiments to test for interactions between ARH, the LDLR, and proteins involved in clathrin-mediated endocytosis. The phosphotyrosine binding domain of ARH interacted with the internalization sequence (NPVY) in the cytoplasmic tail of LDLR in a sequence-specific manner. Mutations in the NPVY sequence that were previously shown to decrease LDLR internalization abolished in vitro binding to ARH. Recombinant ARH bound purified bovine clathrin with high affinity (K D, ∼44 nm). The interaction between ARH and clathrin was mapped to a canonical clathrin box sequence (LLDLE) in ARH and to the N-terminal domain of the clathrin heavy chain. A highly conserved 20-amino acid sequence in the C-terminal region of ARH bound the β2-adaptin subunit of AP-2. Mutation of a glutamic acid residue in the appendage domain of β2-adaptin that is required for interaction with the adapter protein β-arrestin markedly reduced binding to ARH. These data are consistent with the hypothesis that ARH functions as an adaptor protein that couples LDLR to the endocytic machinery. Mutations in the phosphotyrosine binding domain protein ARH cause autosomal recessive hypercholesterolemia, a disorder caused by defective internalization of low density lipoprotein receptors (LDLR) in the liver. To examine the function of ARH, we used pull-down experiments to test for interactions between ARH, the LDLR, and proteins involved in clathrin-mediated endocytosis. The phosphotyrosine binding domain of ARH interacted with the internalization sequence (NPVY) in the cytoplasmic tail of LDLR in a sequence-specific manner. Mutations in the NPVY sequence that were previously shown to decrease LDLR internalization abolished in vitro binding to ARH. Recombinant ARH bound purified bovine clathrin with high affinity (K D, ∼44 nm). The interaction between ARH and clathrin was mapped to a canonical clathrin box sequence (LLDLE) in ARH and to the N-terminal domain of the clathrin heavy chain. A highly conserved 20-amino acid sequence in the C-terminal region of ARH bound the β2-adaptin subunit of AP-2. Mutation of a glutamic acid residue in the appendage domain of β2-adaptin that is required for interaction with the adapter protein β-arrestin markedly reduced binding to ARH. These data are consistent with the hypothesis that ARH functions as an adaptor protein that couples LDLR to the endocytic machinery. Approximately 70% of cholesterol in the circulation is transported in low density lipoproteins (LDL). 1The abbreviations used are: LDL, low density lipoprotein; APP, amyloid precursor protein; ARH, autosomal recessive hypercholesterolemia; FH, familial hypercholesterolemia; GST, glutathione S-transferase; LDLR, LDL receptor; Ni-NTA, nickel nitrilotriacetic acid; PTB, phosphotyrosine binding; PVDF, polyvinylidene difluoride 1The abbreviations used are: LDL, low density lipoprotein; APP, amyloid precursor protein; ARH, autosomal recessive hypercholesterolemia; FH, familial hypercholesterolemia; GST, glutathione S-transferase; LDLR, LDL receptor; Ni-NTA, nickel nitrilotriacetic acid; PTB, phosphotyrosine binding; PVDF, polyvinylidene difluorideLDL receptor (LDLR)-mediated endocytosis in the liver is the primary pathway for the clearance of circulating LDL (1Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. 8th Ed. The Metabolic and Molecular Bases of Inherited Disease. II. McGraw Hill Inc., New York2001: 2863-2913Google Scholar). Reductions in hepatic LDLR activity result in elevated plasma LDL-cholesterol concentrations and the deposition of cholesterol in body tissues (1Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. 8th Ed. The Metabolic and Molecular Bases of Inherited Disease. II. McGraw Hill Inc., New York2001: 2863-2913Google Scholar). The critical importance of the LDLR pathway in preventing accumulation of LDL is illustrated by the very high plasma levels of LDL and premature atherosclerosis that characterize the following three Mendelian disorders in which LDLR-mediated endocytosis is disrupted: familial hypercholesterolemia (FH), familial defective apolipoprotein B100, and autosomal recessive hypercholesterolemia (ARH). Mutations inLDLR cause FH, the most common single gene disorder of LDL metabolism (1Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. 8th Ed. The Metabolic and Molecular Bases of Inherited Disease. II. McGraw Hill Inc., New York2001: 2863-2913Google Scholar). In FH homozygotes both LDLR alleles are defective, and the clearance of LDL from the circulation is markedly reduced resulting in a 6-fold increase in plasma LDL-cholesterol levels. Fibroblasts and lymphocytes cultured from patients with homozygous FH fail to take up and degrade LDL (1Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. 