Processing by Convertases Is Required for Glypican-3- induced

Processing by Convertases Is Required for Glypican-3-induced Inhibition of Hedgehog Signaling*

Received for publication, September 23, 2014, and in revised form, January 22, 2015 Published, JBC Papers in Press, February 4, 2015, DOI 10.1074/jbc.M114.612705

Mariana Capurro‡, Wen Shi‡, Tomomi Izumikawa‡, Hiroshi Kitagawa§, and Jorge Filmus‡1

From the ‡Biological Sciences, Sunnybrook Research Institute, and Department of Medical Biophysics, University of Toronto,Toronto, Ontario M4N 3M5, Canada and the §Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku,Kobe 658-8558, Japan

Background: Glypican-3 (GPC3) is a proteoglycan that is cleaved by convertases and that inhibits Hedgehog signaling.Results: Convertase-resistant GPC3 stimulates Hedgehog signaling, and unlike wild-type GPC3, binds to Patched through itsheparan sulfate chains.Conclusion: Convertase cleavage is required for GPC3-induced inhibition of Hedgehog signaling.Significance: The structure of the GPC3 core-protein determines the binding properties of heparan sulfate chains.

Glypican-3 (GPC3) is one of the six members of the mamma-lian glypican family. We have previously reported that GPC3inhibits Hedgehog (Hh) signaling by competing with Patched(Ptc) for Hh binding. We also showed that GPC3 binds with highaffinity to Hh through its core protein, but that it does not inter-act with Ptc. Several members of the glypican family, includingGPC3, are subjected to an endoproteolytic cleavage by the furin-like convertase family of endoproteases. Surprisingly, however,we have found that a mutant GPC3 that cannot be processed byconvertases is as potent as wild-type GPC3 in stimulating Wntactivity in hepatocellular carcinoma cell lines and 293T cells andin promoting hepatocellular carcinoma growth. In this study, weshow that processing by convertases is essential for GPC3-in-duced inhibition of Hh signaling. Moreover, we show that a con-vertase-resistant GPC3 stimulates Hh signaling by increasingthe binding of this growth factor to Ptc. Consistent with this, weshow that the convertase-resistant mutant binds to both Hh andPtc through its heparan sulfate (HS) chains. Unexpectedly, wefound that the mutant core protein does not bind to Hh. We alsoreport that the convertase-resistant mutant GPC3 carries HSchains with a significantly higher degree of sulfation than thoseof wild-type GPC3. We propose that the structural changes gen-erated by the lack of cleavage determine a change in the sulfationof the HS chains and that these hypersulfated chains mediate theinteraction of the mutant GPC3 with Ptc.

Glypicans are a family of proteoglycans that are bound to theplasma membrane by a glycosylphosphatidylinositol anchor (1,2). Six members of this family have been identified in mammals(GPC1 to GPC6)2 (1). In general, glypicans display heparan sul-

fate (HS)-type glycosaminoglycan (GAG) chains (3). The inser-tion sites for these GAG chains are located close to the C ter-minus, placing them close to the cell surface, and suggestingthat these chains could mediate the interaction of glypicanswith other cell membrane proteins (4). Notably, glypicans donot have domains with obvious homology to characterizeddomains found in other proteins, suggesting that they haveunique functions.

Genetic and biochemical studies have demonstrated thatglypicans can regulate several signaling pathways, includingthose triggered by Hedgehogs (Hhs) (5–7), Wnts (8, 9), bonemorphogenetic proteins (10 –12), and fibroblast growth factors(13). Glypicans can either stimulate or inhibit the interaction ofthese growth factors with their signaling receptors. The func-tion of a glypican in a specific cellular context depends on itsstructural features and on the set of growth factors and growthfactor receptors present in that cellular context.

GPC3 is widely expressed during development (4). Loss-of-function mutations of GPC3 cause the Simpson-Golabi-Beh-mel overgrowth syndrome (14), and Gpc3-null mice displaydevelopmental overgrowth (15). GPC3 regulates embryonicgrowth by inhibiting the Hh signaling pathway (5, 16). Thisinhibition results from the ability of GPC3 to compete withPatched (Ptc), the Hh signaling receptor, for Hh binding. Theability of GPC3 to act as a competitive inhibitor of Hh signalingis due to the fact that this glypican binds to Hh, but not to Ptc(5). Although it is well known that Hh binds with low affinity toHS, the core protein of GPC3 displays high affinity binding toHh (5). Moreover, the interaction of Hh with GPC3 triggers theendocytosis and degradation of the GPC3-Hh complex, reduc-ing the amount of Hh available to bind to Ptc (5, 17).

Contrary to GPC3, glypican-5 (GPC5) stimulates Hh signal-ing (18). This stimulatory activity is based on the ability of thisglypican to increase the binding of Hh to Ptc (18). Consistentwith this, GPC5 interacts with both Hh and Ptc. Notably, bothinteractions are mediated by the GAG chains. The HS chains ofGPC5 display a significantly higher degree of sulfation thanthose of GPC3 (18). Because the negative charge provided bythe sulfate groups is responsible for most of the interactionsinvolving HS chains, this variation in the degree of sulfation of

* This work was supported by the Canadian Institutes of Health Research.1 To whom correspondence should be addressed: Rm. S218, Sunnybrook

Research Institute, 2075 Bayview Ave., Toronto, Ontario M4N 3M5, Canada.Tel.: 416-480-6100, Ext. 3350; Fax: 416-480-5703; E-mail: [email protected].

