Cabello-Verrugio and María-Paz MarzoloEnrique Brandan, Claudio Retamal, Claudio  Endocytic Receptor for DecorinReceptor-related Protein Functions as an The Low Density LipoproteinGlycobiology and Extracellular Matrices:

doi: 10.1074/jbc.M602919200 originally published online August 25, 20062006, 281:31562-31571.J. Biol. Chem. 

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The Low Density Lipoprotein Receptor-related ProteinFunctions as an Endocytic Receptor for Decorin*

Received for publication, March 28, 2006, and in revised form, July 5, 2006 Published, JBC Papers in Press, August 25, 2006, DOI 10.1074/jbc.M602919200

Enrique Brandan1, Claudio Retamal, Claudio Cabello-Verrugio2, and Marıa-Paz Marzolo3

From the Centro de Regulacion Celular y Patologıa “Joaquın V. Luco,” CRCP, Departamento de Biologıa Celular y Molecular,MIFAB, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile

Decorin is a small leucine-rich proteoglycan that modulatesthe activity of transforming growth factor type � and othergrowth factors and thereby influences the processes of prolifer-ation and differentiation in a wide array of physiological andpathological reactions. Hence, understanding the regulatorymechanisms of decorin activity has broad implications. Here wereport that the extracellular levels of decorin are controlled byreceptor-mediated catabolism, involving the low densitylipoprotein receptor family member, low density lipoproteinreceptor-related protein (LRP). We show that decorin is endo-cytosed and degraded by C2C12 myoblast cells and that bothprocesses are blocked by suppressing LRP expression usingshort interfering RNA. The same occurs with CHOcells, but notwith CHO cells genetically deficient in LRP. Finally, we showthat LRP-null CHO cells, transfected to express mini-LRPpolypeptides containing either the second or fourth LRPligand-binding domains, carry out decorin endocytosis andlysosomal degradation. These findings point to LRP-medi-ated catabolism as a new control pathway for the biologicalactivities of decorin, specifically for its ability to influenceextracellular matrix signaling.

Decorin is one of the most studied members of the family ofsmall leucine-rich proteoglycans. Its core protein, which con-stitutes up to 80% of the proteinmoiety, is composed of 12-foldrepeats of a 24-amino acid residue (leucine-rich repeats). Inaddition, decorin carries a single glycosaminoglycan (GAG)4chain at its NH2 terminus. A crystal structure for bovinedecorin has been published (1) that together with earlier x-rayscattering data (2) suggests decorin to be a dimeric protein.

Eachmonomer adopts a curved structure, whereby antiparalleldimerization occurs through the �-sheet on the monomer’sconcave surface.Several different functions, based on the interaction of the

core protein with other proteins, have been established fordecorin, one example being the regulation of extracellularmatrix (ECM) assembly. Decorin regulates collagen fibril for-mation and stabilization and also modulates cell adhesion (3).The interaction of decorin with fibronectin and throm-bospondin leads to the inhibition of fibroblast attachment tothese substrata (4, 5). In addition to the interaction with ECMconstituents, decorin interacts with several growth factors andplasma membrane-located receptors. For instance, it is wellknown that decorin has the ability to form complexes withtransforming growth factor type-� (TGF-�) (6), bind to theinsulin-like growth factor-I (7), and interact with tumor necro-sis factor-� (8). Furthermore, the ectopic expression of decorinhas been shown to retard the growth of various tumor cells, aneffect that can be attained by exogenously applying recombi-nant decorin to a wide variety of cells (9–12). Decorin is alsoknown to cause rapid phosphorylation of the epidermal growthfactor receptor and concurrent activation of the mitogen-acti-vated protein kinase signaling pathway (13). Recent studieshave shown that decorin binds to the insulin-like growth fac-tor-I receptor, inducing its phosphorylation and activation, fol-lowed by receptor down-regulation (7). On the other hand,reducing decorin levels results in a decreased cell responsive-ness to TGF-�, suggesting that decorin is required to activatethe TGF-� signaling pathways (14).Considering the different ECM-related functions exhibited

by decorin, including the accumulation of growth factors andits interaction with matrix constituents as well as several trans-ducing receptors at the cell surface, it is evident that the regu-lation of extracellular decorin concentrations, by varying itsbiosynthesis and degradation rates, is of great physiologicalimportance.Themetabolismof decorin has been studiedmost intensively

in cultured fibroblasts, in which decorin represents the majorproteoglycan species and is secreted into the culture medium,where it follows secretion-recapture cycles (15). Fibroblastsand other cells of mesenchymal origin are known to efficientlyinternalize decorin by receptor-mediated endocytosis (16, 17).Several concerted yet unsuccessful efforts have been made toidentify the endocytic receptor of decorin. Two proteins of 51and 26 kDa, present in endosomes and at the plasma mem-brane, are considered putative decorin receptors (18). How-ever, no functional evidence is as yet available to support this

* This work was supported in part FONDAP-Biomedicine Grants 13980001,MDA 3790, and FONDECYT 1020726. The Millennium Institute for Funda-mental and Applied Biology is financed in part by the Ministerio de Plani-ficacion y Cooperacion (Chile). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

1 Supported in part by an International Research Scholar grant from theHoward Hughes Medical Institute.

2 Supported by DIPUC and CONICYT Fellowship AT-24050108.3 To whom correspondence should be addressed. Tel.: 56-2-6862112; Fax:

56-2-2229995; E-mail: mmarzolo@bio.puc.cl.4 The abbreviations used are: GAG, glycosaminoglycan; Adv-Dcn, adenovirus

containing full-length human decorin; DSS, disuccinimidylsuberate; ECM,extracellular matrix; FBS, fetal bovine serum; LDL, low density lipoprotein;LRP, low density lipoprotein receptor-related protein; RAP, receptor-asso-ciated protein; siRNA, short interfering RNA; TGF-�, transforming growthfactor type �; GST, glutathione S-transferase; DMEM, Dulbecco’s modifiedEagle’s medium; CHO, Chinese hamster ovary; RT, reverse transcription.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 42, pp. 31562–31571, October 20, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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notion, so that the identity of the decorin receptor remains anopen question.The low density lipoprotein (LDL) receptor-related protein,

LRP, is a giant receptor belonging to the LDL receptor family,which binds, endocytoses, andmediates the degradation of sev-eral ligands (19). The receptor’s folding process in the endo-plasmic reticulum requires the participation of the 39-kDareceptor chaperone, RAP (20). This chaperone protein has alsobeen used as a tool and competitor to study novel ligands forLRP, since recombinant RAP binds with high affinity to thereceptor’s ligand-binding domains at the cell surface.Through its large ectodomain, which contains four ligand-

binding domains, LRP binds (among other proteins) multipleECMmolecules, including thrombospondin (21–23), fibronec-tin (24), plasminogen activators (25, 26), matrix metallopro-teinases (27), and connective tissue growth factor (28). Further-more, LRP regulates signaling cascades by binding ECMmolecules, such as fibronectin (29) and thrombospondin (30),and growth factors, such as platelet-derived growth factor(31, 32), connective tissue growth factor (33), and TGF-�(34, 35). Interestingly, several of these molecules also binddecorin (36–39).Given the role of decorin in myoblast differentiation (14, 40)

and the presence of LRP mRNA in human skeletal muscle cells(41), in the present study, we tested whether decorin endocyto-sis in C2C12 mouse myoblasts was affected by the presence ofthe RAP-inhibitable receptor, LRP. Our results showedunequivocally that in LRP-expressing C2C12 myoblasts, theinternalization and degradation of decorin depended on itsinteraction with this endocytic receptor. Furthermore, we alsodemonstrated that in Chinese ovary cells (CHO), LRP was alsoresponsible for decorin endocytosis, involving at least thereceptor’s ligand-binding domains 2 and 4.