8th Ed. The Metabolic and Molecular Bases of Inherited Disease. II. McGraw Hill Inc., New York2001: 2863-2913Google Scholar). Familial defective apolipoprotein B100 is caused by a missense mutation (R3500Q) in apolipoprotein B100 that impairs binding of the LDL particle to the LDLR (2Innerarity T.L. Mahley R.W. Weisgraber K.H. Bersot T.P. Krauss R.M. Vega G.L. Grundy S.M. Friedl W. Davignon J. McCarthy B.J. J. Lipid Res. 1990; 31: 1337-1349Abstract Full Text PDF PubMed Google Scholar). Recently, an autosomal recessive form of hypercholesterolemia that is not due to sequence variations in either the LDLR or apolipoprotein B genes was identified (3Zuliani 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. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 802-809Crossref PubMed Scopus (85) Google Scholar). The rate at which LDL particles are cleared from the plasma by the liver of ARH patients is as low as that in patients with no functional LDLRs (3Zuliani 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. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 802-809Crossref PubMed Scopus (85) Google Scholar). This finding indicates that LDLR function is markedly impaired in the hepatocytes of ARH patients. Immortalized lymphoblasts from ARH patients have more LDLRs on the plasma membrane; however, despite increased cell surface binding of LDL, LDL degradation is decreased by ∼75% compared with lymphoblasts from normal individuals (5Norman D. Sun X.M. Bourbon M. Knight B.L. Naoumova R.P. Soutar A.K. J. Clin. Invest. 1999; 104: 619-628Crossref PubMed Scopus (73) Google Scholar). In contrast, LDLR function is normal or only moderately reduced in fibroblasts from patients with this disorder (3Zuliani 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. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 802-809Crossref PubMed Scopus (85) Google Scholar, 4Arca 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. Lancet. 2002; 359: 841-847Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Autosomal recessive hypercholesterolemia is caused by mutations in a gene that encodes a phosphotyrosine binding (PTB)-domain protein called ARH (6Garcia 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. Science. 2001; 292: 1394-1398Crossref PubMed Scopus (467) Google Scholar). PTB domains are found in several adaptor proteins that link proteins into functional complexes to orchestrate transmembrane signaling and endocytosis (7Guy G.R. Yusoff P. Bangarusamy D. Fong C.W. Wong E.S. Cell. Signal. 2002; 14: 11-20Crossref PubMed Scopus (23) Google Scholar). The PTB domains of adaptor proteins such as SHC, IRS-1, X11, Dab-1, and Dab-2 bind a canonical sequence motif (NPXY) in the cytoplasmic tails of various cell surface receptors (8Forman-Kay J.D. Pawson T. Curr. Opin. Struct. Biol. 1999; 9: 690-695Crossref PubMed Scopus (107) Google Scholar). The cytoplasmic tail of the LDLR contains an NPVY sequence that is required for LDLR-mediated endocytosis (9Davis C.G. van Driel I.R. Russell D.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1987; 262: 4075-4082Abstract Full Text PDF PubMed Google Scholar, 10Chen W.-J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1990; 265: 3116-3123Abstract Full Text PDF PubMed Google Scholar). Accordingly, we proposed that ARH functions as an adaptor protein that binds to the NPVY sequence in LDLR and couples the receptor to the endocytic machinery (6Garcia 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. Science. 2001; 292: 1394-1398Crossref PubMed Scopus (467) Google Scholar). In fibroblasts, LDLRs cluster on the cell surface in clathrin-coated pits that invaginate and detach from the plasma membrane as coated vesicles (1Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. 8th Ed. The Metabolic and Molecular Bases of Inherited Disease. II. McGraw Hill Inc., New York2001: 2863-2913Google Scholar). A naturally occurring mutation that changes the tyrosine of the NPVY motif to cysteine (Y807C) prevents LDLRs from clustering; receptors in cells from this patient are distributed diffusely over the cell surface (11Davis C.G. Lehrman M.A. Russell D.W. Anderson R.G.W. Brown M.S. Goldstein J.L. Cell. 1986; 45: 15-24Abstract Full Text PDF PubMed Scopus (241) Google Scholar). Systematic mutagenesis of the amino acids flanking the Tyr-807 in the cytoplasmic tail of the LDLR indicates that the sequence FDNPVY functions as the internalization signal (9Davis C.G. van Driel I.R. Russell D.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1987; 262: 4075-4082Abstract Full Text PDF PubMed Google Scholar, 10Chen W.