The Modular Adaptor Protein Autosomal Recessive Hypercholesterolemia (ARH) Promotes Low Density Lipoprotein Receptor Clustering into Clathrin-coated Pits

Autosomal recessive hypercholesterolemia is characterized by a cell type-specific defect in low density lipoprotein receptor (LDLR) endocytosis. LDLR-mediated uptake of LDL is impaired in the liver, but not in fibroblasts of subjects with this disorder. The disease is caused by mutations in ARH, which encodes a putative adaptor protein that interacts with the cytoplasmic tail of the LDLR, phospholipids, and two components of the clathrin endocytic machinery, clathrin and adaptor protein-2 (AP-2) in vitro. To determine the physiological relevance of these interactions, we examined the effect of mutations in the ARH on LDLR location and function in polarized hepatocytes (WIF-B). The integrity of the FDNPVY sequence in the LDLR cytoplasmic tail was required for ARH-associated LDLR clustering into clathrin-coated pits. The phosphotyrosine binding domain of ARH plus either the clathrin box or the AP-2 binding region were required for both clustering and internalization of the LDLR. Parallel studies performed in vivo with the same recombinant forms of ARH in livers of Arh-/- mice confirmed the relevance of the cell culture findings. These results demonstrate that ARH must bind the LDLR tail and either clathrin or AP-2 to promote receptor clustering and internalization of LDL. Autosomal recessive hypercholesterolemia is characterized by a cell type-specific defect in low density lipoprotein receptor (LDLR) endocytosis. LDLR-mediated uptake of LDL is impaired in the liver, but not in fibroblasts of subjects with this disorder. The disease is caused by mutations in ARH, which encodes a putative adaptor protein that interacts with the cytoplasmic tail of the LDLR, phospholipids, and two components of the clathrin endocytic machinery, clathrin and adaptor protein-2 (AP-2) in vitro. To determine the physiological relevance of these interactions, we examined the effect of mutations in the ARH on LDLR location and function in polarized hepatocytes (WIF-B). The integrity of the FDNPVY sequence in the LDLR cytoplasmic tail was required for ARH-associated LDLR clustering into clathrin-coated pits. The phosphotyrosine binding domain of ARH plus either the clathrin box or the AP-2 binding region were required for both clustering and internalization of the LDLR. Parallel studies performed in vivo with the same recombinant forms of ARH in livers of Arh-/- mice confirmed the relevance of the cell culture findings. These results demonstrate that ARH must bind the LDLR tail and either clathrin or AP-2 to promote receptor clustering and internalization of LDL. Approximately 70% of plasma cholesterol circulates as a constituent of human low density lipoproteins (LDL). 3The abbreviations used are: LDLlow density lipoproteinARHautosomal recessive hypercholesterolemiaCCPclathrin-coated pitLDLRlow density lipoprotein receptorPTBphosphotyrosine bindingPBSphosphate-buffered salineBSAbovine serum albuminPFAparaformaldehydemAbmonoclonal antibodyFHfamilial hypercholesterolemiaFDBfamilial defective apolipoprotein B-100. The primary route of clearance of LDL from the plasma is by LDL receptor (LDLR)-mediated endocytosis in the liver. The critical role of the LDLR pathway is illustrated by the very high plasma levels of LDL that characterize three genetic disorders in which LDLR-mediated endocytosis is disrupted: familial hypercholesterolemia (FH), familial defective apolipoprotein B-100 (FDB), and autosomal recessive hypercholesterolemia. FH is an autosomal co-dominant disorder caused by mutations in the LDLR gene (1Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw Hill, New York2001: 2683-2913Google Scholar). Individuals with homozygous FH inherit two defective LDLR alleles and have markedly reduced LDL clearance, resulting in a ∼6-fold increase in plasma LDL-cholesterol levels. Fibroblasts and lymphocytes cultured from patients with homozygous FH also fail to internalize and degrade LDL. FDB is an autosomal dominant phenocopy of FH caused by missense mutations in apolipoprotein