Research Article 5305 Mesd binds to mature LDL--related -6 and antagonizes ligand binding

Yonghe Li1,*,‡, Jianglei Chen2, Wenyan Lu1, Lynn M. McCormick1, Jianjun Wang2 and Guojun Bu1,3 1Department of Pediatrics, St Louis Children’s Hospital, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA 2Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, 224 Neckers Hall, Carbondale, IL 62901, USA 3Departments of Cell Biology and Physiology, St Louis Children’s Hospital, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA *Present address: Biochemistry and Molecular Biology Department, Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35255, USA ‡Author for correspondence (e-mail: [email protected])

Accepted 17 August 2005 Journal of Cell Science 118, 5305-5314 Published by The Company of Biologists 2005 doi:10.1242/jcs.02651

Summary Wnt co-receptors LRP5 and LRP6 are two members of absent in sequences from invertebrates, is necessary and the low-density receptor family. Receptor- sufficient for binding to mature LRP6, and is required for associated protein is not only a specialized chaperone but LRP6 folding. We also found that LRP6 is not a also a universal antagonist for members of the low-density constitutively active endocytosis receptor and binding of lipoprotein receptor family. Here we test whether Mesd, a the receptor-associated protein to LRP6 partially competes newly identified chaperone for members of the low-density for Mesd binding. Finally, we demonstrated that Mesd lipoprotein receptor family, also binds to mature receptors antagonizes ligand binding to LRP6 at the cell surface. at the cell surface and antagonizes ligand binding. We Together our results show that in addition to serving as a found that Mesd binds to cell surface LRP5 and LRP6, but folding chaperone, Mesd can function as a receptor not to other members of the low-density lipoprotein antagonist by inhibiting ligand binding to mature LRP6. receptor family. Scatchard analysis revealed that Mesd binds cell surface LRP6 with high affinity (Kd ~3.3 nM). Interestingly, the C-terminal region of Mesd, which is Key words: LRP6, LDLR family, Wnt signaling, Mesd, RAP, DKK1

Journal of Cell Science Introduction (Brennan et al., 2004; Tamai et al., 2004). LRP6-deficient mice The canonical is involved in various are perinatal lethal and exhibit mid/hindbrain defects, posterior differentiation events during embryonic development and can truncation and abnormal limb patterning. These phenotypes are lead to tumor formation when aberrantly activated (Orford et similar to those observed in mice carrying in Wnt al., 1997; Wodarz and Nusse, 1998; Giles et al., 2003; Lustig , specifically Wnt1, Wnt3a and Wnt7a (Pinson et al., and Behrens, 2003; He et al., 2004). The low-density 2000). LRP5-deficient mice have normal embryogenesis, grow lipoprotein-receptor (LDLR)-related protein-5 (LRP5) and to adulthood and are fertile, but show defects in bone accrual, LRP6 are two members of the expanding LDLR family (Herz eye development, and lipoprotein and glucose metabolism and Bock, 2002; Schneider and Nimpf, 2003; He et al., 2004). (Kato et al., 2002; Fujino et al., 2003; Magoori et al., 2003). Recent studies have demonstrated that these two receptors are The 39 kDa receptor-associated protein (RAP) is a indispensable elements of the canonical Wnt pathway by specialized molecular chaperone for the members of the LDLR interacting with several components of the Wnt signaling family, and functions intracellularly as a molecular chaperone pathway. The extracellular domain of LRP5/6 can bind at least to facilitate receptor folding and trafficking within the early four types of secreted including Wnts, Dickkopfs secretory pathway (Bu, 2001). RAP is also a unique receptor (DKKs), Wise, and the connective-tissue growth factor (Pinson antagonist for members of the LDLR family. RAP, which et al., 2000; Tamai et al., 2000; Wehrli et al., 2000; Wu et al., binds with high affinity to LRP, megalin, very-low-density 2000; Bafico et al., 2001; Bo et al., 2001; Semenov et al., 2001; lipoprotein receptor (VLDLR), and E receptor Brott and Sokol, 2002; Itasaki et al., 2003; Mercurio et al., 2 (apoER2), and with a lower affinity to the LDLR, is able to 2004). The cytoplasmic domains of LRP5/6, upon receptor inhibit the binding of most currently known ligands of LDLR activation by Wnt proteins, recruit the cytosolic scaffold family members (Bu, 2001). protein axin to the membrane, therefore preventing it from More recently, two groups have identified a novel participation in the degradation of ␤- (Mao et al., 2001; specialized chaperone for members of the LDLR family, Liu et al., 2003; Tolwinski et al., 2003; Tamai et al., 2004). termed Mesd (mesoderm development) in mouse (Hsieh et al., Recently, it as been reported that a PPPSP motif, which is 2003) and Boca in Drosophila (Culi and Mann, 2003). This reiterated five times in the LRP5/6 cytoplasmic tail, is new chaperone (195 amino acids in mouse) was discovered due necessary and sufficient to trigger Wnt/␤-catenin signaling to its requirement for the folding of LRP5/LRP6, co-receptors 5306 Journal of Cell Science 118 (22)