2 The abbreviations used are: GPC, glypican; HS, heparan sulfate; GAG, glyco-saminoglycan; Hh, Hedgehog; Ptc, Patched; Shh, Sonic Hedgehog; Dlp,Dally-like protein; HCC, hepatocellular carcinoma(s); AP, alkaline phospha-tase; EF, elongation factor; 2AB, 2-aminobenzadine; HexUA, glucuronicacid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 12, pp. 7576 –7585, March 20, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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the GAG chains may explain the differential interaction ofGPC3 and GPC5 with Ptc. However, it should be noted thatGPC3 displays two GAG chains, and GPC5 displays four GAGchains. This difference in the number of GAG chains could alsohave an impact in the binding properties of GPC3 and GPC5.

In contrast to the inhibitory effect of GPC3 on Hh signaling,it is now well established that this glypican can stimulate bothcanonical and non-canonical Wnt activity (19, 20). GPC3 isexpressed by most hepatocellular carcinomas (HCC) (21), andit promotes HCC growth by stimulating canonical Wnt signal-ing (8). Studies in the Gpc3-null mice have also demonstratedthat this glypican regulates Wnt signaling in normal embryonictissues (19).

Several members of the glypican family, including GPC3, aresubjected to an endoproteolytic cleavage by the furin-like con-vertase family of endoproteases (22). The internal cleavage sitegenerates an �40-kDa N-terminal subunit, as well as an �30-kDa C-terminal subunit that carries the GAG chains. Thesesubunits remain linked to each other by one or more disulfidebonds. Surprisingly, however, a mutant GPC3 that cannot beprocessed by convertases is as potent as wild-type GPC3 instimulating Wnt activity in HCC cell lines and 293T cells and inpromoting HCC growth (23).

To better understand the structural requirements for theregulatory function of GPC3 in Hh signaling, we have investi-gated whether GPC3 needs to be processed by convertases toinhibit the Hh signaling pathway. We provide here experimen-tal evidence showing that the cleavage by convertases is essen-tial for GPC3-induced inhibition of Hh signaling. Moreover, weshow that a convertase-resistant GPC3 mutant stimulates Hhsignaling.

EXPERIMENTAL PROCEDURES

Cell Lines and Plasmids—293T and NIH 3T3 cells (obtainedfrom the ATCC) were cultured in DMEM supplemented with10% FBS at 37 °C in a humidified atmosphere with 5% CO2.293T cells were transfected with Lipofectamine 2000, and NIH3T3 cells were transfected with Lipofectamine LTX-Plus (Invit-rogen). All conditioned media were prepared in 293T cellstransfected with the indicated expression vectors and collected48 h after transfection in serum-free conditions, with theexception of ShhN conditioned medium, which was collected 6days after transfection in the presence of 2% FBS. Expressionvectors for GPC3 and GPC3�GAG in EF, GPC3-AP andGPC3�GAG-AP in APtag-2, ShhN in pcDNA, Sonic Hh-alka-line phosphatase (Shh-AP) in APtag-4, and HA-tagged Ptc inmurine stem cell virus (5); GPC3 RR-AA in EF (23); and GPC5in pCMV (18) were previously described. To generate the GPC3RR-AA�GAG in EF, the GAG attachment sites (Ser494 andSer508) were mutated to alanine by site-directed mutagenesis,and the mutations were verified by sequencing. This non-gly-canated GPC3 RR-AA was still processed by convertases, andadditional mutations were required to generate a non-glyca-nated convertase-resistant GPC3. These mutations involvedchanging three additional arginine residues (Arg387, Arg388,and Arg389) and four lysine residues (Lys371, Lys374, Lys394, andLys396) to alanine, generating GPC3 5R4K-9A�GAG, namedGPC39A�GAG. These additional seven mutations were also

introduced in the GPC3 RR-AA expression vector to generateGPC3 5R4K-9A, named GPC3 9A. The GPC3 RR-AA-AP,GPC3 9A�GAG-AP, and GPC3 9A-AP vectors were preparedby inserting these cDNAs into the BspE1 site of the pAP-tag2vector (Gene Hunter Corp.).

Hh Reporter Assay—NIH 3T3 cells were seeded in 6-wellplates (250,000 cells/well) and cotransfected with a luciferasereporter driven by an Hh-responsive promoter (0.4 �g), theindicated amounts of GPC3 variants or GPC5 expression vec-tors, and �-galactosidase (50 ng). One day after transfection,cells were transferred to a 24-well plate at 50% confluence, andon the following day, ShhN or control conditioned medium(diluted 1/10 in DMEM 2% FBS) was added for 48 h. A lucifer-ase assay was then performed, and the luciferase activity wasnormalized based on the �-galactosidase activity. Glypicanexpression levels in the lysates were verified by Western blotanalysis.

