Biochimica et Biophysica Acta - Polytechnique Montréal 11 August 2010 Received in revised form 19...

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The TGF-β co-receptor, CD109, promotes internalization and degradation of TGF-β receptors Albane A. Bizet a , Kai Liu a,1 , Nicolas Tran-Khanh b , Anshuman Saksena a , Joshua Vorstenbosch a , Kenneth W. Finnson a , Michael D. Buschmann b , Anie Philip a, a Division of Plastic Surgery, Department of Surgery, McGill University, Montreal, QC, Canada b Department of Biomedical and Chemical Engineering, Ecole Polytechnique, Montreal, QC, Canada abstract article info Article history: Received 11 August 2010 Received in revised form 19 January 2011 Accepted 24 January 2011 Available online 2 February 2011 Keywords: TGF-β receptor CD109 Internalization Degradation Caveolae Transforming growth factor-β (TGF-β) is implicated in numerous pathological disorders, including cancer and mediates a broad range of biological responses by signaling through the type I and II TGF-β receptors. Internalization of these receptors via the clathrin-coated pits pathway facilitates SMAD-mediated signaling, whereas internalization via the caveolae pathway is associated with receptor degradation. Thus, molecules that modulate receptor endocytosis are likely to play a critical role in regulating TGF-β action. We previously identied CD109, a GPI-anchored protein, as a TGF-β co-receptor and a negative regulator of TGF-β signaling. Here, we demonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109 increases binding of TGF-β to its receptors and enhances their internalization via the caveolae. In addition, CD109 promotes localization of the TGF-β receptors into the caveolar compartment in the presence of ligand and facilitates TGF-β-receptor degradation. Thus, CD109 regulates TGF-β receptor endocytosis and degradation to inhibit TGF-β signaling. © 2011 Elsevier B.V. All rights reserved. 1. Introduction TGF-β 5 , a multifunctional growth factor with three different subtypes, TGF-β1, β2, β3, controls numerous cellular processes such as growth, differentiation, migration and extracellular matrix depo- sition, and thus plays a crucial role in development and homeostasis [1]. Dysregulation of its signaling pathway has been implicated in tissue brosis and cancer, underscoring the critical importance of a tight regulation of TGF-β action. TGF-β signaling is transduced by a pair of transmembrane serine/ threonine kinases known as type I (TGFBR1) and II (TGFBR2) receptors [2]. The binding of TGF-β to TGFBR2, a constitutively active kinase, results in the phosphorylation of TGFBR1. The activated TGFBR1 then propagates the signal by phosphorylating its intracellular substrates SMAD2 and SMAD3, which in turn form complexes with SMAD4. The complexes accumulate in the nucleus, where they regulate gene expression [3]. In addition to the type I and II receptors, many cell types also express the TGF-β co-receptors betaglycan and endoglin [4]. Our group has recently identied another TGF-β co-receptor, CD109, in skin cells [5]. CD109 is a 180 kDa glycosylphosphatidylinositol (GPI)- anchored protein that belongs to the α2-macroglobulin/complement family [6]. CD109 binds the TGF-β1 subtype with high afnity, forms a heteromeric complex with the TGF-β signaling receptors and inhibits TGF-β signaling and responses in a variety of cell types [5]. Moreover, recent reports have shown that CD109 expression is deregulated in basal-like breast carcinoma [7], squamous cell carcinoma [811] and melanoma [5] and that CD109 is mutated in colorectal cancer [12]. Despite its important role in TGF-β signaling and its potential signicance in cancer progression, its mechanism of action is not yet understood. Receptor endocytosis is a pivotal regulatory mechanism in signal transduction. TGF-β receptors are internalized via both clathrin- and caveolae-dependent pathways [13]. Internalization of the TGF-β receptors via the clathrin-coated pits has been linked with signaling via SMAD2/3 and receptor recycling [1417]. In contrast, TGF-β receptor localization in caveolae is associated with downregulation of SMAD2/3 signaling and receptor degradation following ubiquitination by the E3-ubiquitin ligase Smurf2 [13,18]. Because CD109 is a negative regulator of TGF-β signaling, we investigated whether CD109 exerts this effect by modulating caveolae-mediated internalization of the TGF-β receptors or receptor degradation. Biochimica et Biophysica Acta 1813 (2011) 742753 Abbreviations: TGF-β, transforming growth factor-β; Smurf, SMAD ubiquitination regulatory factor; GPI, glycosylphosphatidylinositol; MβCD, methyl-β-cyclodextrin; EEA-1, early endosome antigen-1; HA, hemagglutinin Corresponding author at: Montreal General Hospital, 1650 Cedar Ave., Rm C9-158, McGill University, Montreal, QC, H3G 1A4, Canada. Tel.: +1 514 934 1934 x44535; fax: +1 514 934 8289. E-mail address: [email protected] (A. Philip). 1 Present address: Department of Colorectal Surgery, Tianjin Medical University Cancer Hospital, Tianjin, PR China. 0167-4889/$ see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2011.01.028 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr

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Biochimica et Biophysica Acta 1813 (2011) 742–753

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r.com/ locate /bbamcr

The TGF-β co-receptor, CD109, promotes internalization and degradation ofTGF-β receptors

Albane A. Bizet a, Kai Liu a,1, Nicolas Tran-Khanh b, Anshuman Saksena a, Joshua Vorstenbosch a,Kenneth W. Finnson a, Michael D. Buschmann b, Anie Philip a,⁎a Division of Plastic Surgery, Department of Surgery, McGill University, Montreal, QC, Canadab Department of Biomedical and Chemical Engineering, Ecole Polytechnique, Montreal, QC, Canada

Abbreviations: TGF-β, transforming growth factor-βregulatory factor; GPI, glycosylphosphatidylinositol; MEEA-1, early endosome antigen-1; HA, hemagglutinin⁎ Corresponding author at: Montreal General Hospita

McGill University, Montreal, QC, H3G 1A4, Canada. Tefax: +1 514 934 8289.