-J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1990; 265: 3116-3123Abstract Full Text PDF PubMed Google Scholar). Nuclear magnetic resonance studies reveal that peptides containing the NPVY sequence of LDLR adopt a reverse turn conformation, with the asparagine in position i and the tyrosine in position i + 3 of the turn (12Bansal A. Gierasch L.M. Cell. 1991; 67: 1195-1201Abstract Full Text PDF PubMed Scopus (185) Google Scholar). In contrast, peptides derived from receptors that are defective in internalization do not adopt the reverse turn (12Bansal A. Gierasch L.M. Cell. 1991; 67: 1195-1201Abstract Full Text PDF PubMed Scopus (185) Google Scholar). These findings suggest that the NPVY sequence plays a conformational role that is critical for endocytosis of the LDLR, presumably by mediating binding to adaptor proteins. The identities of the proteins that bind the FDNPVY sequence in the LDLR tail are not known. The adaptor protein AP-2 has been shown to bind other receptors that are internalized via clathrin-coated pits, but studies using plasmon resonance to test for direct interactions between AP-2 and the FXNPXY motif of the LDLR tail have yielded contradictory results (13Boll W. Ohno H. Songyang Z. Rapoport I. Cantley L.C. Bonifacino J.S. Kirchhausen T. EMBO J. 1996; 15: 5789-5795Crossref PubMed Scopus (236) Google Scholar, 14Boll W. Rapoport I. Brunner C. Modis Y. Prehn S. Kirchhausen T. Traffic. 2002; 3: 590-600Crossref PubMed Scopus (66) Google Scholar). Peptides containing the sequence FDNPVY will bind the terminal domain of clathrin in vitro (15Kibbey R.G. Rizo J. Gierasch L.M. Anderson R.G. J. Cell Biol. 1998; PubMed Scopus Google but is not this interaction is to internalization of the In this we ARH with the LDLR tail and with the of clathrin-coated pits, clathrin and AP-2. data that conserved in ARH direct interactions between ARH and the LDLR, ARH and the terminal domain of the clathrin heavy and ARH and the β2-adaptin subunit of AP-2. These findings are consistent with the hypothesis that ARH is a adaptor protein that couples LDLR to the endocytic machinery. A was from A was from A the of the clathrin heavy was from were a to the C-terminal of the was from were from other and were from The sequence of the ARH protein was from the sequence previously (6Garcia 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. Science. 2001; 292: 1394-1398Crossref PubMed Scopus (467) Google Scholar). and sequence that sequence with ARH were from the data using from and was reverse and to the were used to the ARH of The protein from the were using the on the the ARH protein and a of ARH proteins ARH ARH ARH ARH ARH ARH and ARH were using a at the and a by an at the were using the in with of and of The were and and into the and with a sequence The were used to A containing amino acids of the bovine clathrin N-terminal domain to was by of the into the and of the 1991; PubMed Scopus Google Scholar). A containing the cytoplasmic tail of the LDLR to was from of containing of the β2-adaptin subunit of AP-2 to and were from mutations were into the and tail using a mutagenesis were by of the used for are of containing the ARH and the were into of with or and at for in a was by for and for the and were for at by and in of binding for the or in for were using was by and the were with binding or with on a at The were three with either the binding or The were in of and for at to the proteins. The and of the proteins was by and of containing of the protein were with an of protein in of binding and and for on a at were by for and the were by and were three with binding in of and and at for of the and from the were on to with in and using clathrin or LDLR was purified from bovine P. A. Anderson R.G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google and with using the by the A of of ARH bound to or were clathrin in A and bovine were at for on an and clathrin were by for of the a of A were in binding was using and from the of the The and proteins that and sequence with ARH, of the amino acids were in proteins. of sequence were the N-terminal amino the PTB domain to a acid sequence to the clathrin box motif of other proteins Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google and a acid sequence to of ARH. PTB domains in several SHC, and Dab-1, bind receptor tails containing the motif (8Forman-Kay J.D. Pawson T. Curr. Opin. Struct. Biol. 1999; 9: 690-695Crossref PubMed Scopus (107) Google Scholar). To the PTB domain of ARH binds the LDLR, pull-down experiments were using ARH peptides and a protein of the acid cytoplasmic tail of the LDLR to ARH and a containing the PTB domain bound the LDLR tail In contrast, no interaction was between the LDLR tail and an ARH the PTB The of interaction of ARH with the LDLR tail was by on the of the PTB domain of bound to a from the amyloid precursor protein Z. J. J. EMBO J. PubMed Scopus Google Scholar). In the of the with the PTB form a that is to the of the PTB domain by and and in are to to and in ARH. the sequence in the with a by the that the and and the and of the PTB The of the ARH PTB domain with the primary sequence of not A of the PTB domain of ARH on the of the domain of is shown in The mutation of in ARH to either or a protein that to bind the LDLR cytoplasmic tail To the tyrosine of the NPVY sequence in the LDLR tail is required for interaction with the PTB domain of ARH, this residue was to either a cysteine or an These were shown previously to LDLR internalization in cells (9Davis C.G. van Driel I.R. Russell D.