The Adaptor Protein β-Arrestin2 Enhances Endocytosis of the Low Density Lipoprotein Receptor
2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês
10.1074/jbc.m309450200
ISSN1083-351X
AutoresJiao‐Hui Wu, Karsten Peppel, Christopher Nelson, Fang‐Tsyr Lin, Trudy A. Kohout, William E. Miller, Sabrina T. Exum, Neil J. Freedman,
Tópico(s)Advanced Proteomics Techniques and Applications
ResumoEndocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor β-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of β-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether β-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, β-arrestin1 and β-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by ∼70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only β-arrestin2, and not β-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected β-arrestin1/β-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of β-arrestin2, but not β-arrestin1, LDLR endocytosis was reduced. Moreover, β-arrestin2–/– MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, β-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to β-arrestin2, and the efficiency of LDLR endocytosis in cells expressing β-arrestin2 physiologically. We conclude that β-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism. Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor β-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of β-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether β-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, β-arrestin1 and β-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by ∼70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only β-arrestin2, and not β-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected β-arrestin1/β-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of β-arrestin2, but not β-arrestin1, LDLR endocytosis was reduced. Moreover, β-arrestin2–/– MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, β-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to β-arrestin2, and the efficiency of LDLR endocytosis in cells expressing β-arrestin2 physiologically. We conclude that β-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism. In human beings, supraphysiologic levels of plasma low density lipoprotein (LDL) 1The abbreviations used are: LDL, low density lipoprotein; LDLR, LDL receptor; MEF, mouse embryonic fibroblast; ARH, autosomal recessive hypercholesterolemia; AP-2, adaptor protein-2; GST, glutathione S-transferase; LDLRct, LDLR cytoplasmic tail domain; siRNA, small interfering RNA; RNAi, RNA interference; LPDS, lipoprotein-deficient serum; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; [125I]sarile, 125I-[Sar1,Ile8]angiotensin II; FPLC, fast protein liquid chromatography; CHO, Chinese hamster ovary; HEK, human embryonic kidney; PBS, phosphate-buffered saline; IDL, intermediate-density lipoprotein.1The abbreviations used are: LDL, low density lipoprotein; LDLR, LDL receptor; MEF, mouse embryonic fibroblast; ARH, autosomal recessive hypercholesterolemia; AP-2, adaptor protein-2; GST, glutathione S-transferase; LDLRct, LDLR cytoplasmic tail domain; siRNA, small interfering RNA; RNAi, RNA interference; LPDS, lipoprotein-deficient serum; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; [125I]sarile, 125I-[Sar1,Ile8]angiotensin II; FPLC, fast protein liquid chromatography; CHO, Chinese hamster ovary; HEK, human embryonic kidney; PBS, phosphate-buffered saline; IDL, intermediate-density lipoprotein. cholesterol are associated with virtually all cases of atherosclerosis (1Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4306) Google Scholar). Approximately 75% of plasma LDL clearance occurs through endocytosis of the low density lipoprotein receptor (LDLR), predominately in the liver (2Kesaniemi Y.A. Witztum J.L. Steinbrecher U.P. J. Clin. Invest. 