E-mail address: [email protected] (A. Philip).1 Present address: Department of Colorectal Surger

Cancer Hospital, Tianjin, PR China.

0167-4889/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.bbamcr.2011.01.028

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 August 2010Received in revised form 19 January 2011Accepted 24 January 2011Available online 2 February 2011

Keywords:TGF-β receptorCD109InternalizationDegradationCaveolae

Transforming growth factor-β (TGF-β) is implicated in numerous pathological disorders, including cancer andmediates a broad range of biological responses by signaling through the type I and II TGF-β receptors.Internalization of these receptors via the clathrin-coated pits pathway facilitates SMAD-mediated signaling,whereas internalization via the caveolae pathway is associated with receptor degradation. Thus, molecules thatmodulate receptor endocytosis are likely to play a critical role in regulating TGF-β action.Wepreviously identifiedCD109, a GPI-anchored protein, as a TGF-β co-receptor and a negative regulator of TGF-β signaling. Here, wedemonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109increases binding of TGF-β to its receptors and enhances their internalization via the caveolae. In addition, CD109promotes localization of the TGF-β receptors into the caveolar compartment in the presence of ligand andfacilitates TGF-β-receptor degradation. Thus, CD109 regulates TGF-β receptor endocytosis and degradation toinhibit TGF-β signaling.

; Smurf, SMAD ubiquitinationβCD, methyl-β-cyclodextrin;

l, 1650 Cedar Ave., Rm C9-158,l.: +1 514 934 1934 x44535;

y, Tianjin Medical University

ll rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

TGF-β5, a multifunctional growth factor with three differentsubtypes, TGF-β1, β2, β3, controls numerous cellular processes suchas growth, differentiation, migration and extracellular matrix depo-sition, and thus plays a crucial role in development and homeostasis[1]. Dysregulation of its signaling pathway has been implicated intissue fibrosis and cancer, underscoring the critical importance of atight regulation of TGF-β action.

TGF-β signaling is transduced by a pair of transmembrane serine/threoninekinasesknownas type I (TGFBR1)and II (TGFBR2) receptors [2].The binding of TGF-β to TGFBR2, a constitutively active kinase, results inthe phosphorylation of TGFBR1. The activated TGFBR1 then propagatesthe signal by phosphorylating its intracellular substrates SMAD2 andSMAD3, which in turn form complexes with SMAD4. The complexesaccumulate in the nucleus, where they regulate gene expression [3].

In addition to the type I and II receptors, many cell types alsoexpress the TGF-β co-receptors betaglycan and endoglin [4]. Ourgroup has recently identified another TGF-β co-receptor, CD109, inskin cells [5]. CD109 is a 180 kDa glycosylphosphatidylinositol (GPI)-anchored protein that belongs to the α2-macroglobulin/complementfamily [6]. CD109 binds the TGF-β1 subtype with high affinity, forms aheteromeric complex with the TGF-β signaling receptors and inhibitsTGF-β signaling and responses in a variety of cell types [5]. Moreover,recent reports have shown that CD109 expression is deregulated inbasal-like breast carcinoma [7], squamous cell carcinoma [8–11] andmelanoma [5] and that CD109 is mutated in colorectal cancer [12].Despite its important role in TGF-β signaling and its potentialsignificance in cancer progression, its mechanism of action is not yetunderstood.

Receptor endocytosis is a pivotal regulatory mechanism in signaltransduction. TGF-β receptors are internalized via both clathrin- andcaveolae-dependent pathways [13]. Internalization of the TGF-βreceptors via the clathrin-coated pits has been linked with signalingvia SMAD2/3 and receptor recycling [14–17]. In contrast, TGF-βreceptor localization in caveolae is associated with downregulation ofSMAD2/3 signaling and receptor degradation following ubiquitinationby the E3-ubiquitin ligase Smurf2 [13,18].

Because CD109 is a negative regulator of TGF-β signaling, weinvestigated whether CD109 exerts this effect by modulatingcaveolae-mediated internalization of the TGF-β receptors orreceptor degradation.

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2. Materials and methods

2.1. Cell lines

The human keratinocyte cell line HaCaT, kindly provided by P.Boukamp (Heidelberg, Germany), Mv1Lu cells and human embryonickidney 293 cells purchased from the American Type Culture Collection,were cultured as described previously [5]. HaCaT clones stablyexpressing CD109 (or its empty vector, EV) were selected and culturedin the presence of 0.5 mg/ml Geneticin (Invitrogen, Carlsbad, CA, USA).

2.2. Transient transfections and siRNA treatment

Transfections of different combinations of the following plasmids:CD109 or its empty vector (pCMVSport6), caveolin-1 (gift from C.Hardin, University of Missouri), Dyn2K44A (gift from S. Egan,University of Toronto), TGFBR2 and TGFBR1-WT (gifts from J. Wrana,University of Toronto) were performed using Superfect (Qiagen,Mississauga, ON, Canada). Alternatively, cells were transfected withCD109 siRNA (cat.#129083), caveolin-1 siRNA (cat.#10297) or anegative control siRNA (cat.#4611) (Ambion, Austin, TX, USA) usingLipofectamine 2000 (Invitrogen).

2.3. Affinity labeling

293 cells or HaCaT cells were incubated with 100 pM125I-TGF-β1

for 3 h at 4 °C, the ligandwas cross-linked to the receptors with a non-permeable cross-linker BS3 (Pierce, Rockford, IL) and the cells wereincubated at 37 °C for 0 to 8 h prior to lysis as previously described[19]. Protein concentrations were determined using a Lowry proteinassay (Bio-Rad, Mississauga, ON, Canada) and the lysates wereanalyzed by SDS-PAGE/autoradiography. Densitometry was per-formed using the ImageJ software.