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1987; 262: 4075-4082Abstract Full Text PDF PubMed Google Scholar). mutations markedly reduced the binding of the LDLR cytoplasmic tail to ARH In contrast, the of tyrosine with no on the interaction between ARH and the LDLR tail LDLR was previously shown to LDL in cells (9Davis C.G. van Driel I.R. Russell D.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1987; 262: 4075-4082Abstract Full Text PDF PubMed Google Scholar). These data suggest that the interaction between ARH and the LDLR tail an residue at position and that of this residue is not required for of ARH to the LDLR cytoplasmic tail an amino acid in the residue of the NPVY cytoplasmic tail proteins were with to as The were and in The were on and tail proteins and the peptides were by as in the to of ARH (LLDLE) to the clathrin box motif that binding of various proteins to the terminal domain of the clathrin heavy Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). To test for interaction between ARH and we pull-down experiments using ARH and a protein of and the terminal domain of clathrin ARH and ARH bound the clathrin terminal domain of the ARH protein the clathrin box or of the of the clathrin box with abolished binding to of both the acid and glutamic acid with abolished To the affinity of the interaction between ARH and was on and with concentrations of clathrin purified from bovine that ARH bound clathrin with high affinity ∼44 nm). These data that the clathrin box motif of ARH binding to the N-terminal domain of the clathrin heavy and that ARH binds clathrin with high ARH binds bovine clathrin with high bovine clathrin P. A. Anderson R.G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google was with on as The binding the of bound clathrin at The shown in the indicates that the has a of The which the internalization of bind to both clathrin and to a of and J. M.S. 1998; PubMed Scopus Google Scholar). studies that both β-arrestin J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and J.L. J. Biol. Chem. 2002; Google bind to the appendage domain and of β2-adaptin and that mutation of either of glutamic acids and in this sequence To ARH with we pull-down experiments using ARH and proteins ARH bound of β2-adaptin but not to a containing the N-terminal of the Mutation of residue in β2-adaptin to abolished binding to ARH, mutation of residue reduced but not These data that ARH binds the domain on β2-adaptin that is bound by the and that this interaction at of the glutamic acid in the appendage domain of To the region of ARH required for interaction with pull-down experiments were using a of ARH proteins proteins that bound This region of ARH is highly conserved between and but no sequence with the β2-adaptin binding region in the not conserved and in this region of ARH to the in that with and of Mutation of not binding to mutation of to abolished The finding of this is that ARH binds to the LDLR cytoplasmic tail as as to of the clathrin-coated the heavy of and the β2-adaptin subunit of the adaptor protein AP-2. The of ARH that the interactions with three proteins are highly conserved and and in other proteins that binding to and (8Forman-Kay J.D. Pawson T. Curr. Opin. Struct. Biol. 1999; 9: 690-695Crossref PubMed Scopus (107) Google Scholar, Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. 2002; PubMed Scopus Google Scholar). Mutations in in the LDLR cytoplasmic tail that endocytosis binding to ARH. data the hypothesis that ARH functions as an adaptor protein that the LDLR to the endocytic machinery of the coated ARH has a of at of that The N-terminal of the protein of a highly conserved acid sequence by a residue PTB The PTB domains of several proteins bind containing the motif or to other that adopt a reverse turn (7Guy G.R. Yusoff P. Bangarusamy D. Fong C.W. Wong E.S. Cell. Signal. 2002; 14: 11-20Crossref PubMed Scopus (23) Google Scholar). In this we that the PTB domain of ARH binds the cytoplasmic tail of the LDLR in vitro and that the of the NPVY sequence is required for of the tyrosine in the NPVY motif with or cysteine markedly reduced the interaction between ARH and the LDLR the of at this position not the interaction between the proteins. These data suggest that the PTB domain of ARH binds to the LDLR cytoplasmic tail and that this binding an amino acid in the position of the NPVY PTB domains were for interaction with several PTB X11, and bind (8Forman-Kay J.D. Pawson T. Curr. Opin. Struct. Biol. 1999; 9: 690-695Crossref PubMed Scopus (107) Google Scholar). A high has been for the PTB domain of bound to a acid of the precursor protein that contains the sequence Z. J. J. EMBO J. PubMed Scopus Google Scholar). of the ARH PTB domain on the that the FDNPVY sequence of the LDLR cytoplasmic tail as an of the of the ARH PTB highly conserved and of the ARH PTB domain are to form a that the of the FDNPVY Mutation of in ARH to or