Cell Binding Assay—293T cells transfected with the indicatedexpression vectors were transferred to 8 °C and incubated for2 h with Shh-AP or AP conditioned media containing the sameamount of AP activity. Unbound ligand was removed with fourwashes of PBS, and the cells were lysed in 10 mM Tris-HCl, pH8, 1% Nonidet P-40. Lysate aliquots with equal amounts of pro-tein were heated at 65 °C for 10 min to inactivate the cellularphosphatases, and the AP activity was then measured with aSIGMAFAST p-nitrophenyl phosphate tablet set. Glypicanexpression levels in the whole cell lysates were assessed byWestern blot analysis using the anti-GPC3 mAb 1G12. Whenindicated, endogenous Ptc was immunoprecipitated using therabbit anti-Ptc polyclonal antibody H-267 (Santa Cruz Biotech-nology) before measuring the AP activity in the precipitatedmaterial.

Coimmunoprecipitation—293T cells were transfected withGPC3 RR-AA and Shh expression vectors or vector controls(EF and pcDNA, respectively). Cells were lysed in radioimmu-noprecipitation assay buffer, the lysates were precleared withprotein G-Sepharose during 1 h at 4 °C, and GPC3 was immu-noprecipitated with the 1G12 mAb. The presence of Shh in theprecipitated material was assessed by Western blot with therabbit anti-Shh polyclonal antibody H-160 (Santa CruzBiotechnology).

Pulldown Assay—293T cells were transfected with HA-tagged Ptc or vector control and lysed in radioimmunoprecipi-tation assay buffer, and the lysates were subsequently incubatedwith the 12CA5 anti-HA mAb (Roche Applied Science) andprotein G-Sepharose (Sigma Aldrich). Beads were then blockedwith 5% BSA in PBS containing 0.1% Triton X-100 for 90 min atroom temperature, and aliquots containing equal amounts ofbeads were incubated for 1 h with the indicated AP-glypican orAP conditioned media. After four washes with 20 mM Hepes,pH 7.4, 150 mM NaCl, 0.25% Tween 20, the AP activity bound tothe beads was determined as described above.

Analysis of GAG Chains—NIH-3T3 cells were transientlytransfected with GPC3-AP and GPC3 RR-AA. Two days aftertransfection, the AP activity of each conditioned medium wasmeasured. This activity, which reflects the expression levels ofthe transfected glypicans, was similar for GPC3 (1.326 A405/�lof medium) and GPC3 RR-AA (1.182 A405/�l of medium). AP-

GPC3 Requires Convertase Cleavage to Inhibit Hedgehog

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tagged glypicans were purified from conditioned media by ananion-exchange chromatography on DEAE-Sepharose fol-lowed by affinity chromatography with the anti-AP mAb cou-pled to agarose (Sigma-Aldrich). Briefly, DEAE-Sepharose gelwas added to the conditioned media, and after an overnightincubation at 4 °C, the suspension was collected in an emptycolumn and washed with PBS, and the bound material waseluted with 2 M NaCl in PBS. The eluate was then diluted 4-foldwith water and loaded onto the anti-AP-agarose column. Afterwashing the column with 0.5 M NaCl in PBS, the AP-taggedglypicans were eluted with 100 mM triethylamine, pH 11.5, andimmediately neutralized with 1 M NaH2PO4. Finally, the glypi-can preparations were desalted by using Ultracel� 10K (Milli-pore). Purified glypicans were then digested with a mixture ofheparitinase (HSase) I and III. The digest was labeled with2-aminobenzadine (2AB) and subjected to anion-exchangeHPLC on an amine-bound silica PA-03 column (Pack PA; YMCCo., Ltd.).

Localization of GPC3—NIH 3T3 cells were transfected withthe indicated HA-tagged glypicans, and on the following day,they were plated on poly-L-lysine-treated coverslips. Cells werethen starved in serum-free medium during 5 h to favor ciliaformation and fixed with 4% paraformaldehyde. For immuno-staining, cells were permeabilized with 0.1% Triton X-100 inPBS for 15 min and blocked for 30 min in 5% nonfat dry milk inPBS. Primary cilia were visualized with the anti-acetylatedtubulin mAb (T7451, Sigma-Aldrich), and glypicans weredetected with the anti-HA rat mAb 3F10 (Roche Applied Sci-ence) and the corresponding fluorescein-conjugated secondaryantibodies. Confocal images were generated using a scanninglaser microscope LSM510 version 3.2 SP2 (Carl Zeiss) and aZeiss LSM image browser. Image analysis was performed usingthe program ImageJ, as reported previously (17). Briefly, amask, which was constructed by manually outlining cilia in theimage of acetylated tubulin staining, was applied to GPC3/GPC3 RR-AA-stained images to measure the fluorescenceintensity at cilia. The average fluorescence intensity of otherregions of the cell was obtained by measuring several represen-tative regions on the cell by moving the mask. After subtractingbackground from both fluorescence intensities obtained above,the ratio of fluorescence intensity in the cilium to that outsideof the cilium was calculated.

RESULTS

GPC3 Cleavage by Convertases Is Required for GPC3-inducedInhibition of Hh Signaling—We have previously generated amutant GPC3 (GPC3 RR-AA) that cannot be processed by con-vertases by replacing Arg355 and Arg358 in the intraproteolyticprocessing site RQYR with alanine residues (23). This mutantwas as potent as wild-type GPC3 in stimulating Wnt signalingin HCC cell lines and 293T cells (23).

To determine the role of convertase cleavage in the inhibi-tory activity of GPC3 on Hh signaling, we investigated the effectof the GPC3 RR-AA mutant in an Hh reporter assay in NIH 3T3cells. We have previously used these cells to demonstrate thatGPC3 inhibits Hh signaling (5).