EXPERIMENTAL PROCEDURES

Reagents—Plasmids encoding LRP minireceptors, whichinclude the ligand-binding domains 2 (mLRP2) and 4 (mLRP4)have been described previously (42, 43). Annealed LRP-1-spe-cific siRNA as well as control siRNA were obtained fromAmbion (Austin, TX), with LRP siRNA sequences described inRef. 44. GST andGST-RAPwere produced as described in (45).Rabbit anti-LRP antibody was kindly provided by Dr. GuojunBu and used as prescribed by Marzolo et al. (42). Mouse anti-LRP raised against the cytoplasmic domainwas purchased fromCalbiochem, and mouse anti-�-tubulin was from Sigma.Adenoviral vector containing the full-length cDNA for humandecorin (Adv-Dcn) has already been described (14). Decorincore protein was obtained from R&D Systems, and full-lengthdecorin and biglycan were purchased from Sigma. To deter-mine the amount of chondroitin and dermatan sulfate in thecommercial decorin, decorin was radiolabeled with 125I asexplained below and treated with chondroitinase ABC and AC.Both treatments digestedmost if not all of the GAGs associatedto decorin, indicating its chondroitin sulfate nature. The cross-linker agent disuccinimidylsuberate (DSS) was from Pierce.Cell Culture and Transfection—The mouse skeletal muscle

cell line C2C12 (ATCC) (46) was grown and induced to differ-entiate, as described in Ref. 47. The U87 glioblastoma cell line

was cultured inminimal essentialmediumwith 0.1% nonessen-tial amino acids and 1mM sodium pyruvate.Wild-type Chinesehamster ovary cells (CHO-K1) were cultured in Dulbecco’smodified Eagle’smedium (DMEM)with 10% fetal bovine serum(FBS), whereas LRP-deficient cells (LRP-null CHO) (48) werecultured in F-12 medium with 10% FBS. Clonal cell lines,derived from LRP-null CHO cells expressing LRP minirecep-tors, were obtained by transfection, using 2 �g of plasmid DNAand Lipofectamine Plus transfection reagent (Invitrogen) in35-mm dishes, according to the supplier’s protocol. Cells werescreened and analyzed by Western blot and immunofluores-cence. Selected clones were then maintained in wild-typemedium containing 0.4 mg/ml G418.RNA Isolation and Reverse Transcription (RT)-PCR—Total

RNAwas isolated frommyoblast cultures using Trizol (Invitro-gen). For RT reactions, 4 �g of total RNA were treated withDNase I for 15 min at room temperature. Subsequently, sam-ples were incubated with random hexamers and the Maloneymurine leukemia virus reverse transcriptase kit for 10 min at25 °C, 60 min at 37 °C, and finally 10 min at 70 °C. Aliquots of 1�l of cDNA were used as a template for standard PCR proce-dures. The primers used in PCR reactions were as follows: LRPforward, 5�-AGTGCTGCCCAGACACAGCTCAAGTGTG-3�; LRP reverse, 5�-CACGATCTTGCTATCCACCAGCTTG-GTG-3�; glyceraldehyde-3-phosphate dehydrogenase forward,5�-CGGTGTGAACGGATTTGGC-3�; glyceraldehyde-3-phosphate dehydrogenase reverse, 5�-GCAGTGATGGC-ATGGACTGT-3�.

FIGURE 1. Decorin is endocytosed by C2C12 myoblasts. A, C2C12 myo-blasts were incubated with [35S]decorin at 37 °C for 3 h in the absence (Ctrl) orpresence of either decorin (Dcn) or heparin (Hep). After this, cells were ana-lyzed to determine the extent of [35S]decorin endocytosis (black bars) anddegradation (gray bars). Endocytosis is expressed as the clearance of[35S]decorin (16): volume/mg of protein/h. Degradation values, expressed aspercentages, were normalized against control cells. B, C2C12 myoblasts wereincubated with [125I]decorin core protein (Dcn core) for 3 h at 37 °C, in theabsence (Control) or presence of unlabeled decorin core protein (Dcn), hepa-rin (Hep), biglycan (Big), or chloroquine (Chloroq). Degraded decorin core pro-tein is expressed as fmol of [125I]decorin/105 cells.

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C2C12 Infection with Recombinant Adenovirus, Adv-Dcn,and Metabolic Labeling—C2C12 myoblasts were plated at adensity of 30,000 cells/cm2 in 6-well plates. After 4 h,myoblastswere infected with 500 plaque-forming units/cell of Adv-Dcn(14) in DMEM, containing 2% heat-inactivated FBS. After 90min of incubation, standard medium was added, and incuba-tion continued for an additional 24 h, after which cells weremetabolically labeled for 18 h in sulfate and serum-freeDMEM/F-12, containing 100 �Ci/ml [35S]H2SO4 (25 mCi/ml;PerkinElmer Life Sciences). This conditionedmediumwas thenremoved, concentrated, and partially purified on a DEAE-Sephacel column, pre-equilibrated in 10 mM Tris-HCl, pH 7.4,0.2 M NaCl, and 0.1% Triton X-100. Column-bound sampleswere incubated with heparitinase, in appropriate buffer for 4 hat 37 °C, in order to degrade any heparan sulfate proteoglycanspresent in the conditioned medium. The DEAE-Sephacel wasincubated with 1 M NaCl, and the eluate was dialyzed againstphosphate-buffered saline. In experiments using decorin coreprotein, the carrier-free decorin core protein (R&D Systems)was radiolabeled with Na[125I] using chloramine T (49).siRNA Transfection—For transfection experiments, cells

were plated in 6-well plates and incubated until reaching 70%confluence. Cells were subsequently incubated for 6 h in 800 �lof Opti-MEM I, containing 75 nM LRP siRNA or control siRNAplus 8 �l of Lipofectamine 2000 (Invitrogen). Following thistransfection period, FBS was added to the medium, and the cellscultured for a further 12 h. The medium was then changed to

growthmedium, and cells were incu-bated up to 72 h post-transfection,after which decorin endocytosis, deg-radation, or cross-linking assays werecarried out. The effect of siRNA onthe level of LRP synthesis in C2C12myoblasts was evaluated by immuno-blotting against LRP (50).Cross-linking and Competition