abolished binding to the LDLR cytoplasmic This finding that the ARH PTB domain a binding to that in X11, SHC, and of the ARH sequence a highly conserved motif (LLDLE) that to the clathrin box sequence Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). box in several coated proteins binding to the heavy of clathrin Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). and studies have that clathrin box bind to in a between the of the N-terminal of the clathrin heavy Kirchhausen T. S. A. PubMed Scopus Google Scholar). This contains a that the of the of the clathrin box and a that the of the and Kirchhausen T. S. A. PubMed Scopus Google Scholar). and in the clathrin terminal domain with the residue of the clathrin box Kirchhausen T. S. A. PubMed Scopus Google Scholar). Recombinant ARH bound to a protein containing the N-terminal amino acids of the clathrin heavy chain. The clathrin box in ARH was required for clathrin binding; of the or the with abolished binding to the clathrin terminal ARH bound to purified clathrin with high affinity (K D, ∼44 with previously for the F. J.L. 1996; PubMed Scopus Google and AP-2 S. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). These data suggest that ARH contains a functional clathrin box sequence that in the or of LDLR to clathrin-coated pits by ARH. endocytic proteins bind to both clathrin and AP-2. domains on the of the AP-2 interactions with other proteins. The appendage domain of AP-2 binds to proteins containing the motif the subunit binds to proteins containing the internalization signal T. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The β2-adaptin subunit of AP-2 a domain and a appendage domain that are by a region Y. EMBO J. 19: PubMed Google Scholar). The region contains a clathrin box that the binding of AP-2 to clathrin Y. EMBO J. 19: PubMed Google Scholar). The appendage domain to as the contains the β-arrestin binding region J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). studies that glutamic acid and in the appendage domain with conserved in the J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J.L. J. Biol. Chem. 2002; Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). pull-down experiments that ARH with the appendage domain of β2-adaptin and that this interaction of the glutamic acid in β2-adaptin that binding to Mutation of the other glutamic acid residue decreased but not binding to ARH. The region of ARH that is required for binding to β2-adaptin is from the LDLR and clathrin binding domains and is highly conserved and A conserved in this region is required for binding to β2-adaptin and with in These results suggest that ARH bind to the region of AP-2 that is required for of receptors to clathrin-coated The finding that conserved in ARH interactions to in other endocytic adaptor proteins is consistent with (6Garcia 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. Science. 2001; 292: 1394-1398Crossref PubMed Scopus (467) Google and cell (5Norman D. Sun X.M. Bourbon M. Knight B.L. Naoumova R.P. Soutar A.K. J. Clin. Invest. 1999; 104: 619-628Crossref PubMed Scopus (73) Google studies that a role for ARH in LDLR endocytosis. the binding studies were with proteins in do not direct that ARH with the LDLR, and β2-adaptin in or that interaction with proteins is for ARH Recombinant β-arrestin binds to clathrin in vitro with an affinity to that for ARH, mutagenesis studies suggest that this binding not required for internalization of the β-arrestin receptor J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and β-arrestin have no in signaling L.M. K. Science. 1999; PubMed Scopus Google Scholar). studies will required to that ARH with the LDLR, and AP-2 in and that interactions are required for LDLR endocytosis. of the role of ARH in LDLR internalization has been by the of an cell in which LDLR function is in cultured fibroblasts, which critical in the FH, have been in ARH, LDL in fibroblasts from ARH patients is normal or only moderately impaired 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. Lancet. 2002; 359: 841-847Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). is not ARH is required for LDLR function in cell such as but not in is that adaptor protein the role of ARH in fibroblasts but not in the of LDLR internalization in hepatocytes and most LDLRs are in coated pits in fibroblasts but to on the plasma membrane in hepatocytes PubMed Scopus Google Scholar, M. Brown M.S. Goldstein J.L. Anderson R.G. J. Cell Biol. 1990; PubMed Scopus Google Scholar). ARH a function in hepatocytes that is not required in Recently, LDL degradation was shown to markedly reduced in lymphocytes from ARH patients (5Norman D. Sun X.M. Bourbon M. Knight B.L. Naoumova R.P. Soutar A.K. J. Clin. Invest. 1999; 104: 619-628Crossref PubMed Scopus (73) Google that ARH is required for LDLR-mediated endocytosis in lymphocytes from ARH patients a cell in which to the by which ARH LDLR-mediated endocytosis. and for