for the Wnt/Wg signaling pathway. In the mouse, the containing 10% fetal bovine serum. Culture conditions of U87, MCF- consequences of Mesd deficiency resemble that seen in - 7, and human aortic smooth muscle cells have been described before deficient mutants. However, the presence of more extensive (Li et al., 2003). HEK293 cells were from ATCC, and cultured in defects also in extra embryonic tissues as well as abnormal cell DMEM containing 10% fetal bovine serum. proliferation in the epiblast of Mesd-deficient embryos suggests that signals involving either other Wnt proteins or Preparation of recombinant Mesd protein other LDLR family members are also affected. Full-length mouse Mesd cDNA was kindly provided by Bernadette Similarly to other ER chaperones, Mesd also carries an ER Holdener (State University of New York at Stony Brook, Stony Brook, retention signal (KDEL in Drosophila, REDL in mammals) NY). The wild-type and mutant forms of mouse Mesd were generated at its C-terminus and localizes to the ER by by polymerase chain reactions, and subcloned into the expression immunohistochemistry (Iadonato et al., 1993; Culi and Mann, vector pET-30a(+) (Novagen) at the EcoRI and HindIII restriction 2003). All members of the LDLR family have at least one six- sites. The integrity of the subcloned DNA sequence was confirmed by ␤ DNA sequencing. Recombinant proteins were overexpressed from bladed -propeller domain, which is immediately followed by pET-30(+)Mesd in E. coli. BL21(DE3) producing a recombinant an (EGF) repeat. Mesd is specifically fusion protein with a polyhistidine metal-binding tail at the N- required for the maturation of these ␤-propeller/EGF modules terminus, and purified with His-Bind Kits from Novagen according to through the secretory pathway (Culi et al., 2004). In the the manufacturer’s protocol. All the recombinant Mesd proteins lack absence of Mesd, LRP5/LRP6 form aggregates in the ER and the Mesd signal peptide. fail to reach the cell surface. In the present studies, we tested whether Mesd, like RAP, Western blotting also binds to cell surface receptors, and antagonizes ligand To examine the expression of the LDLR family members, cells binding of the LDLR family members. Our data show that cultured in six-well plates were lysed with 0.5 ml lysis buffer Mesd is capable of binding mature LRP6 at the cell surface and (phosphate-buffered saline containing 1% Triton X-100 and 1 mM antagonizing ligand binding. PMSF) at 4°C for 30 minutes. Equal quantities of protein were subjected to SDS-PAGE under non-reducing conditions. Following transfer to Immobilon-P membrane, successive incubations with Materials and Methods primary antibody and horseradish peroxidase-conjugated secondary Materials antibody were carried out for 60 minutes at room temperature. The Human recombinant DKK1 protein and mouse recombinant Wnt3a immunoreactive proteins were then detected using the ECL system. ␤ protein were from R&D Systems. Human recombinant RAP protein To examine the cytosolic -catenin level, cells in six-well plates was expressed in a glutathione S-transferase (GST) expression vector were treated with Mesd at various concentrations for 90 minutes at and isolated as described previously (Bu et al., 1993). Monoclonal 37°C. After washing in ice-cold PBS, cells were collected and anti-Myc antibody 9E10 was from Roche. Monoclonal antibody 8G1 homogenized in a glass Dounce homogenizer in buffer consisting of against human LRP was from Research Diagnostics. Monoclonal anti- 100 mM Tris-HCl pH 7.4, 140 mM NaCl, 2 mM DTT, 2 mM PMSF, ϫ TM ␮ HA antibody has been described before (Li et al., 2000). Polyclonal and 1 Complete protease inhibitors (500 l/well). The rabbit anti-LDLR was produced by immunizing rabbits with homogenate was centrifuged for 10 minutes at 500 g, and the

Journal of Cell Science supernatant was further centrifuged at 100,000 g at 4°C for 90 recombinant human LDLR1-294 fragment. Peroxidase-labeled anti- mouse antibody and ECL system were from Amersham Life Science. minutes. The resulting supernatant was designated the cytosolic fraction. The ␤-catenin levels were then examined by western blotting Plasmid pcDNA3.1C-Myc-hLRP5 containing the full-length human using ␤-catenin-specific antibody from Cell Signaling Technology. LRP5 cDNA and plasmid pCS-Myc-hLRP6 containing the full-length The immunoreactive proteins were detected using the ECL system. human LRP6 cDNA were from Cindy Bartels (Case Western Reserve Films showing immunoreactive bands were scanned with a Kodak University, Cleveland, OH) and Christof Niehrs (Deutsches Digital Science DC120 Zoom Digital Camera and band intensities Krebsforschungszentrum, Heidelberg, Germany), respectively. were analyzed with Kodak Digital Science1D Image Analysis Carrier-free Na125I was purchased from NEN Life Science Products. Software. IODO-GEN was from Pierce. Proteins were iodinated by using the IODO-GEN method as described previously (Li et al., 2000). Luciferase reporter assay ␮ Cell lines and cell culture HEK293 cells were plated into six-well plates. For each well, 0.1 g of the TOP-FLASH TCF luciferase construct (Upstate Biotechnology) LRP6-transduced HT1080 cells and the control cells have been was cotransfected with 0.8 ␮g Mesd-expressing vector, 0.8 ␮g Mesd described before (Li et al., 2004), and were cultured in DMEM ␤ ␮ mutant-expressing vector, or empty vector. A -galactosidase- medium containing 10% fetal bovine serum and 350 g/ml G418. The expressing vector (Promega, Madison, WI) was included as an LRP-null CHO cells stably transfected with human LDLR-related internal control for transfection efficiency. After 48 hours, cells were protein (LRP) minireceptor mLRP4, mLRP4 tail mutant lysed and both luciferase and ␤-galactosidase activities were mLRP4tailess (mLRP4 without the cytoplasmic tail), human LDLR- determined with assay kits (Promega). The luciferase activity related protein 1B (LRP1B) minireceptor mLRP1B4, human VLDLR, was determined with a luminometer using the Dual Luciferase Assay or human apoER2 have been described before (Li et al., 2000; Li et system (Promega). Luciferase activity was normalized to the activity al., 2001; Liu et al., 2001), and were cultured in Ham’s F-12 medium of the ␤-galactosidase. containing 10% fetal bovine serum and 350 ␮g/ml G418. A set of genetically derived murine embryonic fibroblasts (MEF) from mouse embryos deficient for LRP and/or LDLR were obtained from Joachim Ligand binding and degradation Herz, University of Texas Southwestern Medical Center at Dallas Cells (2ϫ105) were seeded into 12-well dishes 1 day prior to assay. (Willnow and Herz, 1994; Narita et al., 2002). These are MEF-1 Ligand-binding buffer (minimal Eagle’s medium containing 0.6% (WT), MEF-2 (LRP-deficient), MEF-3 (LDLR-deficient), and MEF- BSA with a different concentration of radioligand, 0.6 ml/well) was 4 (LRP and LDLR-double-deficient), and are cultured in DMEM added to cell monolayers, in the absence or the presence of 500 nM Mesd binds to mature LRP6 5307