2.4. Immunoprecipitation and western blot

Lysates from 293 cells were immunoprecipitated with a mousemonoclonal anti-caveolin-1 antibody (clone 2297, BD Biosciences,Mississauga, ON, Canada). Lysates from HaCaT cells were immuno-precipitated with mouse monoclonal anti-CD109 (gift from R&Dsystems, Minneapolis, MN, USA) or a rabbit polyclonal anti-caveolin-1antibody (BD Biosciences), while a mouse monoclonal anti-histoneH1 or a rabbit polyclonal anti-HA antibodies (both from Santa CruzBiotechnology, Santa Cruz, CA, USA) were used as control IgG.Western blot analyses were conducted with the following antibodies:mouse monoclonal anti-CD109 (TEA 2/16, BD Biosciences), anti-EEA-1(BD Biosciences), anti-SMAD3 (FL-425) and anti-actin (H-300)antibodies (both form Santa Cruz Biotechnology), rabbit polyclonalanti-TGFBR1 and anti-phospho-SMAD3 antibodies (both from CellSignaling Technologies, Danvers, MA, USA).

2.5. Internalization assay

Internalization assay was performed as described in Ref.[20] with some modifications. Briefly, cells were treated with2.5 mM MβCD (Sigma Aldrich, Oakville, ON, Canada) for 1 h at 37 °Cprior to internalization assay. Cells were then incubated with100 pM

125I-TGF-β1 (Perkin Elmer, Waltham, MA, USA) with or

without 10 nM unlabeled TGF-β1 (to determine non-specific binding)for 2 h at 4 °C and transferred to 37 °C for the indicated time.Surface ligand was removed with 150 mM NaCl, 0.1% acetic acid, 2 Murea. Cells were then solubilized in order to extract internalizedligand.

2.6. Sucrose gradient fractionation

Preparation of membrane fraction from HaCaT cells was per-formed as described in Ref. [21]. The twelve fractions collected wereanalyzed by western blot for EEA-1 and caveolin-1. The fractionscontaining EEA-1 or caveolin-1 were pooled and equal amountsof protein were analyzed by SDS-PAGE/autoradiography or westernblot.

2.7. Immunofluorescence microscopy

293 cells were incubated with 250 pM biotinylated-TGF-β1 (R&D,Minneapolis, MN, USA) for 2 h at 4 °C, then with 10 μg/ml streptavi-din-AF647 for 1 h at 4 °C and placed at 37 °C in DMEM, 10% FBS, for30 min. Cells were fixed with 4% paraformaldehyde and permeabi-lized with methanol. After blocking non-specific sites with 10%normal goat serum, cells were stained using an anti-CD109 antibodyfollowed by an Alexa Fluor 488 (AF488) conjugated anti-mouse IgG(Invitrogen) or by a Cy5-conjugated Fab fragment goat anti-mouseIgG (Jackson ImmunoResearch Lab, West Grove, PA, USA). Cells werethen stained with a Cy3 conjugated rabbit anti-caveolin-1 or AF488conjugated mouse anti-HA antibodies (Invitrogen). Images wereacquired with a LSM 510 META Axioplan 2 confocal laser scanningmicroscope (Carl Zeiss, Toronto, ON, Canada) as described previously[22]. Background was removed by median filtering [23] or bydeconvolution using Huygens Professional Software (Fig. 1D andSupplementary Fig. S2D).

2.8. Plasma membrane preparation

Cell surface proteins were extracted using the Hook Cell SurfaceProtein Isolation kit (G-Biosciences, St. Louis, MO, USA). Briefly, cellsurface proteins were labeled with Hook-sulfo-NHS-SS-Biotin andlysed. An aliquot was analyzed by western blot for actin to make surethat equal amount of protein was used. Biotinylated proteins from theremaining lysates were isolated using streptavidin-agarose column,eluted and analyzed by western blot.

2.9. Statistical methods

Numerical results are represented as means of n≥3 independentexperiments±SEM. Statistical significance between two groups wasdetermined by two-tailed Student t test. Comparisons within morethan two groups weremade by one-way analysis of variance. Multiplecomparisons were made by Holm–Sidak test (post-hoc).

3. Results

3.1. CD109 associates with caveolin-1 at endogenous concentration

We sought to determine whether CD109 exerts its negative effectson TGF-β signaling bymodulating TGF-β receptor localization into thecaveolae, a process shown to enhance TGF-β receptor degradation.Wefirst examined whether CD109, a GPI-anchored protein, can associatewith caveolin-1, a major component of the caveolae. Co-immunopre-cipitation experiments reveal that CD109 associateswith caveolin-1 in293 cells co-transfected with both CD109 and caveolin-1 (Fig. 1A). Inthe absence of caveolin-1 transfection, (i.e. when caveolin-1 expres-sion is undetectable [24]), CD109 is not co-immunoprecipitated(Fig. 1A), demonstrating the specificity of the association.

In untransfected HaCaT cells, caveolin-1 is co-immunoprecipitatedby an anti-CD109 antibody (Fig. 1B), confirming that the interactionbetween CD109 and caveolin-1 is not an artifact of overexpressionand that it occurs at endogenous concentrations of caveolin-1 andCD109 (Fig. 1B). Similarly, an anti-caveolin-1 antibody co-immuno-precipitates small but detectable amounts of CD109 (Supplementary

744 A.A. Bizet et al. / Biochimica et Biophysica Acta 1813 (2011) 742–753

Fig. S1 and Fig. 1C). Importantly, the association between CD109 andcaveolin-1 is enhanced after TGF-β treatment (Fig. 1C).

Immunofluorescence microscopy of HaCaT cells (Fig. 1D) showsthat caveolin-1 (in red) displays a punctate staining pattern at theplasma membrane, with some punctate staining in the cytoplasm,which may represent neo-synthesized or internalized caveolin-1.Endogenous CD109 (blue) localizes mainly at the plasma membranein both non-caveolar and caveolin-1 positive compartments. Thecolocalization of CD109 with caveolin-1 (in purple, Fig. 1D) suggeststhat CD109 is able to associate with caveolin-1 in the same cellularcompartment, supporting the results obtained in Fig. 1A–C.