We first confirmed that, as in 293T and other cell lines (23),GPC3 is cleaved by convertases in NIH 3T3 cells, and this cleav-

age is blocked in the GPC3 RR-AA mutant (Fig. 1A). Next, NIH3T3 cells were transfected with an expression vector where theluciferase gene expression is driven by an Hh-responsive pro-moter. As described previously (5), the transient expression ofwild-type GPC3 in these cells inhibits the luciferase activityinduced by Shh-containing conditioned medium in a dose-de-pendent manner (Fig. 1B). However, this inhibitory activity wasnot observed with the convertase-resistant GPC3 mutant. Infact, a modest but significant stimulatory effect was found (Fig.1C). It should be noted that the stimulatory effect is more evi-dent at lower GPC3 RR-AA expression levels, and it tends todisappear at higher expression levels. However, the convertase-resistant mutant was never able to inhibit Hh signaling, even atvery high levels of expression where wild-type GPC3 displays astrong Hh-suppressive activity (see Fig. 5). As an additionalcontrol, we also performed a Hh luciferase reporter assay in thepresence of increasing amounts of GPC5 (Fig. 1D). As reportedpreviously (18), GPC5 stimulated Hh signaling in a dose-depen-dent manner. This stimulation was observed even at highexpression levels.

GPC3 RR-AA Increases the Binding of Hh to Ptc—We havepreviously shown that wild-type GPC3 inhibits the binding ofHh to Ptc (5). Based on the results of the Hh reporter assaydescribed above, we hypothesized that the convertase-resistantmutant would display an opposite effect on the Hh/Ptc interac-tion. To investigate this hypothesis, NIH 3T3 cells were tran-siently transfected with GPC3 RR-AA and were then incubatedwith a Shh-AP fusion protein at 4 °C. After removing theunbound material, cells were lysed, Ptc was immunoprecipi-tated, and the amount of Shh-AP that coimmunoprecipitatedwith Ptc was quantified by measuring the AP activity in theprecipitated material. We found that at low levels of expression,GPC3 RR-AA significantly stimulates in a dose-dependentmanner the binding of Hh to Ptc (Fig. 2). As control, we showedthat in the same assay, expression of wild-type GPC3 inhibitsthe binding of Shh-AP to Ptc (Fig. 2) Consistent with the lucif-erase assay results, the stimulatory effect of GPC3 RR-AA onthe binding of Shh-AP to Ptc was not observed at higher levelsof expression.

GPC3 RR-AA Interacts with Both Hh and Ptc—Next, weinvestigated the mechanism by which GPC3 RR-AA stimulatesthe binding of Hh to Ptc. We previously reported that GPC3binds with high affinity to Hh but that it does not interact withPtc (5). Based on this, we have proposed that GPC3 inhibitsHh-Ptc binding by acting as a competitive inhibitor. GPC5, onthe other hand, stimulates Hh signaling and interacts with bothHh and Ptc (18). We hypothesized therefore that GPC3 RR-AAshould also interact with Hh and Ptc. As a first approach tostudy the interaction of GPC3 RR-AA with Hh, we performed acoimmunoprecipitation assay in 293T cells. These cells weretransiently transfected with expression vectors for GPC3RR-AA and Shh and lysed, and GPC3 RR-AA was immunopre-cipitated. The presence of Shh in the precipitated material wasthen assessed by Western blot. Fig. 3A shows that Shh coimmu-noprecipitated with GPC3 RR-AA. We also analyzed the GPC3RR-AA-Shh interaction in intact cells by performing a cellbinding assay. GPC3 RR-AA- or vector control-transfected293T cells were incubated with conditioned medium contain-




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ing Shh-AP, or AP alone as control, for 2 h at 8 °C. The unboundmaterial was then washed with PBS, cells were lysed, and thecell-bound AP activity was determined. Fig. 3B shows thatShh-AP binds significantly more to the GPC3 RR-AA-trans-fected cells than to control cells.

To investigate the interaction between GPC3 RR-AA andPtc, we performed a pulldown assay. To this end, protein-Gbeads covered with Ptc or control beads were incubated withconditioned media containing equal activities of AP, GPC3 RR-AA-AP, or GPC3-AP fusion proteins. After washing, theamount of AP that remained bound to the beads was measured.As shown in Fig. 3C, there was a significant specific binding ofGPC3 RR-AA-AP to the Ptc-containing beads. Notably, a muchlower binding of GPC3-AP to Ptc-containing beads was alsodetected (Fig. 3C). A pulldown assay was then repeated with

various dilutions of the corresponding AP conditioned media,revealing that GPC3 RR-AA-AP displays a higher bindingcapacity to Ptc than GPC3-AP in all the dilutions tested (Fig.3D). It should be noted that we have previously shown relativelylow levels of interaction between GPC3 and Ptc in bindingassays (18). Because GPC3 is normally outside of the cilium,where Ptc is activated, we have considered that the GPC3/Ptcinteraction detected in pulldown assays was not physiologicallyrelevant (18).