Assays—Briefly, cells were incu-bated with 220 pM [125I]decorin for4 h at 4 °C in KRH buffer (128 mMNaCl, 5 mM KCl, 5 mM MgSO4, 1.3mM CaCl2, 50 mM Hepes, pH 7.4)supplemented with 0.5% bovineserum albumin (KRH-bovine serumalbumin). In competition experi-ments, cells were co-incubated with[125I]decorin and either recombi-nant decorin core protein (22 nM),bovine decorin containing GAGs(22 nM), heparin (100 �g/ml), GST(1 �M), or GST-RAP (1 �M). Thecells were then sequentially washedin cold KRH-bovine serum albuminand KRH. For cross-linking assays,cells were incubated with cross-linker agent DSS in KRH buffer for30 min at 4 °C. The reaction wasstopped by adding a buffer of 10mMTris-HCl, pH 7.4, containing 250

mM sucrose (49). In immunoprecipitation experiments, DSSwas omitted. Cells were lysed in 50 mM Tris-HCl, pH 7.4, 0.1 MNaCl, 0.5% Triton X-100, containing a mixture of proteaseinhibitors and 1 mM phenylmethylsulfonyl fluoride. Equalamounts of protein (80 �g) from precleared extracts were sep-arated by SDS-PAGE in 3–8% gradient gels, and gels werefinally dried and exposed under a PhosphorImager.Confocal Immunofluorescence Microscopy—LRP expression

and distribution in C2C12 was analyzed by confocal micros-copy. Cells were grown on glass coverslips. For intracellularprotein staining, cells were fixed in 3% paraformaldehyde, per-meabilized with 0.05% Triton X-100 (51), and incubated for 1 hwith 1:100mouse anti-LRP antibody, directed against the cyto-plasmic tail of human LRP-1 (Calbiochem). The incubationbuffer was 50 mM Tris-HCl, pH 7.7, 0.1 M NaCl, and 2% bovineserum albumin. After buffer removal and several washes withthe above buffer, bound antibodies were detected by incubatingthe cells for 30 min with 1:100 affinity-purified fluorescein iso-thiocyanate-conjugated anti-mouse antibodies (Pierce). Afterrinsing, the slides were viewed under a Pascal Zeiss laser-scan-ning confocal microscope (LSM-5).Immunoprecipitation and Immunoblot Analyses—For immu-

noprecipitation assays,myoblasts were lysed in 50mMTris-HCl,pH 7.4, 0.1 M NaCl, 0.5% Triton X-100 buffer, containing amixture of protease inhibitors and 1 mM phenylmethylsulfonylfluoride. Equal amounts of protein (150 �g) from preclearedextracts were immunoprecipitated overnight at 4 °C with 5 �g

FIGURE 2. Decorin endocytosis is inhibited by RAP in skeletal muscle cells. A, myoblasts were incubatedwith [35S]decorin at 37 °C for 3 h, in the absence (Control) or presence of either GST or GST-RAP. After this, cellswere analyzed to determine [35S]decorin endocytosis (black bars) and degradation (gray bars), as for Fig. 1.Endocytosis is expressed as the clearance of [35S]decorin (i.e. volume/mg of protein/h). Degradation values,expressed as percentages, were normalized against control cells. B, myoblasts were incubated with[125I]decorin core protein (Dcn core) at 37 °C for different time periods in the presence of GST-RAP (white circles)or alone (black circles). Internalized and degraded decorin levels were determined as for Fig. 1.

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of rabbit anti-LRP, as previously described (50), followed byincubation for 2 h at 4 °C with 20 �l of protein A-agarose beads(Pierce). Equal volumes of immunoprecipitated protein weresubjected to SDS-PAGE in 3–8% gradient gels, which werethen dried and exposed to phosphorimaging or subjected toLRP immunoblot analysis.For immunoblot assays, cell extracts were prepared in the

same Tris buffer as above, containing protease inhibitors andphenylmethylsulfonyl fluoride. Aliquots were separated bySDS-gel electrophoresis in 3–8% gradient polyacrylamide gels,electrophoretically transferred onto nitrocellulose membranes(Schleicher & Schuell) and probed with either rabbit anti-LRP(1:1000) or mouse anti-�-tubulin (1:5000) antibodies (Sigma).All immunoreactions were visualized by enhanced chemilumi-nescence (Pierce).Decorin Endocytosis and Degradation Assays—The rates of

[35S]decorin endocytosis and degradation were determined inmyoblast cultures, in the absence of serum, as described (16).Briefly, cells were grown in 6-well plates until reaching 80%confluence, after which [35S]decorin (200,000 cpm) was added(in a total volumeof 1ml) and left for 3 h at 37 °C in the presenceor absence of bovine decorin containing GAGs (100 nM),decorin core (100 nM), biglycan (100 nM), heparin (100 �g/ml),chondroitin sulfate (100 nM), GST (1 �M), or GST-RAP (1 �M).In some experiments, cells were treated with LRP siRNA, priorto adding labeled decorin. Since proteoglycan endocytosis isfollowed by intralysosomal degradation and the concomitantrelease of inorganic sulfate into the culture medium, endocyto-sis of [35S]decorin can be measured by determining theamounts of [35S]sulfate present both in the intracellular andculture medium. Soluble [35S]sulfate corresponding to inor-ganic sulfate was determined after precipitating [35S]decorinwith 70% ethanol. Endocytosis can hence be expressed as aclearance rate: the volume of decorin-cleared medium as afunction of time and cellular protein level. Degradation wasdefined as the sum of the intra- and extracellular levels of eth-anol-soluble radioactivity over the total amount of endocytoseddecorin.For [125I]decorin core protein degradation assays, cells were

seeded 18 h before the assay, at a density of 30,000 cells/cm2 in12-well plates. They were then washed with phosphate-buff-ered saline, depleted for 1.5 h in serum-free medium, andwashedwith bindingmedium (DMEM/F-12mediumwith 0.5%bovine serum albumin and 5mMCaCl2). Incubations were car-ried out at 37 °C using 400 �l of binding medium containing 1nM [125I]decorin and either competitors or controls, as indi-cated. Decorin degradation was calculated as the amount ofnon-trichloroacetic acid-precipitable radioactivity recoveredin the medium after incubation, and cpm readings were thentransformed to fmol using iodinated decorin-specific activity.