1983; 71: 950-959Crossref PubMed Scopus (114) Google Scholar). The well characterized endocytosis of the LDLR through clathrin-coated pits depends upon association of the 50-amino acid cytoplasmic tail of the LDLR with components of the cellular endocytic machinery. Although mutagenesis studies of the LDLR have delineated cytoplasmic tail domain residues necessary for LDLR endocytosis (3Davis 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, 4Chen W.J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1990; 265: 3116-3123Abstract Full Text PDF PubMed Google Scholar), proteins with which the LDLR cytoplasmic tail interacts have only recently been identified. The clathrin heavy chain terminal domain itself can interact with peptides from the LDLR cytoplasmic tail but with relatively low affinity (5Kibbey R.G. Rizo J. Gierasch L.M. Anderson R.G. J. Cell Biol. 1998; 142: 59-67Crossref PubMed Scopus (79) Google Scholar). The N-terminal domain of the autosomal recessive hypercholesterolemia (ARH) protein also binds to the LDLR cytoplasmic domain, whereas the C-terminal domain of ARH binds to both clathrin and AP-2 (6He G. Gupta S. Yi M. Michaely P. Hobbs H.H. Cohen J.C. J. Biol. Chem. 2002; 277: 44044-44049Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Thus, ARH appears to link the LDLR to the endocytic machinery, in a process that seems to be required for endocytosis of the LDLR in hepatocytes and lymphocytes (6He G. Gupta S. Yi M. Michaely P. Hobbs H.H. Cohen J.C. J. Biol. Chem. 2002; 277: 44044-44049Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 7Garcia 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 (463) Google Scholar). Whether other adaptor-type proteins may be involved in LDLR endocytosis remains to be determined.Candidate clathrin adaptor proteins for the LDLR could include the β-arrestins, which play important roles in the endocytosis of heptahelical G protein-coupled receptors. β-Arrestin1 and β-arrestin2 were initially characterized as ubiquitously expressed proteins involved in heptahelical G protein-coupled receptor desensitization (8Attramadal H. Arriza J. Aoki C. Dawson T. Codina J. Kwatra M.M. Snyder S. Caron M. Lefkowitz R. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar). With their N-terminal domains (9Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), the β-arrestins bind stoichiometrically to agonist-activated heptahelical receptors, and they do so with greater affinity after the receptors have been phosphorylated by G protein-coupled receptor kinases (10Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). The binding of a β-arrestin to the receptor inhibits receptor/heterotrimeric G protein interaction (8Attramadal H. Arriza J. Aoki C. Dawson T. Codina J. Kwatra M.M. Snyder S. Caron M. Lefkowitz R. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar) but can also initiate signaling via c-Src and other kinases (11Miller W.E. Lefkowitz R.J. Curr. Opin. Cell Biol. 2001; 13: 139-145Crossref PubMed Scopus (278) Google Scholar). To mediate G protein-coupled receptor internalization, β-arrestins link receptors to clathrin (12Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar) and AP-2 (13Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar), both of which are bound by the β-arrestin C-terminal domain. Recent data generated in β-arrestin-deficient cells suggest that particular receptors can interact preferentially with specific β-arrestin isoforms (14Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar).Endocytosis of G protein-coupled receptors in clathrin-coated pits is agonist-dependent (15von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar), like the binding of β-arrestins to G protein-coupled receptors (16Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). In contrast, coated pit-mediated endocytosis of the LDLR is constitutive in fibroblasts (17Anderson R.G. Brown M.S. Beisiegel U. Goldstein J.L. J. Cell Biol. 1982; 93: 523-531Crossref PubMed Scopus (139) Google Scholar). However, because ARH serves a clathrin adaptor function for the LDLR much like that served by the β-arrestins for heptahelical receptors (6He G. Gupta S. Yi M. Michaely P. Hobbs H.H. Cohen J.C. J. Biol. Chem. 2002; 277: 44044-44049Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), we tested the hypothesis that β-arrestins could act as LDLR adaptor proteins, and thereby enhance endocytosis of the LDLR.MATERIALS AND METHODSLipoprotein Metabolism in Mice—All animal care conformed to the "Guide for the Care and Use of Laboratory Animals" issued by the National Institutes of Health. The generation of β-arrestin2–/– mice has been described previously (18Bohn L.M. Lefkowitz R.J. Gainetdinov R.R. Peppel K. Caron M.G. Lin F.T. Science. 