GPC3 RR-AA-Shh and -Ptc Interactions Are Mediated by theGAG Chains—We have previously reported that the wild-typeGPC3 core protein binds with high affinity to Shh (5). To inves-tigate whether the core protein of the GPC3 RR-AA mutantalso interacts with high affinity with Shh, we generated a con-vertase resistant mutant GPC3 that cannot be glycanated. To

FIGURE 1. GPC3 cleavage by convertases is required for GPC3-induced inhibition of Hh signaling. A, the GPC3 RR-AA mutant cannot be cleaved byconvertases in NIH 3T3 cells. Cells were transfected with the indicated HA-tagged vectors. Two days after transfection, cells were lysed, and GPC3 wasimmunoprecipitated (IP) with the 1G12 anti-GPC3 antibody. The immunoprecipitated material was then run through an SDS-PAGE under reducing conditionsand analyzed by Western blot (WB) with the 12CA5 anti-HA antibody. The top asterisk indicates the IgG heavy chain, and the lower asterisk indicates anonspecific band. The short arrow indicates the full-length non-glycanated protein that has not yet reached the cell surface, and therefore cannot be cleaved.The long arrow indicates the cleaved N terminus that carries the HA tag. The two cleaved fragments represent glycosylation variants. It should be noted that theanti-HA antibody is not good at detecting the glycanated protein. B–D, NIH 3T3 cells were transfected with the indicated amounts of wild-type GPC3 (B), GPC3RR-AA mutant (C), or GPC5 (D) expression vectors or vector control (EF/pCMV), along with a luciferase reporter vector driven by a Hh-responsive promoter and�-galactosidase. Cells were incubated for 48 h with ShhN or control conditioned medium, and a luciferase assay was then performed. Bars represent luciferaseactivity after normalization by �-galactosidase (mean � S.D. of triplicates). Each transfection was repeated three times with similar results. One representativeexperiment is shown. Statistical analysis comparing each data set with the correspondent control or between the indicated data sets was performed with theunpaired t test. Significance is indicated as: * � p � 0.05; ** � p � 0.01; and *** � p � 0.001. E, Western blot analysis of GPC3 expression levels in the transfectedNIH 3T3 cells. Arrowhead: GPC3 core protein; bracket: glycanated GPC3.




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this end, serine residues 494 and 508 corresponding to the GAGinsertion sites were mutated to alanine in the GPC3 RR-AAexpression vector. Unexpectedly, this non-glycanated RR-AA(GPC3 RR-AA�GAG) was still cleaved by convertases (data notshown), probably as a result of a greater exposure of additionalprotease cleavage sites on the non-glycanated GPC3 RR-AA.To abolish these cleavages, additional mutations were intro-duced in three other sites displaying convertase consensussequences to generate the GPC3 9A�GAG. The Shh bindingcapacity of this convertase-resistant core protein was then eval-uated by a Shh-AP cell binding assay as described above. Fig. 4Ashows that, unlike cells expressing the wild-type GPC3 coreprotein, the cells expressing GPC3 9A�GAG did not displayspecific binding to Shh-AP. We conclude therefore that theinteraction of the convertase-resistant GPC3 mutant with Shhis mediated by the GAG chains. By repeating the binding withdifferent concentrations of Shh-AP, we also found that theGPC3 RR-AA mutant has a reduced binding affinity to Shh-APas compared with wild-type GPC3 (Fig. 4C).

Next we investigated whether, as in the case of GPC5, theinteraction of GPC3 RR-AA with Ptc is also mediated by theGAG chains. To this end, we performed a Ptc pulldown assaywith a fusion protein that includes the GPC3 9A�GAG mutantand AP (GPC3 9A�GAG-AP). Unlike GPC3 RR-AA-AP, GPC39A�GAG-AP did not bind to the Ptc-covered beads (Fig. 4D),indicating that the GAG chains of GPC3 RR-AA mediate theinteraction with Ptc. As it was shown in Fig. 3C, a lower GAG-mediated interaction of wild-type GPC3 with Ptc was alsodetected.

To confirm that the lack of interaction of the GPC39A�GAG mutant with Hh and Ptc is due to the absence of theGAG chains, and not the consequence of the additional muta-tions, a glycanated GPC3 mutant containing the same muta-tions as GPC3 9A�GAG (GPC3 9A) was generated. Thismutant was as potent as GPC3 RR-AA in stimulating Hh activ-ity in NIH 3T3 cells, and it had a similar capacity to interactwith Hh and Ptc (Fig. 4, E–G). Altogether, from these experi-ments, we conclude that GPC3 RR-AA interacts with Hh andPtc and that the GAG chains are essential for these interactions.

GPC3 9R-A�GAG Does Not Stimulate Hh-induced Lucifer-ase Activity—If the binding of the convertase-resistant mutantGPC3 RR-AA to Hh and Ptc is required for the GPC3 RR-AA-induced stimulation of Hh signaling, it would be expected thatthe GPC3 9A�GAG cannot stimulate Hh signaling. To test this,we performed an Hh luciferase reporter assay in NIH 3T3 cells.As shown previously (5), the transient expression of wild-typeGPC3 or the non-glycanated GPC3 (GPC3�GAG) inhibits theShh-induced luciferase activity in a dose-dependent manner,whereas GPC3 RR-AA displays a stimulatory effect at low levelsof expression (Fig. 5). As expected, we found that GPC39A�GAG does not stimulate Hh signaling (Fig. 5).