RESULTS

Decorin and Decorin Core Protein Are Internalized andDegraded by C2C12 Myoblasts—It has been proposed thatdecorin is endocytosed by several cell types, although the recep-tor responsible for this process has yet to be identified. In orderto investigate this issue, we analyzed the endocytosis of decorinin C2C12 myoblasts. Fig. 1A shows that [35S]decorin, isolated

and purified from C2C12 cells transfected with recombinantadenovirus containing the human decorin sequence, is indeedendocytosed. When expressed as the volume of cleared radio-active ligand per unit of time and protein concentration, myo-blasts cleared about 50 �l/mg/h of [35S]decorin. These valuesare almost twice those reported for decorin clearance in fibro-blasts by Hausser et al. (16). Fig. 1A (left) also shows that uponthe addition of cold commercial decorin, isolated from carti-lage, [35S]decorin clearance was inhibited by about 60%. Deg-radation of [35S]decorin by myoblasts was close to 80% of thetotal amount of decorin that was internalized (Fig. 1A, right).Again, these values are higher than those described for fibro-blasts (16). Moreover, it has been previously demonstrated that[35S]decorin endocytosis and degradation decrease in the pres-ence of heparin (52). In order to determine whether these pro-cesses were dependent on the GAG chains present in decorin,we repeated the experiments using a decorin core proteindevoid of GAGs. Fig. 1B shows that this 125I-labeled dec-orin core protein was efficiently degraded in a process that wasinhibited by unlabeled decorin core protein, commercial bigly-

FIGURE 3. Core protein and glycosaminoglycan chain of decorin both par-ticipate in its endocytosis and degradation through LRP. Myoblasts wereincubated with [35S]decorin at 37 °C for 3 h, in the absence (Ctrl) or presenceof cold decorin (Dcn full), decorin core protein (Dcn core), chondroitin sulfate(CS), GST-RAP (RAP), decorin core plus chondroitin sulfate (Dcn core � CS), ordecorin plus GST-RAP (Dcn full � RAP). After this, cells were analyzed to deter-mine [35S]decorin endocytosis (A) and degradation (B). Endocytosis and deg-radation of decorin are expressed as explained in the legend of Fig. 1.

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can isolated from cartilage, heparin, and also the lysosomalinhibitor, chloroquine. These results indicate that C2C12myo-blasts were able to internalize and degrade whole decorin mol-ecules as well as decorin core protein.DecorinClearance andDegradationAre Inhibited by RAP—To

gain more insight into the possible endocytic receptor(s)involved in the uptake of decorin, C2C12 myoblasts were incu-bated with [35S]decorin in the presence of GST-RAP, whichprevents the association of several ligands to members of theLDL receptor family. Upon co-incubation with RAP, both[35S]decorin clearance and degradation were strongly inhib-ited, whereas incubatingwithGST alone had no effect (Fig. 2A).Fig. 2B shows the kinetics of [125I]decorin core protein inter-nalization and degradation. A plateau for internalization wasattained after 200 min of incubation, whereas degradation of[125I]decorin core protein continued up to 300 min. Both pro-cesses were inhibited by GST-RAP, suggesting that a member

of the LDL receptor family could beinvolved in the process of decorinendocytosis. In order to assign therole of the core protein and theGAGs in the internalization anddegradation process of decorin,inhibited by RAP, we evaluated therole of the core, the GAGs andboth together in the uptake anddegradation of [35S]decorin. Fig. 3shows that the whole unlabeleddecorin molecule inhibited boththe clearance and degradation of[35S]decorin to the same extent asRAP. The core alone, as well aschondroitin sulfate, was less effec-tive in inhibiting the clearance anddegradation. Interestingly, whenadded together, the inhibition wassimilar to that observed using thewhole decorin, suggesting thatboth moieties participate in theinteraction with LRP and in itssubsequent degradation.Myoblasts Express LRP That

Interacts with Decorin—We nextevaluated whether C2C12 myo-blasts expressed LRP by carryingout RT-PCR assays for LRP usingmRNA isolated from C2C12 myo-blasts and from U87 cells as a posi-tive control, given that in these cells,the trafficking and function of LRPhave been well characterized (53).C2C12 myoblasts were seen toexpress LRPmRNA (Fig. 4A) as wellas the protein, after performingWestern blots (Fig. 4B). Finally, wetested for the presence of LRP byindirect immunofluorescence usinga confocalmicroscope, using a poly-

clonal antibody against human LRP. As seen in Fig. 4C, C2C12myoblasts were positive for LRP, with the majority of receptorsbeing intracellular, reflecting a high endocytic activity. Theseresults clearly demonstrated the expression of LRP in C2C12myoblasts. Interestingly, we also found that LRP protein levelsdiminished during skeletal muscle differentiation, as seen byWestern blotting (Fig. 4D).To evaluate the possible interaction between decorin and

LRP at the molecular level, myoblasts were incubated with125I-labeled decorin core protein at 4 °C to avoid endocyto-sis, and the cells were lysed and immunoprecipitated withantibodies against the extracellular domain of LRP. Afterseparation by SDS-PAGE, autoradiography revealed thatdecorin co-immunoprecipitated with the LRP-antibodycomplexes (Fig. 5A). The same figure shows that this inter-action was inhibited by the addition of excess unlabeleddecorin core protein, full-length decorin isolated from car-

FIGURE 4. C2C12 myoblasts express LRP, the levels of which decrease during myogenesis. A, total RNA wasobtained from C2C12 myoblasts and either treated (�RT) or not treated (�RT) with reverse transcriptase.RT-PCR analyses were performed using specific primers aimed at detecting either LRP or glyceraldehyde-3-phosphate dehydrogenase mRNA. Total RNA from U87 cells was used as a positive control. B, Western blotanalyses were performed to determine the protein levels of LRP in C2C12 myoblast extracts. U87 cell extractswere used as positive controls, and tubulin protein levels are shown as loading controls. C, cells were perme-abilized and processed for confocal indirect immunofluorescent staining, using anti-LRP antibodies and fluo-rescein isothiocyanate-conjugated secondary antibodies (a) and controls without anti-LRP antibodies (b). Thebar corresponds to 10 �m. D, Western blots for LRP were performed on the cell extracts of myoblasts inducedto differentiate for 0, 2, 4, or 6 days. U87 cell extracts were used as positive controls, and for all cells, tubulinprotein levels provided a loading control.

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tilage, and GST-RAP, whereas GST alone had no effect. Toanalyze the direct interaction between decorin core proteinand LRP at the cell surface, C2C12myoblasts were incubatedwith [125I]decorin core protein at 4 °C for 3 h, after whichexcess labeled decorin was removed, a cross-linker agent(DSS) was added, and cell lysates were immunoprecipitatedagainst LRP. SDS-PAGE followed by autoradiography (Fig.5B) showed that decorin core protein interacted with LRPand migrated as a high molecular weight complex. To deter-mine whether RAP was able to affect the formation of thiscomplex, the same cross-linking experiment was carried outin the presence of GST-RAP. As seen in Fig. 5C, GST-RAPinhibited the formation of the high molecular weight com-plex, in contrast to controls or the incubation of myoblastswith GST alone (seen in Fig. 5A). Together, these results notonly show that myoblasts express LRP but also show thatdecorin specifically interacts with this receptor.