1999; 286: 2495-2498Crossref PubMed Scopus (779) Google Scholar). Derived from matings between β-arrestin2+/– founder mice, the β-arrestin2–/– and +/+ mice used in this study (n = 30) were littermates and were hybrids of the C57Bl/6J and 129/SvJ genetic backgrounds. Serum cholesterol measurements were performed on ∼200-μl blood samples, obtained from tail cuts performed under methoxyflurane anesthesia. Blood samples were first obtained after 4 weeks on a Paigen diet (by weight: 15% fat, 1.25% cholesterol, and 0.5% cholic acid (19Paigen B. Morrow A. Holmes P. Mitchell D. Williams R. Atherosclerosis. 1987; 68: 231-240Abstract Full Text PDF PubMed Scopus (785) Google Scholar), Dyets, Inc., #615038). After initial blood sampling, mice were fed a normal, low fat diet (Purina Breeder Chow), and blood was sampled again after 8 weeks. Thus, our serum lipid studies constitute a dietary crossover study. The mice were supplied with only water for 12 h prior to blood sampling (19Paigen B. Morrow A. Holmes P. Mitchell D. Williams R. Atherosclerosis. 1987; 68: 231-240Abstract Full Text PDF PubMed Scopus (785) Google Scholar).Blood was allowed to clot for 2 h at room temperature. The thrombus was pelleted at 4 °C, and serum was removed. All mice were bled on the same day, and each batch of 30 samples (high and low fat diet) was assayed simultaneously. Lipid analyses were performed by the Duke University Medical Center Endomet Laboratory, which is standardized for human lipoprotein analyses by the Centers for Disease Control and Prevention. LDL cholesterol was measured with the Liquid Select™ LDL Direct Assay (Equal Diagnostics), and HDL cholesterol was measured with the Roche Applied Science direct HDL-cholesterol method. In pilot studies with mouse plasma divided into aliquots, we found that lipoprotein cholesterol results from these assays correlated very closely with those obtained by fast performance liquid chromatography (FPLC) fractionation using a Superose 6 HR10/30 column (Amersham Biosciences) and the Infinity™ Cholesterol Reagent kit (Sigma); however, LDL cholesterol determined by the LDL Direct assay corresponded to LDL plus IDL cholesterol determined by FPLC fractionation. Plasma lipoprotein fractionation by FPLC was performed on 100-μl samples of mouse plasma as described previously (20Knouff C. Hinsdale M.E. Mezdour H. Altenburg M.K. Watanabe M. Quarfordt S.H. Sullivan P.M. Maeda N. J. Clin. Invest. 1999; 103: 1579-1586Crossref PubMed Scopus (230) Google Scholar).Plasmid Constructs—The LDLR cDNA was obtained from the American Type Culture Collection (21Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (970) Google Scholar). This construct was epitope-tagged at its N terminus with the FLAG™ octapeptide, as described previously (22Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). From the first amino acid of the mature LDLR (Ala) onward (21Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (970) Google Scholar), the remainder of the LDLR sequence was left intact. The resulting construct was subcloned into pcDNA I (Invitrogen). This FLAG-LDLR construct behaved identically to the native LDLR in LDL uptake experiments (data not shown), and is referred to as "LDLR" throughout the text. To create S833D and S833A mutations in the full-length LDLR, we employed two sets of complementary 46-mer oligonucleotides, which included nucleotides 2545–2583 of the human LDLR sequence (containing a 5′-XhoI site) (21Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (970) Google Scholar) as well as 3′ EcoRI and XbaI sites. In these oligonucleotides, nucleotides 2560–2562 were mutated to either GCC (Ala) or GAC (Asp). After annealing, these oligonucleotides were ligated into a XhoI/XbaI-cut LDLR/pcDNA I construct.A GST fusion protein encompassing the 50 amino acids of the LDLR cytoplasmic tail (LDLRct) was created by cassette PCR of the human LDLR, and subcloning the PCR