B-100 that impair binding of LDL 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). Autosomal recessive hypercholesterolemia is caused by mutations in ARH, a putative adaptor protein that is required for normal clearance of LDL from the circulation (3Garcia 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 (490) Google Scholar). Subjects with autosomal recessive hypercholesterolemia, like those with FH and FDB, are hypercholesterolemic and have markedly reduced rates of clearance of circulating LDL (4Harada-Shiba M. Tajima S. Yokoyama S. Miyake Y. Kojima S. Tsushima M. Kawakami M. Yamamoto A. Arterioscler. Thromb. 1992; 12: 1071-1078Crossref PubMed Scopus (42) Google Scholar, 5Zuliani 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 (86) Google Scholar); however, unlike FH, the defect in LDLR endocytosis is cell type-specific. Although LDL degradation is reduced in the liver (5Zuliani 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 (86) Google Scholar) and in immortalized lymphocytes (6Norman D. Sun X.M. Bourbon M. Knight B.L. Naoumova R.P. Soutar A.K. J. Clin. Investig. 1999; 104: 619-628Crossref PubMed Scopus (74) Google Scholar, 7Wilund K.R. Yi M. Campagna F. Arca M. Zuiliani G. Fellin R. Ho Y.-K. Hobbs H.H. Cohen J.C. Hum. Mol. Genet. 2002; 11: 3019-3030Crossref PubMed Scopus (95) Google Scholar), cultured fibroblasts from patients with autosomal recessive hypercholesterolemia internalize and degrade LDL at normal or only modestly reduced rates (8Zuliani G. Vigna G.B. Corsini A. Maioli M. Romagnoni F. Fellin R. Eur. J. Clin. Investig. 1995; 25: 322-331Crossref PubMed Scopus (49) Google Scholar). low density lipoprotein autosomal recessive hypercholesterolemia clathrin-coated pit low density lipoprotein receptor phosphotyrosine binding phosphate-buffered saline bovine serum albumin paraformaldehyde monoclonal antibody familial hypercholesterolemia familial defective apolipoprotein B-100. of the are in Arh-/- mice the of are on the C. Cohen J.C. Hobbs H.H. J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). lymphocytes of subjects with ARH, a of to the plasma resulting in a increase in cell receptor Cohen J.C. Hobbs H.H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). These that ARH is required for the normal of LDLR from the cell to is that ARH to LDLR to clathrin-coated pits. ARH the of the LDLR as a to or a role in the of endocytosis Cohen J.C. Hobbs H.H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. 2002; PubMed Scopus Google Scholar, Sun X.M. M. M. 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The results that the the clathrin and the AP-2 binding of ARH in a to promote the of LDLR to clathrin-coated and the clearance of circulating LDL by the liver. cell culture used in these studies were from The antibody used for and was from and the monoclonal antibody to the LDLR was from Brown J. Biol. Full Text PDF PubMed Google Scholar). were used in these an antibody the in ARH, which not or ARH K.R. Yi M. Campagna F. Arca M. Zuiliani G. Fellin R. Ho Y.-K. Hobbs H.H. Cohen J.C. Hum. Mol. Genet. 2002; 11: 3019-3030Crossref PubMed Scopus (95) Google Scholar), and an antibody the recombinant human protein that with The monoclonal was from and monoclonal was by and were from Molecular and were from were paraformaldehyde from were an from Science. a cell human fibroblasts and hepatocytes D. C. M. J. Biol. PubMed Scopus Google Scholar); the were a from were cultured at with in with bovine serum and as G. D. M. J. Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar) were on a of and The mice were on a and on and To hypercholesterolemia, the Arh-/- mice were normal with cholesterol and for the mice were by of and of was was into the as a in of J. S. A. PubMed Scopus Google Scholar). from mice were on the cholesterol the were ARH from the recombinant was to LDLR endocytosis in the Arh-/- mice of was to the were from the for of plasma cholesterol levels. The livers were and of ARH was performed on to ARH were by the of and and were performed to and of LDLR and are an immortalized cell from the of hepatocytes with human fibroblasts D. C. M. J. Biol. PubMed