unlabeled RAP or 500 nM unlabeled Mesd, followed with incubation for 0-4 hours at 4°C. Thereafter, overlying buffer containing unbound ligand was removed, and cell monolayers were washed and lysed in low-SDS lysis buffer (62.5 mM Tris-HCl pH 6.8, 0.2% SDS, 10% v/glycerol) and counted. The protein concentration of each cell lysate was measured in parallel dishes that did not contain the ligands. Ligand degradation was performed using the methods as described (Li et al., 2000). Briefly, 2ϫ105 cells were seeded into 12-well dishes 1 day prior to assay. Pre-warmed assay buffer (minimal Eagle’s medium containing 0.6% BSA with radioligand, 0.6 ml/well) was added to cell monolayers in the absence or the presence of unlabeled 500 nM RAP or 500 nM Mesd, followed by incubation for 4 hours at 37°C. Thereafter, the medium overlying the cell monolayers was removed and proteins were precipitated by addition of BSA to 10 mg/ml and trichloroacetic acid to 20%. Degradation of radioligand was defined as the appearance of radioactive fragments in the overlying medium that were soluble in 20% trichloroacetic acid.

Kinetic analysis of endocytosis LRP6-transduced HT1080 cells were plated in 12-well plates at a density of 2ϫ105 cells/well and used after overnight culture. Cells were rinsed twice in ice-cold assay buffer (minimal Eagle’s medium containing 0.6% BSA), and 125I-anti-HA IgG was added at 1 nM final concentration in cold assay buffer (0.5 ml/well). The binding of 125I- anti-HA IgG was carried out at 4°C for 90 minutes with gentle rocking. Unbound 125I-anti-HA IgG was removed by washing cell monolayers three times with cold assay buffer. Ice-cold stop/strip solution (0.2 M acetic acid, pH 2.6, 0.1 M NaCl) was added to one set of plates without warming up and kept on ice. The remaining plates were then placed in a 37°C water bath and 0.5 ml assay buffer prewarmed to 37°C was quickly added to cell monolayers to initiate internalization. After each time point, the plates were quickly placed on ice and the assay buffer was replaced with cold stop/strip solution. 125 Fig. 1. Mesd binds to mature LRP6 at the cell surface with high I-anti-HA IgG that remained on the cell surface was stripped by 125 incubation of cell monolayers with cold stop/strip solution for a total affinity. (A) Time course of I-Mesd (5 nM) binding to LRP6- of 20 minutes (0.75 ml for 10 minutes, twice) and counted. Cell transduced HT1080 cells and the control cells. Assay was carried out for the indicated periods at 4°C in the absence (total) or presence of monolayers were then solubilized with low-SDS lysis buffer and 125 125 500 nM Mesd (non-specific). (B) Saturation binding of I-Mesd to