fragment into pGEX2T (Amersham Biosciences) with BamHI (5′) and EcoRI (3′). To create S833D, Y807A, and S833D/Y807A mutations in the GST/LDLRct construct, we used subcloning and cassette PCR similar to that for the wild type construct, but with oligodeoxynucleotides encoding the indicated mutations. Fidelity of PCR-amplified and synthetic oligonucleotide-generated DNA sequences was verified by dideoxy sequencing. Plasmids encoding native rat β-arrestin1 and rat β-arrestin2 (23Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), as well as a K44A ("dominant negative") rat dynamin I mutant (24Miller W.E. Maudsley S. Ahn S. Khan K.D. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2000; 275: 11312-11319Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) have been described previously. FLAG-tagged constructs encoding rat β-arrestin1 and β-arrestin2 (24Miller W.E. Maudsley S. Ahn S. Khan K.D. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2000; 275: 11312-11319Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) were subcloned into pcDNA3.1/Hygro (Invitrogen).Cell Culture and Transfection—CHO ldlA cells, which lack functional LDLRs, were generously provided by Monty Krieger and grown as described before (25Kingsley D.M. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5454-5458Crossref PubMed Scopus (131) Google Scholar). Transfections were performed with LipofectAMINE®, in Opti-MEM™ medium (Invitrogen), according to the manufacturer's instructions. Human embryonic kidney (HEK) 293 cells were cultivated and transfected as described (22Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Mouse embryonic fibroblasts (MEFs) from either wild type, β-arrestin2–/–, or β-arrestin1–/–/β-arrestin2–/– mice were created as described by Kohout et al. (14Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar), and grown in DMEM/10% FBS/100 U/ml penicillin/100 μg/ml streptomycin. To compare MEFs expressing no β-arrestin isoform with genetically identical MEFs expressing only β-arrestin1 or only β-arrestin2, we used stable transfection in a β-arrestin1–/–/β-arrestin2–/– cell line. These cells were transfected with LipofectAMINE® (Invitrogen) and either pcDNA3.1/Hygro (Invitrogen), FLAG-tagged β-arrestin1/pcDNA3.1/Hygro, or FLAG-tagged β-arrestin2/pcDNA3.1/Hygro. Stably transfected lines were selected with and maintained in growth medium containing hygromycin at 250 μg/ml and screened by immunoblotting for β-arrestins. HEK 293 cells were transfected with siRNAs specific for β-arrestin1, β-arrestin2, or neither β-arrestin isoform, exactly as described (26Ahn S. Nelson C.D. Garrison T.R. Miller W.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1740-1744Crossref PubMed Scopus (188) Google Scholar), and split into assay dishes 72 h later.Immunoprecipitations—The day after transfection, ldlA cells were seeded into 100-mm dishes in Ham's F12 supplemented with 10% lipoprotein-deficient newborn calf serum (LPDS medium). The following day, LPDS medium was replaced with fresh medium containing 5% LPDS lacking or containing LDL at 200 μg of protein/ml, and incubated for 10 min at 37 °C. Cells were then washed twice with PBS, and proteins were cross-linked with the reducible cross-linking agent dithiobis(succinimidylpropionate) (Pierce), as described (22Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Cells were then scraped into ice-cold 500 mm NaCl/50 mm Tris-Cl, pH 7.4 (25 °C) with protease inhibitors (buffer A) (23Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), and then disrupted by using a tight-fitting Dounce homogenizer. Membranes from disrupted cells were pelleted (40,000 × g, 30 min, 4 °C), and washed in buffer A. Solubilized membranes were then processed for immunoprecipitation with M2 anti-FLAG™ IgG, as described (22Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Immune complexes were heated in Laemmli buffer for 30 min at 37 °C, to reduce intermolecular cross-links before SDS-PAGE and immunoblotting, which were performed as described previously (22Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar).Immunoblotting—To assay endogenous β-arrestin expression in MEFs, cells were lysed in 10 mm