The GAG Chains of GPC3 RR-AA Display a Higher Degree ofSulfation than Those of Wild-type GPC3—It is well establishedthat the structure of a protein core of a glypican can have animpact in the type, size, and modifications of their GAG chains(24, 25). Thus, the conformational changes generated by thelack of cleavage into two glypican subunits could alter theaccess of one or more HS-synthesizing enzymes to the site ofGAG synthesis. It is also known that the binding specificity ofHS chains is predominantly determined by the degree and typeof sulfation. In fact, we have previously reported that the HSchains of GPC5, which mediate the high affinity interaction ofthis glypican with Ptc, display a significantly higher degree ofsulfation than those of GPC3, which do not interact with Ptc(18). Because the interaction between the convertase-resistantGPC3 and Ptc is mediated by the GAG chains, we hypothesizedthat the differential interaction of GPC3 RR-AA and GPC3 withPtc is a consequence of a different sulfation profile in their GAGchains. To investigate this possibility, we decided to comparethe sulfation profile of the GPC3 and GPC3 RR-AA GAGchains. To this end, GPC3 RR-AA-AP and GPC3-AP fusionproteins were purified from the conditioned medium of tran-siently transfected NIH 3T3 cells and digested with a mixture ofheparitinase I and III. The disaccharides generated by the diges-tion were then labeled with 2AB, separated by anion-exchangeHPLC, and identified by comparing the position of the elutedpeaks with 2AB-labeled standard HS disaccharides. We foundthat that the proportion of non-sulfated disaccharides (0S) inGPC3-AP is significantly higher than in GPC3-RR-AA-AP (Fig.6). Consistent with this finding, the HS chains purified fromGPC3 RR-AA-AP display a higher proportion of sulfated disac-charides (Fig. 6). This result suggests that the higher sulfation ofthe HS chains of GPC3 RR-AA is responsible for the largerbinding capacity of GPC3 RR-AA to Ptc as compared with thatof GPC3.

GPC3 RR-AA-AP Localizes to the Primary Cilium—It is nowwell established that in mammalian cells, Hh signaling is trig-

FIGURE 2. Convertase-resistant GPC3 stimulates the binding of Hh to Ptc.NIH 3T3 cells were transfected with increasing amounts of wild-type GPC3(GPC3), convertase-resistant mutant GPC3 (RR-AA), or vector control (EF). Cellswere then incubated with Shh-AP or AP during 2.5 h at 4 °C. After washing outthe unbound material, endogenous Ptc was immunoprecipitated, and theamount of Shh-AP bound to the immunoprecipitated Ptc was determined bymeasuring AP activity. Top panel, bars represent the relative amount ofShh-AP bound to Ptc (mean � S.D. of duplicates) after subtraction of thebinding measured for AP alone. The experiment was repeated three timeswith similar results. Statistical analysis comparing each data set with the cor-respondent control or between the indicated data sets was performed withthe unpaired t test. Significance is indicated as: *** � p � 0.001.The amount ofimmunoprecipitated Ptc1 from each sample was assessed by Western analy-sis of the immunoprecipitated material (bottom panel).




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gered at the primary cilium where Hh-Ptc interaction takesplace (26). We have previously reported that GPC5, a glypicanthat stimulates Hh signaling and interacts with Ptc, can befound at the ciliary membrane (18). Conversely, we could notdetect GPC3 at this location. We therefore decided to investi-gate whether the convertase-resistant GPC3 RR-AA also local-izes to the primary cilium. To this end, NIH 3T3 cells weretransfected with GPC3 RR-AA, fixed, and immunostained forGPC3 and acetylated tubulin as a marker of the primary cilium.We found that in cells expressing GPC3 RR-AA levels thatstimulate Hh signaling, this mutated glypican can be clearlydetected at the primary cilium (Fig. 7A). As reported previously(18), we could not detect wild-type GPC3 at this location (Fig. 7B).

DISCUSSION

In this study, we show not only that the convertase process-ing of GPC3 is required for GPC3-induced inhibition of Hhsignaling, but also that a convertase-resistant GPC3 has anunexpected stimulatory effect on Hh signaling. Notably, unlikewild-type GPC3, the convertase-resistant mutant is able to

interact with Ptc. Another important change that we found inthis mutant is that its interaction with Shh is mediated by theGAG chains, and not by the core protein as we previouslyreported for wild-type GPC3 (5). It should be noted that onlytwo amino acids were mutated in the primary sequence ofGPC3 to abolish the processing by convertases. Thus, to explainthe drastic changes in the ability of the convertase-resistantmutant to interact with Shh and Ptc, we propose that the lack ofcleavage into two subunits induces a structural change inGPC3. As shown in Fig. 8, this structural change in the GPC3mutant core protein could alter or hide the Hh binding site. Inaddition, this structural change could also induce a significantchange in the sulfation pattern of the GAG chains (see below).