3.2. CD109 and TGF-β receptors colocalize in caveolae

We have previously shown that CD109 binds directly to TGFBR1 [5].We therefore sought to determine whether TGF-β receptors co-localizewith CD109 in caveolae. Because the endogenous levels of TGF-βreceptors in HaCaT cells are too low to allow detection of theinternalized ligand, 293 cells were transfected with TGFBR1/TGFBR2and caveolin-1 and labeledwith biotin-TGF-β, followed by streptavidin-AF647 treatment. After 30 min at 37 °C to allow receptor and ligandinternalization, the cellswere immunostained for CD109 and caveolin-1(Fig. 1E). Importantly, a significant amount of triple colocalization(visualized in white) is observed between the internalized biotin-TGF-β(in green), CD109 (in blue), and caveolin-1(in red) (Fig. 1E). Theproportion of internalized biotin-TGF-β that is not associated withCD109 and caveolin-1 (Fig. 1E) may represent molecules that areinternalized via the clathrin pathway (associated with SMAD2/3signaling) or that are in recycling compartments. In the absence ofTGF-β treatment, CD109 localizes mainly at the plasma membrane,where it partially co-localizes with caveolin-1 (Fig. 1F), as observed inuntransfected HaCaT cells (Fig. 1D). When the cells were incubated at4 °C instead of 37 °C, no biotin-TGF-β staining was detected (data notshown), indicating that at 4 °C, the receptors are not internalized.Interestingly, in the absence of TGF-β receptor transfection, low levels ofco-localization between CD109 and caveolin-1 could be observed(Fig. 1G and Supplementary Fig. S2A). It is possible that the interactionbetween the GPI-anchored CD109 and the integral membrane proteincaveolin-1 is mediated via endogenous TGF-β receptors in that case,although the contribution of other transmembrane proteins or lipidmolecules cannot be ruled out.

The notion that the biotin-TGF-β staining is specific for biotin-TGF-β bound to TGFBR1/TGFBR2 complex and not to other TGF-β bindingmolecules under our detection settings is supported by the followingresults: (1) when TGFBR1/TGFBR2 are not transfected, no staining isobserved for biotin-TGF-β, even in the presence of CD109 (Fig. 1G);(2) biotin-TGF-β colocalizes with HA-tagged TGFBR1 (SupplementaryFig. S2C). This suggests that TGF-β follows the same internalizationroute as TGFBR1, as reported by others [13,20]; (3) HA-TGFBR1 co-localizes with CD109 in caveolae vesicles and at the plasmamembrane (Supplementary Fig. S2D). Together, these data indicatethat biotin-TGF-β can be used to assess TGF-β receptor localizationand suggest that CD109 is able to form a complex with TGF-β-boundTGF-β receptors (activated receptors) in caveolae.

Fig. 1. CD109 associates with caveolin-1 and colocalizes with the TGF-β receptors in caveolaand caveolin-1 or their corresponding empty vector were immunoprecipitated with an anti-IgG band demonstrates equal loading. (B) Cell lysates from non-transfected HaCaT cells we(used as control IgG), followed by western blot for CD109 and caveolin-1. (C) Non-transfectthen subjected to immunoprecipitation with an anti-caveolin-1 antibody or with an anti-HAdensitometry of CD109 levels in the immunoprecipitate is indicated. (D–G) Immunofluorendogenous CD109 (blue). Co-localization of CD109 with caveolin-1 appears in purple. (E) 2TGF-β/streptavidin (green) and incubated for 30 min at 37 °C to allow receptor internalizatioconfocal microscopy. Triple co-localization of caveolin-1, CD109, and biotin-TGF-β appears itransfected as in (E) were left untreated and stained for CD109 (blue) and caveolin-1 (red). Cexperiments is shown. (G) 293 cells transfected with CD109, caveolin-1, TGFBR1/TGFBR2 owhere colocalization between CD109 and caveolin-1 occurs. A representative panel of cells

3.3. CD109 enhances binding of TGF-β to TGF-β receptors and TGF-β-boundreceptor internalization

We then asked if CD109 affects TGF-β-bound receptor internaliza-tion. CD109 overexpression significantly (pb0.05, n=5 independentexperiments) increases the number of cells that have internalizedbiotin-TGF-β (i.e. TGF-β-bound receptors) as compared to empty vector(EV) transfection in 293 cells (Fig. 2A and B). After normalization fortransfection efficiency (as determined by caveolin-1 expression), weobserve that 64% of caveolin-1 positive cells internalize biotin-TGF-β inthe presence of CD109, whereas only 27% incorporate biotin-TGF-β inEV transfected cells (Fig. 2B), suggesting that CD109 increases theinternalization of TGF-β-bound receptors. The amount of internalizedbiotin-TGF-β reflects the levels of receptors that underwent endocyto-sis, and also any recycling and/or degradation that may have occurredduring that time period. Notably, CD109 transfection does not alter thelevel of TGF-β receptors or caveolin-1, as compared to EV transfection(Fig. 3B and data not shown), suggesting that CD109 only affects theamount of internalized TGF-β-bound receptors.

We next examined the effect of CD109 on the internalization of125I-TGF-β at endogenous TGF-β receptor concentration in HaCaT cells.

The125I-TGF-β internalization assay has been shown to reliably reflect

receptor internalization [20,25]. At early time points (15 and 30 min),CD109 enhances

125I-TGF-β internalization (Fig. 2C). The increase of

125I-TGF-β internalization in CD109 overexpressing cells is followed by a

decline after 30 min whereas a decline occurs only after 60 min in EVtransfected cells (Fig. 2C). Thedecline in internalized

125I-TGF-βhas been

reported previously and might be due to ligand depletion and/ordegradation [20]. Importantly, our results suggest that CD109 enhancesligand-bound receptor internalization.