Tris-Cl/2 mm EDTA (pH 8.0) with protease inhibitors (buffer B), and the membrane fraction was pelleted at 20,000 × g for 15 min at 4 °C. Liver samples were obtained from C57Bl/6J mice sacrificed under pentobarbital anesthesia. Fresh liver was perfused with Ringer's Lactate, then minced and homogenized with a small Polytron™ (Brinkman) in buffer B. Insoluble debris was pelleted at 40,000 × g for 30 min at 4 °C. As throughout this report, protein concentrations of liver and cell supernatant fractions were assayed by a modified Lowry method, with IgG as the standard (23Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Forty μg of each specimen was subjected to SDS-PAGE and immunoblotting, as described (23Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar).LDL Uptake Assays—Either 16 h after transfection and 24 h before assay (ldlA cells, 293 cells), or 48 h before assay (MEFs and siRNA-treated 293 cells), cells were split and seeded at equal densities (∼50% confluence, 2.7–4.7 × 104 cells/cm2) into 6-well dishes, in growth medium containing 10% LPDS (in lieu of fetal bovine serum) (27Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1277) Google Scholar). For LDLR-transfected 293 cells, 10% LPDS growth medium was supplemented with 10 μg of cholesterol and 0.1 μg of 25-hydroxycholesterol per milliliter, to down-regulate endogenous LDLRs (3Davis 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). For siRNA-transfected 293 cells, 10% LPDS growth medium was supplemented with 1 μm lovastatin. On the day of assay, separate aliquots of cells were processed for cell surface LDLR assay (see below) at 4 °C, and LDL uptake (endocytosis) assays at 37 °C. LPDS growth medium was replaced with cognate medium containing 5% LPDS and LDL labeled with the fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI-LDL), at a concentration of 3–5 μg of protein/ml ("labeling medium," for "total uptake"). Labeling medium containing unlabeled LDL at 500 μg of protein/ml was used to assess nonspecific uptake of the DiI-LDL. Cells were incubated at 37 °C in 5% CO2 for 5 h (27Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1277) Google Scholar). LDL uptake assays were then terminated by trypsinizing cells, washing twice with PBS, and fixing with 3.6% formaldehyde in PBS. Cellular uptake of LDL was assessed by flow cytometry for cellular DiI fluorescence (28Innerarity T. Pitas R. Mahley R. Methods Enzymol. 1986; 129: 542-565Crossref PubMed Scopus (135) Google Scholar). Specific ([total]–[nonspecific]) uptake of DiI-LDL was divided by the relative cell surface LDLR number (see below), to obtain the Internalization Index for LDLRs in each cell line (3Davis 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). The values for internalization index were then normalized to that obtained for the control cells within each experiment, to facilitate averaging results across independent experiments. For DiI-LDL uptake, the intra-assay coefficient of variation was <5%, and mean nonspecific uptake constituted 25% of mean total uptake.Cell Surface LDLR Assessment—For cells transfected with the N-terminal FLAG™-tagged LDLR construct, cell surface LDLRs were quantitated by cell surface immunofluorescence and flow cytometry, as described (22Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). For MEFs, which expressed only endogenous LDLRs, cell surface receptor number was assessed at 4 °C by ligand binding, with DiI-LDL (27Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1277) Google Scholar). Cells were first detached in PBS/EDTA (Versene, Invitrogen), pelleted, and washed in ice-cold minimal essential medium. Next, cells were resuspended in 200 μl of ice-cold DiI-LDL labeling medium lacking ("total binding") or containing unlabeled LDL at 500 μg of protein/ml ("nonspecific binding"), and incubated at 4 °C for 3 h. Subsequently, cells were pelleted and washed with 1.5 ml of ice-cold HEPES-buffered saline (with 2.5 mm CaCl2), then fixed in this solution with 3.6% formaldehyde. Specific cell surface DiI-LDL binding was then assessed as described above and was used to estimate the relative cell surface LDLR number within each