Considering the ability of the convertase-resistant GPC3mutant to interact with both Ptc and Shh, along with the obser-vation that more Hh is bound to Ptc in the presence of thisGPC3 mutant, our results strongly suggest that GPC3 RR-AAstimulates the Hh signaling pathway by facilitating or stabiliz-ing the interaction between Hh and Ptc. Interestingly, a similar

FIGURE 3. GPC3 RR-AA interacts with both Hh and Ptc. A, GPC3 RR-AA-Shh coimmunoprecipitation. 293T were transfected with the indicated expressionvectors, and GPC3 was immunoprecipitated (IP). Top panel: the presence of Shh in the precipitated material was probed with an anti-Shh antibody. Middle andbottom panels: the amount of ectopic Shh or mutant GPC3 in whole cell lysates was assessed by Western blot (WB). Arrowhead: GPC3 core protein; bracket:glycanated GPC3. B, cell binding assay. 293T cells transfected with the indicated expression vectors were incubated with Shh-AP or AP alone for 2 h at 8 °C. Cellswere then washed and lysed, and the AP activity of aliquots of cell lysates containing equal amounts of protein was determined. Bars represent the mean � S.D.of triplicates. The background binding of AP alone to the cells was subtracted from each measurement. OD, optical density. C and D, pulldown assay.Ptc-covered protein-G beads or control beads were incubated with equal activities of GPC3-AP, GPC3 RR-AA-AP, or AP alone (C) or the indicated dilutions (D).After washing, the AP activity retained by the beads was measured. Bars represent the specific AP activity (mean � S.D. of triplicates) bound to the Ptc beadsafter subtracting the corresponding AP values obtained for the control beads. All the experiments were repeated at least three times. One representativeexperiment is shown. Statistical analysis comparing each data set with the correspondent control or between the indicated data sets was performed with theunpaired t test. Significance is indicated as: * � p � 0.05; ** � p � 0.01; and *** � p � 0.001. CM, conditioned medium.




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mechanism has been proposed to explain GPC3-induced stim-ulation of Wnt signaling (27). We have recently shown thatGPC3 plays a direct role in the activation of Wnt signaling bybinding to both Wnt and its receptor Frizzled (Fz) and by stim-ulating the formation of signaling complexes (27). Because theconvertase-resistant GPC3 is as potent as wild-type GPC3 inthe stimulation of Wnt signaling (23), our data suggest that thestructural changes generated by the lack of convertase cleavagedo not alter GPC3-Wnt and GPC3-Fz binding interactions.Thus, the processing of GPC3 by convertases allows this glypi-can to have the opposite effect in two key signaling pathways.

As mentioned above, like the convertase-resistant GPC3,GPC5 also binds to Hh and Ptc through the GAG chains andstimulates Hh signaling. Interestingly, GPC5 is not processedby convertases. However, the lack of convertase processingcannot be considered a requirement for the Hh stimulatoryactivity of glypicans because the Drosophila glypican Dally-like(Dlp), which also stimulates Hh signaling, is cleaved by conver-tases (6). Interestingly, contrary to what we have found withGPC5 and GPC3 RR-AA, the GAG chains are not required forthe stimulatory activity of Dlp on Hh signaling because the Dlpcore protein interacts with Hh (28). Thus, Hh interacts with thecore proteins of two glypicans that are cleaved by convertases,GPC3 and Dlp. Whether the processing by convertases is alsorequired for the exposure of the Hh-interacting domain in Dlpremains to be studied.

Taken together, our results indicate that in the context of Hhsignaling, GPC3 RR-AA behaves like GPC5. It should be noted,however, that although GPC5 stimulates Hh-induced luciferaseactivity in a dose-dependent manner reaching high levels ofstimulation at higher levels of expression, GPC3 RR-AA dis-

FIGURE 4. The interaction between GPC3 RR-AA-Shh and Ptc is mediated by the GAG chains. A and C, cell binding assay. A, 293T cells were transientlytransfected with expression vectors for wild-type GPC3 (GPC3), non-glycanated GPC3 (GPC3�GAG), convertase-resistant GPC3 RR-AA (GPC3 RR-AA), non-glycanated convertase-resistant GPC3 (GPC3 9A�GAG), or vector control (EF). Cells were then incubated with Shh-AP or AP alone, washed, and lysed, and thebound AP activity of aliquots of cell lysates containing equal amounts of protein was determined. Bars represent the mean � S.D. of triplicates. The backgroundbinding of AP alone was subtracted for each measurement. OD, optical density. B, Western blot analysis of the GPC3 variants expression levels in the cell lysates.Arrowhead: GPC3 core protein; bracket: glycanated GPC3. C, 293T cells transfected with the indicated expression vectors were incubated with different dilutionsof Shh-AP or AP conditioned medium (CM). A cell binding assay was performed as described in A. D, G, GPC3-Ptc pulldown assay. Ptc-covered protein-G beadsor control beads were incubated with equal activities of the indicated AP fusion proteins. After washing, the AP activity retained by the beads was measured.Bars represent the specific AP activity (average � S.D. of triplicates) bound to Ptc beads after subtracting the corresponding AP values obtained for the controlbeads. All experiments were repeated at least three times with similar results. Representative experiments are shown. E, NIH 3T3 cells were transfected with theindicated expression vectors or vector control (EF), along with a luciferase reporter vector driven by an Hh-responsive promoter, and �-galactosidase. Cellswere then stimulated with Shh- or control-conditioned medium. A luciferase assay was then performed. Bars represent fold stimulation induced by Hh(mean � S.D. of triplicates). F, cell binding assay. 293T cells transfected with the indicated expression vectors were incubated with Shh-AP or AP alone for 2 hat 8 °C. Cells were then washed, lysed, and the AP activity of aliquots of cell lysates containing equal amounts of protein was determined. Bars represent themean � S.D. of triplicates. Statistical analysis comparing each data set with the correspondent control or between the indicated data sets was performed withthe unpaired t test. Significance is indicated as: * � p � 0.05; ** � p � 0.01; and *** � p � 0.001.