Scopus Google Scholar). The in a for the of protein G. D. M. J. Biol. PubMed Scopus Google Scholar) the LDLR 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). and are not in this cell D. C. M. J. Biol. PubMed Scopus Google Scholar) these not human LDLR or To determine the cell location of the LDLR in polarized we performed in an antibody that interacts with the LDLR. The were and with the of AP-2 and the of clathrin The plasma LDLR was on the and only with AP-2 or with To determine the location of ARH in were with to ARH and to either AP-2 or three was in the and with the The of ARH on the plasma with AP-2 and that the ARH with the plasma was not to clathrin-coated in these of LDLR and ARH in the for the ARH and the LDLR, we with a recombinant the human LDLR The were with a monoclonal LDLR antibody The were and with to and ARH, and for The human LDLR was only on the with an protein was which is with 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). was the the of and ARH were the of the recombinant LDLR on the cell that of the LDLR protein of ARH the of AP-2 or of the adaptor and AP-2 S.M. 2001; PubMed Scopus Google Scholar, C. G. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). to these we not in the of AP-2 or clathrin with the of recombinant human ARH in The recombinant ARH was cytoplasmic but was also at the plasma The recombinant ARH to with AP-2 and of ARH in of we examined the effect of ARH on the cell of the LDLR. were with or an in the cell or of the LDLR were not we examined the LDLR in both human ARH and LDLR by the with and either or of LDLR with the in LDLR on the of ARH with LDLR in that ARH clustering of LDLR The FDNPVY in the cytoplasmic tail of the LDLR a role in LDLR internalization Brown J. Biol. Full Text PDF PubMed Google Scholar, Brown J. Biol. 1990; Full Text PDF PubMed Google Scholar) and to recombinant ARH in G. S. Hobbs H.H. Cohen J.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). To determine the integrity of the FDNPVY is required for cell clustering of the LDLR in to ARH we with a of the LDLR that ARH and LDLR in G. S. Hobbs H.H. Cohen J.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of ARH LDLR was on the cell in either the LDLR or of ARH in clustering of the LDLR in LDLR but not in the The of ARH and LDLR was in the two of the of ARH to LDLR clustering in the required the integrity of the FDNPVY internalization sequence in the LDLR cytoplasmic of ARH of the LDLR in and LDLR not to determine the of in the ARH and LDLR clustering of LDLR in pits. we performed to the LDLR on the cell with and the of LDLR an the plasma with of the the clathrin-coated to these the into in LDLR and The of on the cell a increase in the of of pit the of ARH in a of LDLR from the pit of the plasma to pits. The of of plasma was reduced in both ARH and the LDLR. These results are with ARH internalization of LDLR, of ARH not the of LDLR in of on the of is the of and pit pit is the of and in a To that the LDLR clustering by of ARH in internalization of we the of LDLR and to those LDLR and the LDLR and the were on the cell in ARH, a of the LDLR was we examined the effect of ARH on the LDLR but ARH, the were on the cell The was the ARH and LDLR ARH in the clustering of LDLR in and LDL of in recombinant or and were in and with and either an or and with for at were and for of ARH to LDLR studies ARH that with the LDLR, the domain of the of and a of AP-2 S. A. 2002; PubMed Scopus Google Scholar, G. S. Hobbs H.H. Cohen J.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). To determine these are required for clustering and internalization of LDLR in we a of recombinant ARH that the of ARH to bind LDLR, and AP-2 the effect of for at in the domain of ARH, that binding of ARH to the LDLR in G. S. Hobbs H.H. Cohen J.C. J. 