Interestingly, CD109 overexpression is associated not only with anincrease of

125I-TGF-β internalization, but also with an increase of

surface-bound125I-TGF-β at 30 min (Fig. 2D). The ratio of internalized/

surface-bound125I-TGF-β (I/S) is higher in CD109 transfected cells, as

compared to EV cells at both 15 and 30 min (Fig. 2E), suggesting thatCD109 accelerates TGF-β-bound receptor endocytosis. Moreover,knocking down CD109 expression using CD109 siRNA or CD109antisense morpholino oligos leads to a decrease in both internalizedand surface-bound

125I-TGF-β at 30 min (Fig. 2F and Supplementary

Fig. S3). These results indicate that endogenous CD109 increases surfacebinding and internalization of TGF-β.

To determine whether the increase in TGF-β binding to the cellsurface is due to an increase in TGF-βbinding to theTGF-β receptors andnot solely due to an increase in TGF-β binding to CD109, we performedaffinity labeling of cell surface receptors at 4 °C followed by SDS-PAGEanalysis. Our results show that CD109 is able to enhance TGF-β bindingto the receptors (Fig. 3A and B). CD109 siRNA transfection inHaCaT cellsdecreases and CD109 overexpression in 293 cells increases the amountof cell surface

125I-TGF-β-bound receptors (Fig. 3A and B). Notably,

CD109 does not affect the total level of TGFBR1, as demonstrated bywestern blot (Fig. 3A and B). Moreover, overexpression of CD109 doesnot alter the cell-surface level of TGF-β receptors (Fig. 3B). These results,together with our previous findings demonstrating that CD109 forms acomplexwith the TGF-β receptors [5], suggest that suchassociation is animportant step in CD109's ability to increase TGF-β binding to this

e. (A–C) Co-immunoprecipitation: (A) Cell lysates from 293 cells transfected with CD109caveolin-1 (cav-1) antibody followed by western blot with an anti-CD109 antibody. There immunoprecipitated with an anti-CD109 antibody or with an anti-histone antibodyed HaCaT cells were treated with or without 25 pM TGF-β for 30 min. Cell lysates wereantibody (used as control IgG), followed by western blot for CD109 and caveolin-1. Theescence: (D) HaCaT cells were immunostained for endogenous caveolin-1 (red) and93 cells transfected with CD109, caveolin-1, TGFBR1/TGFBR2 were stained with biotin-n. After fixation, cells were stained for CD109 (blue) and caveolin (red) and analyzed byn white. A representative cell of n=5 independent experiments is shown. (F) 293 cellso-localization of CD109 with caveolin-1 appears in purple. A representative cell of n=3r their corresponding empty vectors were stained as in (E). The arrow points at a cellis shown (n=3 experiments)

745A.A. Bizet et al. / Biochimica et Biophysica Acta 1813 (2011) 742–753

multimeric TGF-β receptor complex and enhances the internalization ofthis TGF-β bound-complex.

3.4. CD109 enhances TGF-β receptor internalization via the caveolarpathway

Next, we assessed if the CD109-induced increase in TGF-β receptorinternalization could be mediated by the caveolar compartment.

Internalization of biotin-TGF-β in EV transfected 293 cells isprevented by co-transfection of the Dynamin2-K44A mutant(Dyn2K44A) (Fig. 4A), which blocks endocytosis via both clathrinand caveolar pathways [26] and is consistent with previous reportsdemonstrating that TGF-β receptors are internalized via clathrin and/or caveolae pathways [13,27]. Importantly, internalization of biotin-TGF-β is also blocked by co-transfection of the Dynamin2-K44Amutant (Dyn2K44A) when CD109 is overexpressed (Fig. 4A). In the

Fig. 2. CD109 promotes TGF-β internalization. (A–B) Immunofluorescence: 293 cells were stained with biotin-TGF-β/streptavidin (green) and incubated for 30 min at 37 °C to allowreceptor internalization. Cells were fixed and immunostained for CD109 (blue) and caveolin (red) and analyzed by confocal microscopy. (A) Comparison of populations of CD109 andcontrol empty vector (EV) transfected cells. A representative cell showing internalization of biotin-TGF-β is enlarged in the left corner of the overlay. (B) Percentage of transfected(caveolin-1 positive) cells that have internalized biotin-TGF-β (mean of n=5 independent experiments±SEM, *: pb0.05). (C–F)

125

I-TGF-β internalization assay: (C) Left panel:HaCaT cells transfected with CD109 or EVwere treated with 100 pM

125I-TGF-β and incubated at 37 °C for the indicated times. The graph shows the amount of internalized

125I-TGF-β1

(expressed as specific cpm). Right panel: Western blot showing CD109 overexpression in HaCaT cells. (D) Amount of surface-bound and internalized125I-TGF-β after 30 min

incubation at 37 °C in HaCaT cells transfected with CD109 or EV (normalized to surface-bound EV). (E) Ratio of internalized/surface-bound125I-TGF-β (I/S) in HaCaT cells transfected

with CD109 or EV. (F) Left panel: Amount of surface-bound and internalized125I-TGF-β after 30 min incubation at 37 °C in HaCaT cells transfected with CD109 siRNA or control siRNA

(normalized to surface-bound control siRNA). Right panel: Western blot showing CD109 siRNA efficacy. All graphs show the mean of nN3 independent experiments, ± SEM,*: pb0.05.

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presence of Dyn2K44A, the biotin-TGF-β staining appears at theplasma membrane in 81 (± 5.2)% of the cells and co-localizes withCD109 staining, whereas the biotin-TGF-β staining is found at theplasma membrane in only 5.1 (± 4.6)% of the cells in the absence ofDyn2K44A (Fig. 4A). This indicates that CD109 promotes dynamin-dependent internalization of the TGF-β-bound receptors.