Journal of Cell Science counted. The sum of I-anti-HA IgG that was internalized plus that remaining on the cell surface after each assay was used as the LRP6-transduced HT1080 cells and the control cells. Assay was maximum potential internalization. The fraction of internalized 125I- carried out at indicated concentrations for 3 hours at 4°C in the anti-HA IgG after each time point was calculated and plotted. absence (total) or presence (non-specific) of 500 nM Mesd. (C) Scatchard plots of data in B. All values are the average of triple determinations with the s.d. indicated by error bars. Cell surface DKK1 binding and immunodetection Human DKK1 cDNA (clone MGC:868, IMAGE:3508222) was obtained from Invitrogen and subcloned into pcDNA3 (EcoRI/XbaI). To facilitate immunodetection, a c-Myc epitope was included at the cells stably transduced with LRP6 cDNA. Human HT1080 C-terminus. The integrity of the subcloned DNA sequence was cells, which express undetectable levels of LRP6, were confirmed by DNA sequencing. Human DKK1-conditioned media transduced with a viral vector alone (pLNCX2) or with vector were produced by transient transfection of HEK293 cells with containing LRP6 cDNA (Li et al., 2004) and used for 125I-Mesd pcDNADKK1-Myc in serum-free medium, and allowed to bind to binding (Fig. 1A). 125I-Mesd (5 nM) reached maximal binding LRP6-transduced HT1080 cells and control cells at room temperature after 2 hours incubation at 4°C with LRP6-expressing HT1080 for 60 minutes in the absence or presence of 1 ␮M Mesd. Cells were cells (Fig. 1A). Inclusion of excess unlabeled Mesd (500 nM) then fixed in 4% paraformaldehyde, labeled with anti-Myc completely eliminated this binding. No significant 125I-Mesd monoclonal antibody and detected with Alexa-488 goat anti-mouse binding was seen with the control cells (pLNCX2). Saturation IgG. The immunofluorescence was detected by a laser-scanning confocal microscope (Olympus Fluoview 500). of Mesd specific binding was seen at concentrations of >6.4 nM (Fig. 1B). Scatchard analysis of the binding data revealed that Mesd binds LRP6 with a Kd of ~3.3 nM (Fig. 1C). This affinity of Mesd to LRP6 is comparable to that of RAP to LRP Results (Iadonato et al., 1993). Mesd binds to mature LRP6 at the cell surface To determine whether Mesd binds to other members of the The specialized molecular chaperone RAP binds with high LDLR family, we performed 125I-Mesd binding analysis with affinity to most members of the LDLR family at the cell four groups of cells expressing different members of the LDLR surface. To examine whether Mesd also possesses this feature, family (Fig. 2). In the first experiment, we used HEK293 cells we performed cell surface ligand binding experiments with transiently transfected with cDNAs for the LDLR, LRP5, 5308 Journal of Cell Science 118 (22)

Fig. 2. Mesd binds to mature LRP5/6 but not significantly to other members of the LDLR family. (A) Binding of 125I-Mesd (5 nM) to HEK293 cells transient transfected with human HA-tagged LDLR, Myc-tagged LRP5, Myc-tagged LRP6 or control vector. Lower panel, western blot analysis for the expression of the LDLR, LRP5 and LRP6. Equal amounts of cell lysate were applied for each lane. (B) Binding of 125I-Mesd (5 nM) to LRP-null CHO cells stably transfected with LRP minireceptor mLRP4, LRP1B minireceptor mLRP1B4, VLDLR, apoER2 or empty pcDNA3 vector only. (C) Binding of 125I-Mesd (5 nM) to wild-type murine embryonic fibroblasts (MEF-1) or MEF cell lines genetically deficient in LRP (MEF-2), LDLR (MEF-3) or both (MEF-4). Lower panel, western blot analysis of LRP and the LDLR expression in MEF cell lines. (D) Binding of 125I-Mesd (5 nM) to human breast cancer cell line MCF-7, human glioblastoma cell line U87 and human aortic smooth muscle cells (SMC). Lower panel, western blot analysis of LRP expression in these cell lines. Assays were carried out for 4 hours at 4°C in the absence (total) or presence of 500 nM Mesd. Values are the means of triple determinations with the s.d. indicated by error bars. *P<0.01 indicates a significant difference compared with control cells transfected with empty pcDNA3 vector.

LRP6 or empty pcDNA3 vector. In the second experiment, we 2D). Therefore, specific binding of Mesd to CHO and MEF Journal of Cell Science used LRP-null Chinese hamster ovary (CHO) cells stably cells may reflect endogenous LRP5/LRP6 in these cells. transfected with LRP minireceptor mLRP4, LRP1B minireceptor mLRP1B4, apoER2, VLDLR, or empty pcDNA3 vector (Li et al., 2000; Li et al., 2001; Liu et al., 2001). mLRP4 The C-terminal region of Mesd is necessary and is composed of residues 3274-4525 of the full-length LRP, sufficient for LRP6 binding which includes the fourth cluster of ligand-binding repeats and Having established that Mesd binds to mature LRP6 at the cell the entire C-terminus of the receptor. mLRP1B4 is composed surface with high affinity, we then investigated the Mesd of residues 3276-4599 of the full length LRP1B, which sequences that are required for this interaction. Upon sequence includes the fourth cluster of ligand binding repeats and the comparison of mouse Mesd and its homologs from different entire C-terminus of the receptor. mLRP4 and mLRP1B4 species, we found that the first 12 amino acids of mouse Mesd mimic the function and trafficking of LRP and LRP1B, are absent in Caenorhabditis elegans and Caenorhabditis respectively. In the third experiment, we used wild-type murine briggsae, and that mouse Mesd, as well as human Mesd, has embryonic fibroblasts and murine embryonic fibroblasts with an extra ~30 fragment prior to the conserved ER genetic deficiency of LDLR, LRP, or both (Willnow and Herz, retention signal in its C-terminus (Culi and Mann, 2003; Hsieh 1994; Narita et al., 2002). In the fourth experiment, we used et al., 2003). We thus generated two truncated Mesd mutants the human breast cancer cell line MCF-7, human glioblastoma lacking either the N-terminal region, MESD(12-195), or both cell line U87, and human aortic smooth muscle cells (SMC). the N-terminal and C-terminal regions, Mesd(12-155) (Fig. MCF-7 cells express LRP at an undetectable level, whereas 3A). The ability of these mutants to bind to cell surface LRP6 U87 cells and SMC express abundant LRP (Li et al., 2003). was then assessed. Interestingly, we found that although Interestingly, among the members of the LDLR family truncation of the N-terminal 11 amino acids of mouse Mesd examined, only LRP5 specifically binds to Mesd, albeit at had no effect on LRP6 binding, further truncation of the last lower levels compared to LRP6 when these receptors were 40 amino acids completely abolished LRP6 binding (Fig. 3B). expressed at comparable levels (Fig. 2A). Although there is a We then generated a truncated Mesd mutant containing the last suggestion of Mesd binding to LRP when examined in CHO 45 amino acids of C-terminal region (Fig. 3A), and found that and MEF cells (Fig. 2B,C), specific Mesd binding to U87 or the 45 amino acids are necessary and sufficient for binding to SMC, both of which express abundant LRP, was minimal (Fig. mature LRP6 (Fig. 4). Mesd binds to mature LRP6 5309