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Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of either the clathrin box or the AP-2 not of the LDLR in clathrin-coated pits. we recombinant ARH with mutations in both the clathrin box and in the AP-2 binding in LDLR clustering was the integrity of either the clathrin box or the AP-2 binding region is to but of both clustering of the LDLR. of ARH to LDLR determine the ARH required for LDLR clustering were also required for receptor we polarized with recombinant and forms of LDLR and were by the with the at and the were to to receptor LDLR, the on the cell were in both LDLR and of ARH with the LDLR was and ARH to the ARH protein that to LDLR clustering also to receptor The forms of ARH that receptor clustering also internalization These are with clustering of required for receptor internalization in and that both clustering and internalization the at in the cytoplasmic tail of the LDLR. of of ARH in the role of the ARH on LDLR function in we used recombinant to ARH in Arh-/- mice C. Cohen J.C. Hobbs H.H. J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Arh-/- mice are only hypercholesterolemic C. Cohen J.C. Hobbs H.H. J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), the mice were a high cholesterol for resulting in a increase in plasma cholesterol of ARH in these mice was with a in circulating levels of plasma in cholesterol levels in mice a and we the same of recombinant ARH used for the studies in the of recombinant ARH either the or the mutations in the domain to of a effect the plasma of to these recombinant ARH with mutations in the clathrin box or in the AP-2 binding plasma cholesterol to levels to those of mice with the ARH in cholesterol was in with of the recombinant an ARH both mutations and with the of the which ARH function in vivo but not in the of ARH on plasma cholesterol levels in mice those in of this is that ARH clustering of LDLR into clathrin-coated on the of the of ARH, were on the of polarized ARH and LDLR were in these the LDLR in pits. LDLR clustering was by an increase in receptor The in the of LDLR required a internalization sequence in the cytoplasmic tail of the LDLR and an domain with either not a clathrin box or an AP-2 binding in The for ARH function in hepatocytes were for the by studies in Arh-/- The hypercholesterolemia of Arh-/- mice was by of ARH, and the binding required for receptor clustering and internalization in were also required to lipoproteins in these that ARH the clustering of on the cell of hepatocytes by the cytoplasmic tail of the receptor to components of the clathrin-coated pit and and that clustering of the receptor is required for clearance of LDL by the liver. of recombinant LDLR in the cell of of the in these LDLR were on the as 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and a to with AP-2 or clathrin confirmed that of the were of which is to the of LDLR in the livers of mice a human LDLR M. Brown J. Biol. 1990; PubMed Scopus Google Scholar). The of of recombinant ARH in also to that of the of recombinant ARH in not the of LDLR, clathrin or but markedly the of recombinant LDLR and of ARH with LDLR in the in clustering of into as by These that ARH in to promote clustering of recombinant either the of was or of ARH and the recombinant human LDLR. studies have that high of as ARH and have a dominant effect on LDLR clustering and internalization in S. A. 2002; PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of ARH in not with LDLR clustering or The results and studies caused by the in cell used or to in the levels of of ARH and LDLR. cell LDLR are in as in fibroblasts or cell as adaptor like or with internalization of M. Brown J. Biol. 1990; PubMed Scopus Google Scholar, S.M. 2001; PubMed Scopus Google Scholar) and high levels of ARH these cell as of the ARH required to the with of pits. of the in this we were to the sequence of the ARH, the LDLR and in the pits. ARH promote the clustering of LDLR in either by the of to or by in to studies in that at on the F. Biol. 1999; PubMed Scopus Google Scholar). from studies in is with adaptor as to A. S. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). studies an in which clathrin at on the cell but only to adaptor and are M. W. A. R. K. Full Text Full Text PDF PubMed Scopus Google Scholar). cell of clathrin that are and to and M. W. A. R. K. Full Text Full Text PDF PubMed Scopus Google Scholar). The of that with the LDLR, to M. Mol. Biol. 1990; PubMed Scopus Google Scholar). ARH the LDLR in the and and of the receptor to pit endocytosis. The integrity of the FDNPVY sequence in the cytoplasmic tail of the LDLR was required for clustering and internalization of the LDLR in of the in the FDNPVY with which markedly reduced the ARH and the LDLR tail in S. A. 2002; PubMed Scopus Google Scholar, G. S. Hobbs H.H. Cohen J.