Specific Inhibition of LRP ProteinSynthesis Eliminates the Interaction,Endocytosis, and Degradation ofDecorin—To focus on the functionalrelationship between decorin andLRP, C2C12 myoblasts were trans-fected with a specific siRNA for LRP.Fig. 6A shows that LRP expressionwas strongly inhibited by LRP siRNA,as has been previously reported (44),whereas transfection with siRNA-Control hadno effect onLRP levels inthese cells. Under these experimentalconditions,the complex previously formedbetween [125I]decorin core proteinand LRP, did not arise, as indicated inFig. 6B. Given that these results pro-vided strong evidence for the forma-tion of a decorin-LRP complex, wenext investigated whether inhibitingLRP synthesis would have a func-tional impact on decorin endocytosisand degradation. Fig. 7A shows thatstrong inhibition of [35S]decorinclearance occurred in myoblaststransfected with LRP siRNA, in con-trast to cells transfected with siRNA-Control. Moreover, the degradation of[35S]decorin was also strongly inhib-ited by the lack of LRP expression(Fig. 7B). These results all point toLRP as the endocytic receptor fordecorin in C2C12 myoblasts.CHOCells Rely on LRPExpression

in Order to Degrade Decorin CoreProtein—To confirm the resultsobtained in myoblasts, wherebydecorin endocytosis and degrada-tion were inhibited by the absenceof endogenous LRP resulting from

siRNA techniques, we evaluated the ability of a CHO cellline, devoid of LRP expression, to degrade decorin core pro-tein. Fig. 8A shows that, unlike LRP-null CHO cells, wildtype CHO-K1 cells were able to degrade [125I]decorin coreprotein and that this process was strongly inhibited by GST-RAP. As a comparison, the values of core protein degrada-tion and inhibition by GST-RAP are shown for C2C12 myo-blasts as well. In order to characterize the LRP ligand-binding domains involved in decorin binding, we transfectedLRP-null CHO cells with the cDNA encoding for LRP mini-receptors. These contain, at the ectodomains, the second(mLRP2) or the fourth (mLRP4) ligand-binding domains ofLRP and all of the other receptor regions (namely the trans-membrane and cytoplasmic domains). As reported for otherligands (43), we found that both minireceptors were able tomediate decorin internalization and degradation in CHOcells (Fig. 8B). These results unequivocally confirm that LRP

FIGURE 5. [125I]decorin core protein binds to LRP in C2C12 cells. A, myoblasts were incubated with[125I]decorin core protein at 4 °C for 3 h in the absence (Control) or presence of unlabeled decorin core protein(Dcn core), full-length decorin (Dcn full), GST, or GST-RAP. Cells were washed and lysed, and extracts wereimmunoprecipitated using anti-LRP antibodies. Radiolabeled immunoprecipitated proteins were visualized byphosphorimaging (upper panel), and total LRP protein levels were determined by Western blotting (lowerpanel). B, myoblasts were incubated with [125I]decorin core protein at 4 °C for 3 h and then cross-linked,followed by immunoprecipitation with anti-LRP antibodies. Radiolabeled immunoprecipitated proteins werevisualized under a PhosphorImager. C, myoblasts were incubated with [125I]decorin core protein at 4 °C for 3 h,in the absence (Ctrl) or presence of either GST or GST-RAP. After cross-linking, cell extracts were electrophoreti-cally separated by SDS-PAGE. Gels were stained, dried, and exposed under a PhosphorImager to detect radio-labeled proteins (upper panel). Total protein levels are shown by Coomassie Blue staining (lower panel).

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is an endocytic receptor for decorin and that its ability torecognize decorin involves at least two of its ligand-bindingdomains.

DISCUSSION

In this paper, we show for the first time that the multiligandreceptor LRP functions as the endocytic receptor for decorin.To sustain this fact, we examine the inhibition of decorin endo-cytosis and degradation by specifically abolishing the expres-sion of LRP using siRNA and by precluding decorin uptakeusing RAP, an inhibitor of the interaction between LRP andseveral ligands (20). Furthermore, experiments using cells thatdo not express LRP showed these to be unable to take up anddegrade decorin. LRP was also found to interact with decorinthrough the proteoglycan’s core protein. Thus, either thewholeproteoglycan molecule or its core protein were specificallyendocytosed and degraded, and both processes were inhibitedby RAP or depletion of LRP using siRNA.We also show that theendocytosis of decorin resulted from a direct interactionbetween LRP and decorin. Evidence for this was provided bycross-linking assays using radiolabeled decorin and by co-im-munoprecipitating LRP with labeled decorin, noting again thatboth reactions were inhibited by RAP. The interaction between

decorin and LRP was deemed specific, since in the presence ofexogenous competitors, namely the decorin core protein or thefull molecule, the co-immunoprecipitation of LRP and decorinwas reduced. The LRP-mediated clearance and degradationprocesses of sulfated decorin was partially blocked with chon-droitin sulfate alone or the core protein of decorin. However,the inhibitory effect of both elements was additive, suggestingthat the core and the GAGs moieties have a role in the interac-tion with LRP.Experiments measuring the kinetics of decorin internaliza-

tion and degradation suggested that lysosomes were likely to beinvolved in these processes, given that the lysosomal inhibitorchloroquine inhibited the degradation of radiolabeled decorincore protein by �90%. The related proteoglycan biglycan wasalso able to inhibit decorin degradation, confirming previousfindings (54) and suggesting that biglycan could also be endo-cytosed by LRP. The endocytosis of decorin was first describedby Kresse’s group (16, 55), although the receptor required fordecorin binding and subsequent endocytosis was not identified.A protein of 51 kDa, present in endosomes and at the plasmamembrane, has been suggested to act as the decorin receptordue to its high affinity for the decorin core protein (18, 55, 56).However, no data have shown this protein to have a functionalrole in the uptake and degradation of decorin, as it was clearly

FIGURE 6. Decorin binding to LRP decreases in myoblasts treated withLRP siRNA. A, myoblasts were transfected with a 75 nM concentration ofeither control siRNA (si-Ctrl) or LRP siRNA (si-LRP), and control experimentswithout siRNA (Lipo) were also included (44). Western blotting for LRP wasperformed in cell extracts, 72 h after transfection. Tubulin is shown as a pro-tein loading control. B, cross-linking assays were carried out in myoblastsincubated with [125I]decorin core protein at 4 °C for 3 h. Following incubation,cells were washed and treated with the cross-linker DSS, extracts were sepa-rated by SDS-PAGE, and gels were dried and exposed by phosphorimaging(upper panel). Gel staining with Coomassie Blue is shown in the lower panel.

FIGURE 7. Decorin endocytosis and degradation decrease in myoblaststreated with LRP siRNA. As for Fig. 6, myoblasts were transfected with a 75nM concentration of either control siRNA (si-Ctrl) or LRP siRNA (si-LRP), includ-ing control experiments without siRNA (Lipo) (44). 72 h after transfection, cellswere incubated with [35S]decorin at 37 °C for 3 h and then analyzed to deter-mine endocytosis (A) and degradation of [35S]decorin (B), as for Fig. 1. Endo-cytosis is expressed as the clearance of [35S]decorin (volume/mg of protein/h)Percentage degradation values were normalized against control cells.