Mesd⌬C. The steady-state levels of LRP6 were analyzed by western blotting with the anti-MYC antibody (Fig. 5A). As seen in the figure, two forms of the receptor, i.e. the ER form and mature form (containing complex sugar modifications), were seen for LRP6. In the presence of Mesd coexpression, but not of Mesd⌬C coexpression, the amount of the mature form of LRP6 was significantly increased (Fig. 5A). Activation of canonical Wnt signaling leads to the stabilization of ␤-catenin and regulation of transcription through transcription regulators including lymphoid-enhancing factor (LEF)-1 and T-cell factors (TCF). The TOP-FLASH luciferase reporter contains TCF-binding sites and can be directly activated by the ␤-catenin/TCF complex (Korinek et al., 1997). LRP6 is , and only the mature receptor can reach the cell surface and modulate Wnt signaling (Cong et al., 2004). We next examined the effect of Mesd⌬C on Wnt signaling using the TOP-FLASH luciferase reporter assay in HEK293 cells. As expected, Mesd coexpression, but not Mesd⌬C coexpression, significantly enhanced TCF/LEF transcriptional activity (Fig. 5B). Together, these results suggest that the C-terminal region of Mesd Fig. 3. The C-terminal region of Mesd is required for interaction with LRP6. (A) Schematic representation of Mesd mutants. (B) Binding analyses of 125I- is required for LRP6 folding and its signaling Mesd and its mutants (5 nM) to LRP6-transduced HT1080 cells. Assays were function at the cell surface. carried out for 3 hours at 4°C in the absence or presence of 500 nM Mesd or its mutants as indicated. Values are the means of triple determinations with the s.d. indicated by error bars. LRP6 is not a constitutively active endocytosis receptor and mediates a limited level of Mesd degradation The C-terminal region of Mesd is required for LRP6 Cell surface receptors that traffic between the plasma folding membrane and endocytic compartments contain signals within Journal of Cell Science Mesd is a specific chaperone for LRP5 and LRP6. To examine their cytoplasmic tails that allow for efficient recruitment into the role of this C-terminal region of Mesd on receptor folding, endocytic vesicles. In many cases (e.g. LRP and the LDLR), we generated a Mesd mutant (Mesd⌬C), which lacks the C- these signals are constitutively active and mediate continuous terminal region (amino acids 156-191) but retains the ER receptor endocytosis independently of ligand binding. To retention signal (REDL) (Fig. 3A). We next evaluated a examine whether LRP6 is a constitutively active endocytosis potential role for Mesd⌬C on LRP6 folding. HEK293 cells receptor, we performed kinetic analysis of receptor endocytosis were transiently transfected with cDNA for the LRP6 with with HT1080 cells transduced with HA-tagged LRP6. To cotransfection of control vector, or cDNAs for Mesd or eliminate potential effects of LRP6 ligands on its

Fig. 4. The C-terminal region of Mesd is necessary and sufficient for LRP6 binding. (A) Binding analyses of 125I-Mesd and its mutant Mesd (150-195) (5 nM) to LRP6-transduced HT1080 cells and control cells. (B) Binding analyses of 125I-Mesd and its mutant Mesd (150-195) (5 nM) to LRP6-transduced HT1080 cells. Assays were carried out for 3 hours at 4°C in the absence or presence of 500 nM Mesd or its mutant. Values are the means of triple determinations with the s.d. indicated by error bars. 5310 Journal of Cell Science 118 (22)

internalization without ligand stimulation, we investigated LRP6-mediated Mesd uptake and degradation. HT1080 cells transduced with LRP6 exhibited 125I-Mesd degradation at a level of 320 fmoles/mg cell protein after 4 hours of incubation at 37°C, whereas 125I-Mesd binding following 4 hours of incubation at 4°C was detected at a level as high as 1320 fmoles/mg cell protein (Fig. 7). These results suggest that Mesd binding to LRP6 at the cell surface does not trigger significant endocytosis, and consequently little Mesd uptake and degradation can be detected.