FIGURE 5. GPC3 9A�GAG does not stimulate Hh signaling. A, luciferaseassay. NIH 3T3 cells were transfected with the indicated expression vectors orvector control (EF), along with a luciferase reporter vector driven by an Hh-responsive promoter, and �-galactosidase. Cells were then stimulated withShh or control conditioned medium. A luciferase assay was then performed.Bars represent -fold stimulation induced by Hh (mean � S.D. of triplicates). B,Western blot assessment of the expression levels of the indicated GPC3 vari-ants in the cell lysates of transfected cells. Arrowhead: GPC3 core protein;bracket: glycanated GPC3. One representative experiment of four is shown.Statistical analysis comparing each data set with the correspondent controlor between the indicated data sets was performed with the unpaired t test.Significance is indicated as: * � p � 0.05 and ** � p � 0.01.

FIGURE 6. The HS chains of GPC3 RR-AA display a higher degree of sulfa-tion than those of GPC3. Purified GPC3 RR-AA-AP and GPC3-AP weredigested with heparitinase I and II, and the digested material was labeledwith 2AB and analyzed by HPLC. The identity of each peak was determined bycomparison with 2AB-labeled standard HS disaccharides: �HexUA-GlcNAc(0S), �HexUA-GlcNAc(6-O-sulfate) (6S), �HexUA-GlcN(2-N-sulfate) (NS),�HexUA-GlcN(2-N-,6-O-disulfate) (NS,6S), �HexUA(2-O-sulfate)-GlcN(2-N-sulfate) (NS,2S), and �HexUA(2-O-sulfate)-GlcN(2-N-,6-O-disulfate) (TriS). Thebars represent average of triplicates � S.D. Significance is indicated as: * �p � 0.05. (unpaired t test) The disaccharide composition for each sample wasthen calculated and expressed as percentage. The experiment was repeatedthree times. One representative experiment is shown.




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plays a reduced stimulatory activity at higher levels of expres-sion. The reasons for this differential behavior are currentlyunknown.

Another interesting finding of this study is that the conver-tase-resistant GPC3 acquires the ability to interact with Ptc.This interaction is mediated by the HS chains, suggesting thatthe chains carried by GPC3 RR-AA are different from the ones

carried by the wild-type GPC3. It is well established that thebinding specificity of HS chains predominantly depends onthe degree and type of sulfation. Based on this, we compared thesulfation profile of the GPC3 and GPC3 RR-AA HS chains. Wefound that indeed the HS chains of GPC3 RR-AA display asignificantly higher degree of sulfation than those of GPC3, sug-gesting therefore that this higher sulfation level allows GPC3 tointeract with Ptc. This is consistent with our previous observa-tion that the HS chains of GPC5 also display more sulfationthan those of GPC3 (18).

It is well known that the level of sulfation in the HS chains ofa given cell type is determined by the levels of sulfotransferasesand sulfatases (24). In this study, we have compared the HSchains of GPC3 and GPC3 RR-AA purified from the same cellline. Thus, it could be proposed that the differences that wefound in the sulfation of the HS chains are the consequence ofthe structural changes generated by the cleavage of the GPC3core protein by convertases. This modification of the structureof the core protein could alter the accessibility of one or more ofthese HS-modifying enzymes to the site of HS chain synthesis inthe GPC3 RR-AA mutant. Alternatively, the conformationalchange generated by the lack of cleavage of GPC3 could alter itsability to interact with other proteins, and could consequentlychange the Golgi compartment in which GPC3 becomes glyca-nated. It has been proposed that different Golgi compartmentscould harbor different relative amounts of the various enzymesinvolved in the synthesis of GAG chains (29).

Another important observation of this study is that GPC3RR-AA is detected in the primary cilium, where Hh-Ptc inter-action occurs. This finding is consistent with our results thatGPC3 RR-AA interacts with Ptc and Hh and increases the bind-ing of Hh to Ptc. As reported previously (17), we did not detectwild-type GPC3 at this location. It remains to be elucidatedwhether the ability to bind to Ptc is the determinant factor forthe localization of GPC3 in the cilium.

Note Added in Proof—Hiroshi Kitagawa’s contributions to this arti-cle fulfill the JBC authorship criteria, but his authorship was inadver-tently omitted from the version of the article that was published onFebruary 4, 2015 as a Paper in Press.

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FIGURE 7. GPC3 RR-AA localizes to the primary cilium. Upper panels, NIH3T3 cells transfected with GPC3 RR-AA or wild-type GPC3 expression vectorswere immunostained for GPC3 (red) and acetylated tubulin (green). Yellow inthe merge picture indicates co-localization. Insets show a higher magnifica-tion detail of the cilium area. Scale bars: 10 �m. The experiment was repeatedthree times. One representative cell is shown. Lower panel, ratio of fluores-cence intensity in/out of cilia of NIH 3T3 cells transfected with GPC3 or GPC3RR-AA. Bars represent the average of five cells � sd.

FIGURE 8. Diagram of the proposed impact of convertase cleavage on theability of GPC3 to interact with Shh and Ptc.




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Mariana Capurro, Wen Shi, Tomomi Izumikawa, Hiroshi Kitagawa and Jorge FilmusHedgehog Signaling

Processing by Convertases Is Required for Glypican-3-induced Inhibition of

doi: 10.1074/jbc.M114.612705 originally published online February 4, 20152015, 290:7576-7585.J. Biol. Chem.

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