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), also in a of ARH to promote clustering of in the that the domain of ARH and the internalization sequence in the LDLR is required for receptor clustering in but to ARH in the mice was on of the domain of ARH the of which also G. S. Hobbs H.H. Cohen J.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). on this we that two and a that the of the FDNPVY sequence G. S. Hobbs H.H. Cohen J.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of or at the ARH and the LDLR tail in G. S. Hobbs H.H. Cohen J.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) but not clustering and internalization of the LDLR in high of of in for the reduced of this for the LDLR and LDLR The of the to internalization not that the domain is not required of for at in the domain of ARH clustering and internalization of the LDLR in and in livers of Arh-/- clustering of LDLR into clathrin-coated to the domain was with at of two protein in the of the the clathrin box or the AP-2 binding of either the clathrin box or the AP-2 binding not of the LDLR with pits. that clustering of LDLR in clathrin-coated is not on to binding in the the of binding in ARH clustering of LDLR with components of the endocytic These are with the of and Y. J. PubMed Scopus Google Scholar) that the and internalization of clathrin-coated a of with components of the of which with The low of the components to but also the pit to in on the of The results of the the of ARH to LDLR endocytosis in studies have that ARH is for normal LDLR endocytosis in but not in fibroblasts (6Norman D. Sun X.M. Bourbon M. Knight B.L. Naoumova R.P. Soutar A.K. J. Clin. Investig. 1999; 104: 619-628Crossref PubMed Scopus (74) Google Scholar, 7Wilund K.R. Yi M. Campagna F. Arca M. Zuiliani G. Fellin R. Ho Y.-K. Hobbs H.H. Cohen J.C. Hum. Mol. Genet. 2002; 11: 3019-3030Crossref PubMed Scopus (95) Google Scholar). These in the of LDLR in cell are on the cell in clathrin-coated (1Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw Hill, New York2001: 2683-2913Google Scholar), that internalization of from clathrin-coated to is the in LDLR in these LDLR endocytosis is not impaired in fibroblasts (6Norman D. Sun X.M. Bourbon M. Knight B.L. Naoumova R.P. Soutar A.K. J. Clin. Investig. 1999; 104: 619-628Crossref PubMed Scopus (74) Google Scholar, 7Wilund K.R. Yi M. Campagna F. Arca M. Zuiliani G. Fellin R. Ho Y.-K. Hobbs H.H. Cohen J.C. Hum. Mol. Genet. 2002; 11: 3019-3030Crossref PubMed Scopus (95) Google Scholar, G. Vigna G.B. Corsini A. Maioli M. Romagnoni F. Fellin R. Eur. J. Clin. Investig. 1995; 25: 322-331Crossref PubMed Scopus (49) Google Scholar) that ARH is not required for of from to the of in lymphocytes are in Cohen J.C. Hobbs H.H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. 2002; PubMed Scopus Google Scholar, Sun X.M. M. M. C. Naoumova R.P. Soutar A.K. J. Clin. Investig. 2002; PubMed Scopus Google Scholar). the of from the to the cell to in on the plasma of Cohen J.C. Hobbs H.H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), which is to we in to in the density of in pit is in normal and in lymphocytes Cohen J.C. Hobbs H.H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). These in the rates at which are from to and from to ARH promote of from to in both hepatocytes and the of ARH, the on the plasma in of to clathrin-coated by These to for ARH in but not in of ARH and the LDLR in the not only in LDL clustering but also in a of the LDLR from the cell to we have to a in ARH or in the LDLR that receptor clustering from receptor we determine the increase in LDLR internalization is a of receptor clustering in clathrin-coated or ARH also a effect on the internalization of the receptor in and for to and for and for of the used in the to for the