Mesd binding to the cell surface LRP6 does not significantly change the cytosolic ␤-catenin level ␤-catenin is a key molecule in the Wnt/␤-catenin signaling pathway. A cytosolic pool of ␤-catenin interacts with DNA- binding proteins and participates in Wnt signal transduction (Hinck et al., 1994; Gottardi et al., 2001; Klingelhofer et al., 2003). To determine whether Mesd binding to cell surface LRP6 directly regulates Wnt signaling, we studied the effects of Mesd binding on cytosolic ␤-catenin levels in HT1080- LRP6 cells. Thus, LRP6-transduced HT1080 cells were treated with 0.5 to 5 nM Mesd for 2 hours at 37°C, and the cytosolic ␤-catenin levels were examined by western blotting using ␤- Fig. 5. The C-terminal region of Mesd is required for LRP6 folding. catenin antibody. We found that there was no significant (A) HEK293 cells were transiently transfected with the indicated change in the cytosolic ␤-catenin levels upon Mesd treatment cDNAs. Cell lysates were analyzed by SDS-PAGE under reducing (data not shown), indicating that Mesd binding to cell surface conditions and western blotted with anti-FLAG or anti-HA antibodies as indicated. (B) HEK293 cells were cotransfected with LRP6 does not directly modify Wnt signaling. LRP6, MESD, Mesd⌬C or empty pcDNA3 vector and a TCF/LEF transcriptional activity reporter plasmid (TOP-FLASH). The luciferase activity was measured 48 hours after transfection. Values RAP binds to LRP6 and partially competes for Mesd are the means of triple determinations with the s.d. indicated by error binding bars. RAP binds with high affinity to LRP, megalin, VLDLR and apoER2, and with a lower affinity to the LDLR (Bu, 2001). To determine whether RAP also binds LRP6, we performed RAP- Journal of Cell Science internalization, we utilized 125I-anti-HA IgG for LRP6 binding analysis with HT1080 cells stably expressing LRP6 at endocytosis assays. Binding of 125I-anti-HA IgG to HA-tagged 4°C. Control cells, expressing vector alone, exhibited a LRP6 was specific, i.e. the binding of 125I-anti-HA IgG to the moderate level of cell surface 125I-RAP binding, probably HT1080 control cells was minimal when compared to HT1080- mediated by cell surface heparan sulfate proteoglycan and LRP6 cells (Fig. 6A). We used HA-tagged LRP minireceptor endogenous receptors of the LDLR family. The presence of mLRP4 as a positive control and mLRP4tailess (mLRP4 excess unlabeled RAP (500 nM), but not Mesd (500 nm), lacking the cytoplasmic tail) as a negative control for 125I-anti- completely eliminated this binding (Fig. 8A). Compared to the HA IgG endocytosis (Li et al., 2000). Interestingly, we found control cells, LRP6 expressing HT1080 cells displayed ~20% that the endocytosis rate of LRP6 was extremely slow, and was increase of RAP binding, and this increase was abolished by indistinguishable from that of mLRP4tailess (Fig. 6B), excess unlabeled Mesd (Fig. 8A). These results suggest that indicating that LRP6 itself is unable to initiate endocytosis. RAP binds to cell surface LRP6 with a relatively low affinity. Having established that Mesd binds to LRP6 at the cell To determine whether RAP and Mesd bind to identical, surface with high affinity, and that LRP6 does not undergo overlapping, or different sites on the receptors, we performed

Fig. 6. LRP6 is not a constitutively active endocytosis receptor. (A) Anti-HA IgG binding to cell surface HA-tagged LRP6. Binding of 125I-anti-HA IgG (1 nM) to LRP6- transduced HT1080 cells and the control cells was carried out for 90 minutes at 4°C. (B) LRP6 endocytosis. LRP6- transduced HT1080 cells, mLRP4-transfected CHO cells and mLRP4tailess-transfected CHO cells were incubated with 1 nM 125I-anti-HA IgG at 4°C for 90 minutes, and then incubated at 37°C for the indicated times. The amount of internalized anti-HA IgG was determined. Values are the means of triple determinations with the s.d. indicated by error bars. Mesd binds to mature LRP6 5311

Fig. 7. LRP6 exhibits a limited level of Mesd degradation. (A) LRP6-mediated 125I-Mesd (5 nM) degradation in LRP6- transduced HT1080 cells and control cells was carried out for 4 hours at 37°C in the absence or presence of 500 nM Mesd. (B) 125I-Mesd (5 nM) binding to LRP6-transduced HT1080 cells and the control cells was carried out for 4 hours at 4°C in the absence or presence of 500 nM Mesd. Values are the means of triple determinations with the s.d. indicated by error bars.

binding and competition analysis of these two chaperones with 500 nM unlabeled Mesd (Fig. 8B). RAP inhibited 125I-Mesd HT1080 cells stably expressing LRP6. First, we performed binding in a dose-dependent manner with ~60% inhibition binding of 5 nM 125I-Mesd (5 nM) to cell surface LRP6 in the achieved with 500 nM RAP, whereas the same concentration presence of various concentrations of excess unlabeled RAP or of unlabeled Mesd inhibited >90% of 125I-Mesd binding (Fig. 8B). When 125I-Mesd (5 nM) uptake and degradation were performed, 500 nM unlabeled Mesd completely, whereas 500 nM unlabeled RAP only partially, inhibited 125I-Mesd degradation (Fig. 8C). Together, these results suggest that Mesd and RAP probably bind to different, but perhaps adjacent sites on LRP6. The lower affinity of RAP to cell surface LRP6 may also contribute to its lower efficiency in inhibition of Mesd binding.