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shown for LRP in this work. Although our results clearly dem-onstrate that decorin is endocytosed via LRP and subsequentlydegraded, a possible role for the 51-kDa protein in these pro-cesses cannot be discarded.On the other hand, the endocytic receptor of decorin has also

been suggested to contain a binding site for heparin/heparansulfate, after observation of the inhibition of decorin endocyto-sis in the presence of heparin (52, 57). Several LRP ligands, suchas factor IXa and factor VIII complexed with van Willebrandfactor, bind and are also properly presented to the receptor byheparan sulfate proteoglycans (58). This could also be the casefor decorin, since its endocytosis and degradation were inhib-ited by both RAP and heparin. LRP also binds and endocytosesseveral ECM molecules, including fibronectin (24), throm-bospondin (21–23), plasminogen activators (27, 59), andmatrixmetalloproteinases (27), thereby regulating their bioavailabilityand functions. These processes involve, in many cases, the acti-vation of signal transduction pathways related to differentphysiological roles. Thus, LRP participates in events such as cellmigration (30, 60), myofibroblast differentiation (33), growth

inhibition (61), lipoprotein catabo-lism (62), angiogenesis, and metas-tasis (19, 27) among others. There-fore, it is appealing to study thepotential role of LRP in modulatingthe bioavailability and consequentsignaling of decorin specifically dur-ing myogenesis (14). Here we showthat upon differentiation of C2C12myoblasts, LRP expressiondecreased, which would have func-tional implications for several pro-cesses, including ECM formationand composition, essential pro-cesses that are regulated during dif-ferentiation (64). The regulation ofLRP expression is complex anddepends on the cellular context. Forexample, both the overexpressionand down-regulation of LRP havebeen observed in different cancers(65, 66). In adipocytes, LRP is up-regulated by peroxisome prolifera-tor-activated receptor � (67). InPC12 cells, TGF-�2 increasesmRNA and protein expression ofLRP (68), and in retinal pigment epi-thelial cells, LRP-1 mRNA expres-sion is strongly increased upon cellstimulation with TGF-�1, TGF-�2,or vascular endothelial growth fac-tor whereas platelet-derived growthfactor and fibroblast growth factortype 2 elicited only minor effects(69). In vascular smooth musclecells, LDL up-regulates LRP expres-sion (70), and in skeletal muscle,LRP expression is decreased upon

insulin treatment (41). Consequently, it would be interesting tocharacterize the regulatory elements involved in the decrease ofLRP expression reported here during skeletal muscledifferentiation.As already mentioned, decorin is also involved in several cel-

lular processes, such as cell adhesion, migration, proliferation,and signaling (for a review see Ref. 71). Therefore, the synthesisand degradation of decorin and accessory molecules requiredduring such processes must be highly regulated. In skeletalmuscle, we have previously shown that decorin synthesis is lowin myoblasts yet increases during skeletal muscle differentia-tion (51). This observation is concordantwith the requirementsof a functional and organized ECM in order for skeletal muscledifferentiation to proceed successfully (64, 72). It is well knownthat decorin interactswithmanyECMconstituents, the expres-sion of which is also up-regulated duringmyogenesis, includingcollagen type I, II, and IV (73, 74) and fibronectin (75), and ispresent in the correctly organized ECM of skeletal muscle (76,77). Moreover, decorin is known to induce growth arrest andretard the growth of a variety of tumor cells (78, 79). This

FIGURE 8. CHO cells expressing endogenous or recombinant LRP endocytose and degrade decorin. A,LRP-null CHO cells (gray bar) were assayed for [125I]decorin core protein degradation. LRP-expressing C2C12cells (white bars) and CHO-K1 (wild type) cells (black bars), used as controls, were submitted to competitionassays with GST-RAP and GST. B, LRP-null CHO cells, expressing minireceptors containing the second (mLRP2)or fourth (mLRP4) LRP ligand-binding domains, were assayed for [35S]decorin endocytosis and degradation inthe absence (control, black bars) or presence of either full-length decorin (Dcn full, light gray bars) or GST-RAP(dark gray bars). CHO-K1 cells and LRP-null CHO cells were used as controls. As before, endocytosis is expressedas the clearance of [35S]decorin (volume/mg of protein/h), and degradation values, shown as percentages,were normalized against controls, in this case CHO-K1 cells, with the corresponding error bars.

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growth arrest is associated with an induction of p21, a potentinhibitor of cyclin-dependent kinase activity (80). It has alsobeen suggested that the epidermal growth factor receptor couldbe involved, through mobilization of intracellular Ca2�, in apossible mechanism by which decorin causes growth suppres-sion (81). Myoblasts are the source of myogenic cells for theformation of skeletal muscle fibers duringmuscle developmentand regeneration (82, 83). The number of skeletal muscle pre-cursors or satellite cells is generally quite small, and an activeprocess of myoblast proliferation precedes the formation andrepair of injured muscle (84). One can therefore speculate thatthe active clearance of decorin by LRP from the myoblast con-ditioned medium, described in this paper, could result in a lossof decorin inhibition upon myoblast proliferation during thisstage of differentiation. Since LRP protein levels decrease dur-ing skeletal muscle differentiation, it is likely that when prolif-eration ceases, decorin is partially if not totally incorporatedinto the ECM. Interestingly, decorin also binds TGF-�, a stronginhibitor ofmuscle formation (85), andwe have data suggestingthat decorin and biglycan modulate the bioavailability ofTGF-� for its transducing receptors by sequestering TGF-�to the ECM. Indeed, the accumulation of decorin and bigly-can at the ECM has a strong inhibitory effect on the bindingof TGF-� to its transducing receptors and its subsequentsignaling activity.5The binding of growth factors to proteoglycans and the con-

sequent modulation of growth factor activities represent animportant conceptual advance in the field. In myoblasts, wehave shown that the expression of the plasmamembrane-asso-ciated proteoglycans syndecan-1 and -3 is not only down-reg-ulated during skeletal muscle differentiation (63, 86) but is alsocritical in order to present fibroblast growth factor type 2 to itstransducing receptors and thereby modulate myogenesis (47,63). We and others have shown that decorin can stimulateTGF-�-dependent signaling in osteoblasts and nondifferenti-ated myoblasts (14, 40), and it has been suggested that decorinmight interact with certain cell surface proteins or receptors asa means of presenting TGF-� to its transducing receptors. Thefact that LRPbinds decorin inmyoblasts therefore points to thisreceptor as a possible candidate for the modulation of TGF-�activity by decorin, observed previously (14).