Because CD109 is an inhibitor of TGF-β signaling [5], we lookedmore specifically at CD109's ability to increase internalization via

caveolae, a route that has been shown to lead to receptor degradationand signaling downregulation. We demonstrate that pre-treatmentwith methyl-β-cyclodextrin (MβCD), an inhibitor of the caveolae/lipid raft-dependent endocytosis [17,26] blocks CD109's effect on125I-TGF-β internalization in HaCaT cells (Fig. 4B). The increase in

TGF-β internalization mediated by CD109 is also reversed bycaveolin-1 siRNA (Fig. 4C), confirming that CD109 is able to enhancecaveolae-mediated endocytosis. The slight (and non-significant)

Fig. 3. CD109 enhances TGF-β binding to the TGF-β receptors without altering the total or cell surface receptor levels. (A) HaCaT cells transfected with control or CD109 siRNA wereaffinity labeled with 100 pM

125I-TGF-β. Left panel: The amount of cell surface receptors bound to TGF-β is visualized by autoradiography. CD109 is detected as two bands of 180 and

150 kDa, as described previously [5]. Right panel: The same lysates were analyzed for TGFBR1 by western blot. (B) 293 cells transfected with TGFBR1, TGFBR2 and CD109 or its controlEV were affinity labeled with 100 pM

125I-TGF-β. Left panel: Autoradiography showing the amount of cell surface TGF-β-bound receptors. Middle panel: The same lysates were

analyzed for TGFBR1 by western blot. Right panel: Cell surface proteins from 293 cells transfected with TGFBR1/TGFBR2, CD109 or EV were labeled with Hook-sulfo-NHS-SS-Biotin,pulled down with streptavidin and analyzed by western blot with CD109 and TGFBR1 antibodies. Similar amounts of protein were used in the experiment as demonstrated by theactin blot. All results are representative of n=3 independent experiments and all lanes from a panel come from the same blot.

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decrease observed after MβCD treatment or caveolin-1 siRNAtransfection in EV transfected cells suggests that the internalizationof TGF-β via the caveolar pathway at basal conditions is modest inHaCaT cells. This is not consistent with the report showing thatabout 50% of TGF-β receptors localized in caveolae in Mv1Lu cells[13]. To determine whether the discrepancy is due to cell-typespecific differences, we determined whether CD109 regulates TGF-βinternalization into caveolae in Mv1Lu cells. In this cell type, CD109siRNA decreases

125I-TGF-β internalization as expected, in the

absence of MβCD (Fig. 4D). Importantly, treatment of MβCD leadsto a reduction of

125I-TGF-β internalization in control siRNA but not in

CD109 siRNA transfected cells, indicating that, in Mv1Lu cells, a largeproportion of TGF-β is internalized via the caveolae and thatendogenous CD109 mediates this effect. In addition, these resultssupport the argument that the amount of TGF-β internalizing via thecaveolae varies between cell-types. Also, the difference observed incontrol siRNAMv1Lu or EV HaCaT cells in presence of MβCD is due tocell type specificity since no difference was observed between EVand control siRNA transfection in the absence of MβCD (data notshown). Together, our results suggest that CD109 increases theproportion of TGF-β-bound receptors that internalize via the caveolarpathway.

3.5. CD109 promotes TGFBR1 compartmentalization into the caveolae ina ligand-dependent manner

TGF-β receptors have been shown to traffic independently ofligand addition [13,17]. Thus, we determined whether the effect of

CD109 on TGFBR1 trafficking occurs in a ligand-independent manner.By sucrose gradient centrifugation, the raft fractions (fractions 5 and6) were separated from the non-raft fractions (fractions 8 to 12). Thepurity of the raft and the non-raft fraction was verified using the earlyendosome antigen-1 (EEA-1) and caveolin-1, two markers conven-tionally used for the non-raft/endosomal and the raft/caveolarcompartment, respectively [13,21] (Fig. 5A). The analysis of TGFBR1and CD109 localization in the pooled raft and pooled non-raftfractions were performed on the same gel by western blot (Fig. 5B).In the absence of exogenous TGF-β, TGFBR1 is present predominantlyin the non-raft fraction in both CD109 and EV transfected cells. Incontrast, TGF-β treatment leads to a marked increase in the amount ofTGFBR1 in the raft fraction of CD109 transfected cells as comparedto EV transfected cells (Fig. 5B). In addition, TGF-β treatment ofEV transfected cells results in a modest increase in the amount ofTGFBR1 in the raft fraction (Fig. 5B), presumably due to the presenceof endogenous CD109. The low amount of TGFBR1 detected in theraft fraction in HaCaT cells is consistent with our previous results inthis cell type (Fig. 4B) showing that only a small amount of TGF-β-bound receptors is internalized via the lipid raft/caveolar pathwayin EV transfected HaCaT cells. Under basal conditions, CD109localizes mainly in the non-raft compartment, but is also present inthe lipid raft compartment (visualized on amore exposed film), whichagrees with our immunofluorescence data (Fig. 1D). The effect of TGF-β on the pattern of CD109 localization is similar to the one observed forTGFBR1: TGF-β treatment increases the localization of CD109 in the raftcompartment, which is consistent with our co-immunoprecipitationdata (Fig. 1C). Together, these results suggest that CD109 promotes

Fig. 4. CD109 enhances TGF-β internalization via the caveolar pathway. (A) Immunofluorescence: 293 cells transfected with TGFBR1/TGFBR2, CD109 (or its EV) and Dyn2K44A (or itsEV) were treated with biotin-TGF-β/streptavidin (green), incubated for 30 min at 37 °C and stained for CD109 (blue). Colocalization of CD109 and biotin-TGF-β appears in turquoise(overlay). Representative cells are shown. (B–D)

125

I-TGF-β internalization assay: (B) HaCaT cells transfected with CD109 or EVwere treated with or without MβCD prior to incubationwith

125I-TGF-β. The graph shows the amounts of internalized

125I-TGF-β after 30 min incubation at 37 °C (normalized to EV, no treatment). (C) Left panel: Amount of internalized

125I-

TGF-β after 30 min incubation at 37 °C in HaCaT cells co-transfected with CD109 or EV and with caveolin-1 or control siRNA (normalized to EV, control siRNA). Right panel: Westernblot showing caveolin-1 siRNA efficacy. (D) Left panel: Mv1Lu cells transfected with CD109 siRNA or control siRNA were treated with or without MβCD prior to incubation with

125I-

TGF-β. The graph shows the amounts of internalized125I-TGF-β after 30 min incubation at 37 °C (normalized to CD109 siRNA, no treatment). Right panel: Western blot showing

CD109 siRNA efficacy in Mv1Lu cells. All graphs show the mean of n≥3 independent experiments±SEM, *: pb0.05.