Mesd antagonizes ligand binding to LRP6 at the cell surface RAP is a unique receptor antagonist for members of the LDLR family, and is able to inhibit the binding of most known ligands of the LDLR family members. To determine whether Mesd is also able to block LRP6 ligand binding, we examined cell surface DKK1 binding by immunostaining. DKK1 is an LRP6- specific ligand and antagonist. As expected, Myc-tagged DKK1 binds to LRP6 cells (Fig. 9B) but not to the control cells Journal of Cell Science (Fig. 9A). Importantly, the presence of Mesd completely blocked the binding of Myc-DKK1 to LRP6 at the cell surface (Fig. 9C). To confirm the above results, we examined the binding and degradation of 125I-DKK1. LRP6-expressing HT1080 cells exhibited significantly higher levels of 125I-DKK1 binding and degradation than the control cells. The increased DKK1 binding and degradation were abolished by excess unlabeled Mesd, but not by excess unlabeled RAP (Fig. 9D,E). Together, these results indicate that Mesd can specifically block DKK1 binding to LRP6 at the cell surface.

Discussion Mesd binds to LRP5 and LRP6 at the cell surface Recent studies have identified Mesd as a novel ER chaperone Fig. 8. RAP binds to LRP6 and partially competes for Mesd binding. that is specifically required for the correct folding and (A) RAP binds to mature LRP6 at the cell surface. Binding of 125I- trafficking of several members of the LDLR family including RAP (5 nM) to LRP6-transduced HT1080 cells and the control cells LRP5, LRP6, LDLR and LRP (Culi and Mann, 2003; Hsieh et was carried out for 4 hours at 4°C in the absence (total) or presence 125 al., 2003; Culi et al., 2004). However, flies or mice with a of 500 nM RAP, or 500 nM Mesd. (B) Binding of I-Mesd (5 nM) nonfunctional Boca or Mesd gene die during embryogenesis to LRP6-transduced HT1080 cells was carried out for 2 hours at 4°C in the absence (total) or presence of various concentrations of RAP and display phenotypes that are consistent with an inability to or 500 nM Mesd. (C) LRP6-mediated 125I-Mesd (5 nM) degradation transduce a Wg/Wnt signal, suggesting that Mesd is probably was carried out for 4 hours at 37°C in the absence or presence of 500 more important for LRP5/6 than for other members of the nM Mesd or 500 nM RAP. Values are the means of triple receptor folding, trafficking, and maturation. Thus, it is not determinations with the s.d. indicated by error bars. surprising as demonstrated here that Mesd binds to cell surface 5312 Journal of Cell Science 118 (22)

LRP5 and LRP6, but not to other members of the LDLR family. Although mouse Mesd protein shares ~45% identity and ~65-85% similarity with open reading frames predicted in Mesd from different species, mouse Mesd as well as human Mesd has an extra ~30 amino acid fragment prior to the conserved C- terminal ER-retention signal (Culi and Mann, 2003; Hsieh et al., 2003). In the present study, we found that this C-terminal region, uniquely present in vertebrate Mesd, is necessary and sufficient for binding to mature LRP6 at the cell surface. We also found that this region is required for LRP6 folding. It is tempting to speculate that the ability of Mesd to bind mature LRP6 and regulate ligand binding/Wnt signaling might be a unique feature of vertebrate Mesd acquired during evolution. Because fly Mesd (termed Boca) (Culi and Mann, 2003), despite lacking this C-terminal region, is also essential for the folding of arrow (the ortholog of LRP5 and LRP6 in Drosophila), the mechanisms of Mesd chaperone function might have also evolved Fig. 9. Mesd inhibits DKK1 binding to LRP6. (A-C) Serum-free conditioned during evolution. The biological significance of our medium was harvested from HEK293 cells transiently transfected with cDNA findings requires further investigation. for human Myc-DKK1 and allowed to bind to LRP6-transduced HT1080 cells (B,C) and control cells (A) in the absence (A,B) or presence (C) of 1 ␮M Mesd. Cell-surface-bound Myc-tagged DKK proteins were fixed and detected by RAP binds to LRP6 immunofluorescence staining with anti-Myc antibody. (D) DKK1 binding to cell To date, the most dramatic effect observed when surface LRP6 is inhibited by Mesd. Binding of 125I-DKK1 (5 nM) to LRP6- RAP is bound to LRP is the inhibition of binding transduced HT1080 cells or the control cells was carried out for 3 hours at 4°C in the absence (total) or presence of 500 nM RAP or 500 nM Mesd. (E) LRP6- and/or uptake of all known LRP ligands. This 125 ability of RAP distinguishes it from other LRP mediated DKK1 degradation is inhibited by Mesd. LRP6-mediated I-Mesd (5 ligands, which seldom inhibit the binding and/or nM) degradation was carried out for 4 hours at 37°C in the absence or presence of 500 nM RAP or 500 nM Mesd. Values are the means of triple determinations uptake of each other. In addition to LRP, RAP also with the s.d. indicated by error bars. Bar, 10 ␮m. binds to other members of the LDLR gene family and inhibits ligand interactions. Studies have shown Journal of Cell Science that RAP exhibits high-affinity binding (KD ~1-10 nM) for 2002) and a member of the gene family which LRP, megalin, the VLDLR and apoER2, but low affinity (KD including DKK1, DKK2, DKK3 and DKK4. By binding to ~250 nM) for the LDLR (Bu, 2001). In the present study, we LRP6, DKK1 disrupts the binding of LRP6 to the Wnt/ demonstrated that RAP binds to LRP6 at a relatively low ligand-receptor complex, and then inhibits Wnt/␤-catenin affinity and that RAP binding to LRP6 can be blocked by signaling (Semenov et al., 2001). In the present study, we Mesd. We also demonstrated that RAP can partially compete demonstrated that Mesd can specifically block DKK1 binding for Mesd binding to LRP6. Thus, Mesd and RAP probably bind to LRP6 at cell surface. to different, but perhaps adjacent sites on LRP6. Alternatively, Mesd binding to cell surface LRP6 does not significantly binding of one chaperone may alter the conformation of the change the cytosolic ␤-catenin level, indicating that Mesd is receptor such that binding of the other becomes less optimal. unable to modify Wnt signaling by directly binding to LRP6 at the cell surface. However, the ability of Mesd in modulation DKK1 binding to LRP6 suggests that Mesd may Mesd antagonizes ligand binding to LRP6 have a physiological role in the regulation of LRP5/6 function Wnt proteins and DKK proteins are two important groups of at the cell surface. In this regard, it is interesting to note that LRP6 extracellular ligands. By using co-immunoprecipitation, the LRP5 that causes high bone mass (G171V) several group reported that LRP6 can bind Wnt1, Wnt3a and disrupts LRP5 interaction with Mesd (Zhang et al., 2004) but Xenopus Wnt8 (Tamai et al., 2000; Mao et al., 2001; Kato et allows for autocrine Wnt signaling within the intracellular al., 2002; Itasaki et al., 2003; Liu et al., 2003). Perhaps as a compartments. We examined total cellular Mesd expression result of weaker binding between Wnt and LRP6 than that by western blotting in over ten human normal and cancer between Wnt and Frizzled, Wnt-LRP6 binding affinities have cell lines and found that Mesd is universally expressed at a not been reported (He et al., 2004). In the present studies, we similar level in all cell lines tested. However, we were unable were unable to detect consistent and specific binding of Wnt3a to detect Mesd in the concentrated conditioned media of to cell surface LRP6 or any effects of exogenous Mesd protein several human cell lines (data not shown). It is possible that on Wnt3a-induced Wnt signaling (data not shown). DKK1 is regulated secretion of Mesd occurs only under certain a high-affinity ligand for LRP6 (Wu et al., 2000; Bafico et al., physiological or pathophysiological conditions. 2001; Bo et al., 2001; Semenov et al., 2001; Brott and Sokol, Alternatively, Mesd may bind to mature LRP5/6 in certain Mesd binds to mature LRP6 5313