Acknowledgments—We thank Dr. Guojun Bu (Washington Univer-sity School of Medicine, St. Louis, MO) for providing the plasmidencoding mLRP2 and rabbit anti-LRP antibody.

REFERENCES1. Scott, P. G., McEwan, P. A., Dodd, C. M., Bergmann, E. M., Bishop, P. N.,

and Bella, J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 15633–156382. Scott, P. G., Grossmann, J. G., Dodd, C. M., Sheehan, J. K., and Bishop,

P. N. (2003) J. Biol. Chem. 21, 213. Reed, C. C., and Iozzo, R. V. (2002) Glycoconj. J. 19, 249–2554. Merle, B., Durussel, L., Delmas, P., and Clezardin, P. (1999) J. Cell. Bio-

chem. 75, 538–5465. Merle, B.,Malaval, L., Lawler, J., Delmas, P., andClezardin, P. (1997) J. Cell.

Biochem. 67, 75–836. Yamaguchi, Y., Mann, D., and Ruoslahti, E. (1990) Nature 346, 281–2847. Schonherr, E., Sunderkotter, C., Iozzo, R. V., and Schaefer, L. (2005) J. Biol.

Chem. 280, 15767–157728. Tufvesson, E., and Westergren-Thorsson, G. (2002) FEBS Lett. 530,

124–1289. Biglari, A., Bataille, D., Naumann, U., Weller, M., Zirger, J., Castro, M. G.,

and Lowenstein, P. R. (2004) Cancer Gene Ther. 11, 721–73210. Stander, M., Naumann, U., Dumitrescu, L., Heneka, M., Loschmann, P.,

Gulbins, E., Dichgans, J., and Weller, M. (1998) Gene Ther. 5, 1187–119411. Grant, D. S., Yenisey, C., Rose, R. W., Tootell, M., Santra, M., and Iozzo,

R. V. (2002) Oncogene 21, 4765–477712. Reed, C. C., Gauldie, J., and Iozzo, R. V. (2002) Oncogene 21, 3688–369513. Moscatello, D. K., Santra, M., Mann, D. M., McQuillan, D. J., Wong, A. J.,

and Iozzo, R. V. (1998) J. Clin. Invest. 15, 406–41214. Riquelme, C., Larrain, J., Schonherr, E., Henriquez, J. P., Kresse, H., and

Brandan, E. (2001) J. Biol. Chem. 276, 3589–359615. Schmidt, G., Hausser, H., and Kresse, H. (1990) Biochem. J. 266, 591–59516. Hausser, H., Ober, B., Quentin-Hoffmann, E., Schmidt, B., and Kresse, H.

(1992) J. Biol. Chem. 267, 11559–1156417. Truppe, W., and Kresse, H. (1978) Eur. J. Biochem. 85, 351–35618. Hausser, H., Wedekind, P., Sperber, T., Peters, R., Hasilik, A., and Kresse,

H. (1996) Eur. J. Cell Biol. 71, 325–33119. Herz, J., and Strickland, D. K. (2001) J. Clin. Invest. 108, 779–78420. Bu, G., and Marzolo, M. P. (2000) Trends Cardiovasc. Med. 10, 148–15521. Godyna, S., Liau, G., Popa, I., Stefansson, S., and Argraves, W. S. (1995)

J. Cell Biol. 129, 1403–141022. Mikhailenko, I., Kounnas,M. Z., and Strickland, D. K. (1995) J. Biol. Chem.

270, 9543–954923. Chen, H., Sottile, J., Strickland, D. K., andMosher, D. F. (1996) Biochem. J.

318, 959–96324. Salicioni, A. M., Mizelle, K. S., Loukinova, E., Mikhailenko, I., Strickland,

D. K., and Gonias, S. L. (2002) J. Biol. Chem. 277, 16160–1616625. Herz, J., Clouthier, D. E., and Hammer, R. E. (1992) Cell 71, 411–42126. Bu, G., Williams, S., Strickland, D. K., and Schwartz, A. L. (1992) Proc.

Natl. Acad. Sci. U. S. A. 89, 7427–743127. Yang, Z., Strickland, D. K., and Bornstein, P. (2001) J. Biol. Chem. 276,

8403–840828. Segarini, P. R., Nesbitt, J. E., Li, D., Hays, L. G., Yates, J. R., III, and Car-

michael, D. F. (2001) J. Biol. Chem. 276, 40659–4066729. Gonias, S. L., Wu, L., and Salicioni, A. M. (2004) Thromb. Haemost. 91,

1056–106430. Orr, A.W., Elzie, C. A., Kucik, D. F., andMurphy-Ullrich, J. E. (2003) J. Cell

Sci. 116, 2917–292731. Boucher, P., Liu, P., Gotthardt, M., Hiesberger, T., Anderson, R. G., and

Herz, J. (2002) J. Biol. Chem. 277, 15507–1551332. Loukinova, E., Ranganathan, S., Kuznetsov, S., Gorlatova, N., Migliorini,

M. M., Loukinov, D., Ulery, P. G., Mikhailenko, I., Lawrence, D. A., andStrickland, D. K. (2002) J. Biol. Chem. 277, 15499–15506

33. Yang, M., Huang, H., Li, J., Li, D., and Wang, H. (2004) FASEB J. 18,1920–1921

34. Huang, S. S., Leal, S. M., Chen, C. L., Liu, I. H., and Huang, J. S. (2004)FASEB J. 18, 1719–1721

35. Tseng, W. F., Huang, S. S., and Huang, J. S. (2004) FEBS Lett. 562, 71–7836. Schonherr, E., Broszat,M., Brandan, E., Bruckner, P., andKresse,H. (1998)

Arch. Biochem. Biophys. 355, 241–24837. Schmidt, G., Hausser, H., and Kresse, H. (1991) Biochem. J. 280, 411–41438. Winnemoller, M., Schon, P., Vischer, P., and Kresse, H. (1992) Eur. J. Cell

Biol. 59, 47–5539. Nili, N., Cheema, A. N., Giordano, F. J., Barolet, A. W., Babaei, S., Hickey,

R., Eskandarian,M. R., Smeets,M., Butany, J., Pasterkamp, G., and Strauss,B. H. (2003) Am. J. Pathol. 163, 869–878

40. Takeuchi, Y., Kodama, Y., and Matsumoto, T. (1994) J. Biol. Chem. 269,32634–32638

41. Boucher, P., Ducluzeau, P. H., Davelu, P., Andreelli, F., Vallier, P., Riou,J. P., Laville, M., and Vidal, H. (2002) Biochim. Biophys. Acta 1588,226–231

42. Marzolo, M. P., Yuseff, M. I., Retamal, C., Donoso, M., Ezquer, F., Farfan,5 Droguett, R., Cabello-Verrugio, C., Riquelme, C., and Brandan, E. (2006)

Matrix Biol. 25, 332–341.