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TGFBR1entry into the lipid raft and that this effect is enhanced by TGF-βtreatment.

We further examined the ability of CD109 to induce TGF-β-boundreceptor localization to the caveolar compartment by first affinitylabeling the receptors on HaCaT cells with

125I-TGF-β and then

performing sucrose gradient fractionation as describe above. InCD109 transfected cells, the majority of TGF-β-bound TGFBR1localizes into the caveolin-1 positive/lipid raft fraction, whereas inEV transfectants, TGFBR1 resides mainly in the non-raft fraction(Fig. 5C). Similar results were obtained with TGF-β-bound TGFBR2(data not shown). These results indicate that CD109 leads topreferential localization of TGFBR1 into the lipid raft compartment,in the presence of TGF-β.

3.6. Inhibition of TGF-β signaling by CD109 involves caveolae-mediatedinternalization

The results presented so far show that CD109 promotes TGF-βreceptor internalization via the caveolae. We next investigated if thisstep is involved in mediating CD109's inhibitory effect on TGF-βsignaling. Our results show that overexpression of CD109 inhibitsTGF-β-induced SMAD3 phosphorylation (Fig. 6A), as expected [5].This inhibitory effect of CD109 is abrogated by co-transfection ofcaveolin-1 siRNA (Fig. 6A), indicating that CD109' effects on TGF-βsignaling involve the caveolar pathway. Similarly, knocking downCD109 expression using siRNA results in an increase in TGF-β-inducedSMAD3 phosphorylation, and this effect is blocked by co-transfection

Fig. 5. CD109 promotes TGFBR1 entry into the caveolar compartment in the presence of TGF-β. (A) Lysates fromHaCaT cells transfectedwith CD109 or EV and treated without (−) orwith (+) 25 pM TGF-β for 30 min were subjected to sucrose gradient fractionation. The resulting fractions were analyzed by western blot for EEA1 and caveolin-1. (B) Left panel: Thepooled raft (fractions 5–6) and pooled non-raft (fractions 8–12) fractions were analyzed by western blot for CD109 and TGFBR1. A representative western blot is shown. Right panel:Densitometric analysis of TGFBR1 levels. (C) Left panel: HaCaT cells transfected with CD109 or EV were affinity labeled with

125I-TGF-β prior sucrose gradient fractionation. Labeled

TGFBR1 is detected by autoradiography. Coomassie blue staining shows equal protein loading. The same samples were analyzed for EEA-1 and caveolin-1 by western blot. Rightpanel: Densitometric analysis of the autoradiogram.

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of caveolin-1 siRNA (Fig. 6B). This result suggests that downregulationof TGF-β signaling by endogenous CD109 involves the caveolarpathway.

3.7. CD109 accelerates TGF-β receptor degradation

Localization of TGF-β receptors to caveolae has been shown to beassociated not only with a decrease in SMAD2/3 phosphorylation butalso with receptor degradation [13]. Therefore, we tested if CD109modulates TGF-β receptor degradation, by monitoring the levels ofaffinity labeled receptors (Fig. 7A). Consistent with our previousfindings, CD109 overexpression increases the binding of

125I-TGF-β to

its receptors, compared to EV transfection (see time 0). TGFBR1,TGFBR2 and CD109 levels decrease over time, suggesting that theCD109-TGF-β-bound-receptor complex is being degraded (Fig. 7A).Quantification of TGFBR1 levels by densitometry (expressed as apercentage of time 0) reveals that CD109 accelerates degradation ofTGF-β-bound receptor (Fig. 7A, right panel, pb0.05). In addition,stable transfection of CD109 in HaCaT cells (clone CD3-3 and CD2-4)accelerates the degradation of endogenous TGFBR1 and TGFBR2, ascompared to stable transfection of EV (clone EV3-3 and EV1-6)(Fig. 7B and Supplementary Fig. S4, pb0.05). Furthermore, theincreased rate of degradation is prevented by MG132, a proteasome

inhibitor, indicating that CD109 enhances proteasomal degradation ofTGF-β receptors (Fig. 7B). Moreover, HaCaT cells transfected withCD109 siRNA and treated with TGF-β display a significant (pb0.05)increase in TGFBR1 levels, as compared to control siRNA transfectionand this effect of CD109 can be blocked by addition of MG132, but notchloroquine, an inhibitor of lysosomal degradation (Fig. 7C). Theseresults indicate that endogenous CD109 facilitates proteasomaldegradation of TGF-β receptors and confirm that the previous results(Fig. 7A and B) were not an artifact due to CD109 overexpression.

Collectively, our results suggest that CD109 negatively regulatesTGF-β action by promoting TGF-β receptor internalization to thecaveolar compartment and by enhancing TGFBR1 degradation via theproteasomal pathway (Fig. 8).

4. Discussion

Our group has previously identified CD109, a GPI-anchoredprotein, as a novel TGF-β co-receptor [5]. CD109 has high affinityfor TGF-β1, forms a complex with the TGF-β signaling receptors andnegatively regulates TGF-β signaling in vitro [5]. Mutation of CD109 orderegulation of its expression has been reported in many cancers [7–12,28]. Because of CD109's potential relevance as a regulator of TGF-βaction in vivo, we examined themechanism bywhich it regulates TGF-

Fig. 6. CD109's inhibition of TGF-β signaling involves the caveolar pathway. (A–B) Western blot: (A) HaCaT cells transfected with CD109 or its EV, and, caveolin-1 siRNA or controlsiRNA were treated with 15 pM TGF-β. Cell lysates were analyzed by western blot for phosphorylated-SMAD3 (P-SMAD3) and total SMAD3 (T-SMAD3). Bottom panel: Densitometryof P-SMAD3 after 30 min TGF-β treatment (mean of n=4 independent experiments±SEM, *pb0.05). (B) HaCaT cells transfected with CD109 siRNA, caveolin-1 siRNA or controlsiRNA were treated with 15 pM TGF-β for 15 min. Cell lysates were analyzed by western blot as in (A). Bottom panel: Densitometry of P-SMAD3 (mean of n=4 independentexperiments±SEM, *pb0.05).