intracellular compartments (e.g. endosomes) and regulate four ␤-propeller/EGF modules and three ligand-binding autocrine Wnt signaling. Future studies are required to repeats (Brown et al., 1998; Kim et al., 1998). The formation examine these possibilities. of correct disulfide bonds during receptor folding presents a The ability of RAP to universally inhibit ligand binding challenging task for ER chaperones. The function of Mesd (as prompted the use of recombinant RAP as an antagonist in the well as RAP) during folding may be primarily to inhibit study of LRP and other members of the LDL receptor family, indiscriminate disulfide bond formation, in particular including examination of in vivo functions of these receptors. intermolecularly between different receptor molecules during Thus, the ability of Mesd to regulate LRP6 ligand binding and after their translation. Specifically, Mesd is required for the suggests that recombinant Mesd could also be a useful tool in maturation of these ␤-propeller/EGF modules through the the study of ligand-LRP6 interactions. Application of secretory pathway (Culi et al., 2004). recombinant Mesd protein to cells may also allow for detection The majority of ER chaperones bind only to unfolded, of LRP6-mediated Wnt signaling that is inhibited by misfolded or folding intermediates of their target proteins. endogenous DKK. However, RAP remains bound to the LDLR family members to safeguard their trafficking through the early secretory pathway by antagonizing premature ligand binding (Bu et al., LRP6 endocytosis 1995; Willnow et al., 1996). Results from our current studies Traditionally, all members of the LDLR family have been suggest that Mesd may also possess this feature. We showed regarded as cell surface endocytosis receptors that function in that Mesd binds to cell surface LRP6 and LRP5, and that Mesd delivering their ligands to lysosomes for degradation, although antagonizes DKK1 binding to mature LRP6. The possibility recent studies have revealed new important roles for these that Mesd escorts/safeguards mature LRP5/6 in the secretory receptors in signal transduction. A common characteristic of and/or endocytic pathways requires further investigation. the LDLR family members is that at least one copy of the NPXY sequence is found within the cytoplasmic tails of most We are grateful to Bernadette C. Holdener, Christof Niehrs and members of the LDLR family. For the LDLR, this NPXY motif Cindy Bartels for providing the cDNAs of Mesd, LRP5 and LRP6. serves as a signal for receptor endocytosis through coated pits We also thank Alan L. Schwartz and Theodore C. Simon for helpful (Chen et al., 1990). For LRP, the YXXL motif, but not the two discussion and support during the course of this study. This work was supported by a grant from the American Heart Association NPXY sequences, within the cytoplasmic tail of LRP serves as (0330118N) to Y.L., and a grant from the National Institutes of Health the dominant signal for LRP-mediated endocytosis (Li et al., (DK61761) to G.B. G.B. is an Established Investigator of the 2000). LRP belongs to the class of receptors which undergo American Heart Association. constitutive endocytosis in the presence or absence of ligand. This feature may be determined by the constant exposure of its endocytosis signals, and is highlighted by its cell surface References distribution concentrated within clathrin-coated pits. Bafico, A., Liu, G., Yaniv, A., Gazit, A. and Aaronson, S. A. (2001). 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