experiment. The intra-assay coefficient of variation was <7%, and mean nonspecific binding constituted 35–60% of mean total binding. Within a given experiment, cells were used only if cell surface LDLR density was within 30% (transfected cells) to 50% (MEFs) of control cells.LDL and LPDS Preparation—LDL (1.019 < d < 1.055) from normal human plasma and newborn calf lipoprotein-deficient serum (d > 1.21 g/ml) were prepared by vertical spin density gradient ultracentrifugation, as described (27Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1277) Google Scholar, 29Chung B. Segrest J. Ray M. Brunqell J. Hokanson J. Krauss R. Beaudrie K. Cone J. Methods Enzymol. 1986; 128: 181-209Crossref PubMed Scopus (422) Google Scholar). LDL purity was assessed by Coomassie Blue staining of SDS 3–20% gradient gel electrophoresis. DiI-LDL was either purchased from Molecular Probes, Inc., or prepared as described (28Innerarity T. Pitas R. Mahley R. Methods Enzymol. 1986; 129: 542-565Crossref PubMed Scopus (135) Google Scholar).Angiotensin II Receptor Internalization—Fibroblasts in 35-mm wells were incubated with serum-free medium (Dulbecco's modified Eagle's medium/1% bovine serum albumin/20 mm Hepes, pH 7.4) containing either vehicle ("naïve") or 100 nm angiotensin II for 30 min (37 °C), and then washed with 40 mm sodium acetate/150 mm NaCl, pH 5.0 (10 min, 25 °C) to remove cell surface-bound angiotensin II (30Haddad G. Amiri F. Garcia R. Regul. Pept. 1997; 68: 111-117Crossref PubMed Scopus (16) Google Scholar). After three washes with PBS, cells were incubated for 3 h at 4 °C in serum-free medium containing 1 nm [125I]sarile (an angiotensin II receptor antagonist, PerkinElmer Life Sciences), to assess cell surface angiotensin II receptor binding in the absence (total binding) or presence (nonspecific binding) of 500 nm angiotensin II (31Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Cells were then washed thrice, solubilized in 0.1 n NaOH, and aliquoted to gamma counting and Lowry protein assay (31Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Specific binding (total–nonspecific) was used to assess receptor internalization, as: 100 × ([125I]sarile bound to angiotensin II-challenged cells)/([125I]sarile bound to naïve cells). Total binding in each well constituted <10% of the CPM used in each binding assay well, and nonspecific binding averaged 40 ± 10% of total binding.Fusion Protein Production—GST and the GST/LDLRct proteins were made in the Escherichia coli strain BL21 by standard methods (32Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor2001Google Scholar). After elution from glutathione-agarose with reduced glutathione, fusion proteins were concentrated and dialyzed in Centriprep 10 units (Amicon). Purity of the preparations was ∼90%, as determined by SDS-PAGE and Coomassie Blue staining.β-Arrestin2 Purification—The FLAG™-tagged rat β-arrestin2 cDNA (24Miller W.E. Maudsley S. Ahn S. Khan K.D. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2000; 275: 11312-11319Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) in pVL1393 (Invitrogen) was used to make a recombinant baculovirus, with BaculoGold DNA (BD Pharmingen). Spodoptera frugiperda-9 cells infected with the β-arrestin2FL baculovirus were harvested 72 h after infection and lysed in buffer C (50 mm HEPES, pH 7.4, 0.5% Nonidet P-40, 250 mm NaCl, 10% glycerol, 2 mm EDTA, and protease inhibitors). Insoluble debris was pelleted, and the supernatant was mixed with anti-FLAG M2-agarose (Sigma) at 4 °C, 16 h. Beads were loaded into a column and washed extensively with buffer C. β-Arrestin2FL was eluted with 0.1 m glycine, pH 3.5, into a 0.1 volume of 0.5 m Tris, pH 7.5. Fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Purity of the preparation was ∼95%.In Vitro Binding of β-Arrestin2 to the LDLRct—[35S]β-arrestin2 was synthesized by in vitro translation, using the TnT® Quick Coupled Transcription/Translation System (Promega), [35S]methionine/[35S]cysteine (11 mCi/ml, PerkinElmer Life Sciences), and the rat β-arrestin2 expression plasmid (23Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), a
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