LRP Is an Endocytic Receptor for Decorin

31570 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 42 • OCTOBER 20, 2006

at PON

TIFIC

IA U

NIV

ER

SIDA

D on M

arch 17, 2014http://w

ww

.jbc.org/D

ownloaded from

P., Li, Y., and Bu, G. (2003) Traffic 4, 273–28843. Obermoeller-McCormick, L. M., Li, Y., Osaka, H., FitzGerald, D. J.,

Schwartz, A. L., and Bu, G. (2001) J. Cell Sci. 114, 899–90844. Li, Y., Lu, W., and Bu, G. (2003) FEBS Lett. 555, 346–35045. Cuitino, L., Matute, R., Retamal, C., Bu, G., Inestrosa, N. C., andMarzolo,

M. P. (2005) Traffic 6, 820–83846. Yaffe, D., and Saxel, O. (1977) Nature 270, 725–72747. Larrain, J., Carey, D. J., and Brandan, E. (1998) J. Biol. Chem. 273,

32288–3229648. Li, Y., Marzolo, M. P., van Kerkhof, P., Strous, G. J., and Bu, G. (2000)

J. Biol. Chem. 275, 17187–1719449. Lopez-Casillas, F., Riquelme, C., Perez-Kato, Y., Ponce-Castaneda, M. V.,

Osses, N., Esparza-Lopez, J., Gonzalez-Nunez, G., Cabello-Verrugio, C.,Mendoza, V., Troncoso, V., and Brandan, E. (2003) J. Biol. Chem. 278,382–390

50. Obermoeller, L. M., Chen, Z., Schwartz, A. L., and Bu, G. (1998) J. Biol.Chem. 273, 22374–22381

51. Brandan, E., Fuentes, M. E., and Andrade, W. (1991) Eur. J. Cell Biol. 55,209–216

52. Hausser, H., Witt, O., and Kresse, H. (1993) Exp. Cell Res. 208, 398–40653. Bu, G., Maksymovitch, E. A., Geuze, H., and Schwartz, A. L. (1994) J. Biol.

Chem. 269, 29874–2988254. Hausser, H., Schonherr, E., Muller, M., Liszio, C., Bin, Z., Fisher, L., and

Kresse, H. (1998) Arch. Biochem. Biophys. 349, 363–37055. Hausser, H., and Kresse, H. (1991) J. Cell Biol. 114, 45–5256. Hausser, H., Hoppe,W., Rauch, U., and Kresse, H. (1989) Biochem. J. 263,

137–14257. Hausser, H., and Kresse, H. (1999) Biochem. J. 344, 827–83558. Nykjaer, A., and Willnow, T. E. (2002) Trends Cell Biol. 12, 273–28059. Warshawsky, I., Herz, J., Broze, G. J., Jr., and Schwartz, A. L. (1996) J. Biol.

Chem. 271, 25873–2587960. Webb, D. J., Nguyen, D. H., and Gonias, S. L. (2000) J. Cell Sci. 113,

123–13461. Huang, S. S., Ling, T. Y., Tseng, W. F., Huang, Y. H., Tang, F. M., Leal,

S. M., and Huang, J. S. (2003) FASEB J. 17, 2068–208162. Wilsie, L. C., and Orlando, R. A. (2003) J. Biol. Chem. 278, 15758–1576463. Fuentealba, L., Carey, D. J., and Brandan, E. (1999) J. Biol. Chem. 274,

37876–3788464. Osses, N., and Brandan, E. (2002) Am. J. Physiol. 282, C383–C39465. Desrosiers, R. R., Rivard, M. E., Grundy, P. E., and Annabi, B. (2006) Pedi-

atr. Blood Cancer 46, 40–49

66. Benes, P., Jurajda, M., Zaloudik, J., Izakovicova-Holla, L., and Vacha, J.(2003) Breast Cancer Res. 5, R77–R81

67. Gauthier, A., Vassiliou, G., Benoist, F., and McPherson, R. (2003) J. Biol.Chem. 278, 11945–11953

68. Harris-White, M. E., and Frautschy, S. A. (2005) Curr. Drug Targets CNSNeurol. Disord. 4, 469–480

69. Hollborn, M., Birkenmeier, G., Saalbach, A., Iandiev, I., Reichenbach, A.,Wiedemann, P., and Kohen, L. (2004) Invest. Ophthalmol. Vis. Sci. 45,2033–2038

70. Llorente-Cortes, V., Otero-Vinas, M., Sanchez, S., Rodriguez, C., andBadimon, L. (2002) Circulation 106, 3104–3110

71. Iozzo, R. V. (1999) J. Biol. Chem. 274, 18843–1884672. Melo, F., Carey, D. J., and Brandan, E. (1996) J. Cell. Biochem. 62, 227–23973. Bidanset, D. J., Guidry, C., Rosenberg, L. C., Choi, H. U., Timpl, R., and

Hook, M. (1992) J. Biol. Chem. 267, 5250–525674. Scott, J. E., and Hughes, E. W. (1986) Connect. Tissue Res. 14, 267–27875. Asundi, V. K., and Dreher, K. L. (1992) Eur. J. Cell Biol. 59, 314–32176. Brandan, E., Fuentes, M. E., and Andrade, W. (1992) J. Neurosci. Res. 32,

51–5977. Caceres, S., Cuellar, C., Casar, J. C., Garrido, J., Schaefer, L., Kresse, H., and

Brandan, E. (2000) Eur. J. Cell Biol. 79, 173–18178. Santra, M., Mann, D. M., Mercer, E. W., Skorski, T., Calabretta, B., and

Iozzo, R. V. (1997) J. Clin. Invest. 100, 149–15779. Santra, M., Skorski, T., Calabretta, B., Lattime, E. C., and Iozzo, R. V.

(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7016–702080. De Luca, A., Santra, M., Baldi, A., Giordano, A., and Iozzo, R. V. (1996)

J. Biol. Chem. 271, 18961–1896581. Patel, S., Santra, M., McQuillan, D. J., Iozzo, R. V., and Thomas, A. P.

(1998) J. Biol. Chem. 273, 3121–312482. Bischoff, R. (1986) Dev. Biol. 115, 129–13983. Casar, J. C., McKechnie, B. A., Fallon, J. R., Young, M. F., and Brandan, E.

(2004) Dev. Biol. 268, 358–37184. Mouly, V., Aamiri, A., Bigot, A., Cooper, R. N., Di Donna, S., Furling, D.,

Gidaro, T., Jacquemin, V., Mamchaoui, K., Negroni, E., Perie, S., Renault,V., Silva-Barbosa, S. D., and Butler-Browne, G. S. (2005) Acta Physiol.Scand. 184, 3–15

85. Florini, J. R., Roberts, A. B., Ewton, D. Z., Falen, S. L., Flanders, K. C., andSporn, M. B. (1986) J. Biol. Chem. 261, 16509–16513

86. Larrain, J., Cizmeci-Smith, G., Troncoso, V., Stahl, R. C., Carey, D. J., andBrandan, E. (1997) J. Biol. Chem. 272, 18418–18424

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