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β signaling. Here, we show that CD109 increases TGF-β binding to itsreceptors, promotes TGF-β receptor compartmentalization andinternalization into the caveolae and accelerates TGF-β receptorproteasomal degradation. Together, these findings suggest thatdownregulation of TGF-β signaling by CD109 involves these mech-anisms (Fig. 8). In addition, our results suggest that ligand additionmay regulate CD109's effect on TGF-β receptor localization to lipidraft/caveolar compartment.

Caveolae serve as platforms for signaling molecule assembly,endocytosis of receptors, and modulation of signaling [29]. They are asubset of lipid rafts characterized by the insertion of caveolin-1 in themembrane bilayer and are enriched in GPI-anchored proteins. Here,we report that caveolin-1 associates with CD109, suggesting thatCD109 can localize to caveolae. This association is likely indirect andmay involve lipid molecules or transmembrane proteins, such asTGFBR1, which has been shown to interact directly with both CD109[5] and caveolin-1 [18]. Interestingly, immunofluorescence andsucrose gradient analyses reveal that CD109 is present in bothcaveolar and non-caveolar compartments. Whether the non-caveolarcompartment represents a storage area for CD109 or whether CD109has other roles in that compartment is not known.

The ability of CD109 to enhance the binding of TGF-β to itsreceptors on one hand and to inhibit TGF-β signaling on the othersuggests that CD109 may direct the ligand and its receptors into acompartment where they cannot signal. Although our results do notrule out the possibility that CD109 may also play a role in clathrin-mediated endocytosis of TGF-β receptor, our data showing thatinhibitors of caveolae-mediated endocytosis reverse CD109's effect onTGF-β internalization suggest that CD109 facilitates TGF-β-boundreceptor internalization via the caveolae, a compartment associatedwith TGF-β receptor degradation and signaling downregulation[13,18]. Accumulation of receptors in caveolae has been shown totrigger the rapid internalization via caveolae [30]. Thus, CD109 mayfacilitate receptor clustering in the presence of ligand to promote theirinternalization via the caveolar route. Together, our study demon-strates that activated TGF-β receptors can internalize via the caveolae

and that this event is regulated by CD109. Thus, an alteration in CD109concentration, as seen in many cancers, may lead to a change in theproportion of TGF-β receptor internalizing via caveolae, resulting inaberrant TGF-β signaling and action.

Several molecules have been implicated in the regulation of TGF-βreceptor compartmentalization. Hyaluronan has been shown tomodulate TGF-β receptor partitioning to caveolae [21], but thequestion of hyaluronan controlling TGF-β receptor degradationremains open. The TGF-β co-receptors betaglycan and its homologueendoglin have been shown to facilitate TGF-β receptor endocytosisthrough their interaction with β-arrestin2, a component of theclathrin-coated pits [31,32]. However, there is a discrepancy in theresults reported, since internalization via the clathrin pathway leadsto inhibition of TGF-β signaling in one study [31], and delay inreceptor degradation in another study [33]. The proportion of TGF-βreceptors that internalize via the caveolae may vary depending on thecell type (possibly due to a difference in the levels of caveolin,receptors and/or co-receptors), as observed in the current study, andmay explain the discrepancy. Here, we demonstrate that, in contrastto other TGF-β co-receptors, CD109 may regulate TGF-β receptorinternalization and degradation in a ligand-dependent manner.

Although the events downstream of TGF-β receptor internalizationare ligand-dependent [34,35], studies on TGF-β receptor endocytosishave been unable to demonstrate an effect of ligand on TGF-β receptorcompartmentalization or trafficking [13,36]. Our results reveal thatligand may play a role in CD109's effect on TGF-β receptor compart-mentalization. By sucrose gradient fractionation, we show that CD109promotes localization of TGFBR1 into the caveolae, in the presence ofTGF-β. Our findings are in line with the results obtained for severalreceptors, such as insulin, β2adrenergic and EGF receptors, that localizeinto caveolae in a ligand-dependent manner [37–39]. Although themolecular mechanisms involved in TGF-β receptor internalization areless well understood than those of the receptors above, our studyprovides evidence that ligand addition can modulate TGF-β receptorlocalization into caveolae in the presence of CD109. These findingsimplicate a link between ligand-induced TGF-β receptor

Fig. 7. CD109 accelerates TGFBR1 proteasomal degradation. (A–B) Affinity labeling: (A) 293 cells transfected with CD109 or EV and TGFBR1/TGFBR2 were affinity labeled at 4 °C with125I-TGF-β, which was then covalently cross-linked to the receptors, and incubated at 37 °C for 0 to 8 h to allow receptor internalization. Left panel: The labeled receptors were

visualized by autoradiography. Coomassie blue staining shows equal protein loading. Right panel: The amount of labeled TGFBR1 was quantified by densitometry (mean of n=4independent experiments±SEM, * difference between EV and CD109 groups is statistically significant: pb0.05). (B) HaCaT cells stably transfected with CD109 (clone CD3-3) or EV(clone EV3-3) were affinity labeled as in (A), in the presence or absence of 20 μM MG132. Left panel: Labeled TGFBR1 and TGFBR2 were visualized by autoradiography. Coomassieblue staining shows equal protein loading.Middle panel: The amount of labeled TGFBR1 was quantified by densitometry (mean of n=3 independent experiments±SEM, *: pb0.05).Right panel: Western blot showing CD109 overexpression in CD3-3 clone, compared to EV3-3 clone. (C) Western blot: HaCaT cells transfected with control or CD109 siRNA weretreated with 100 pM TGF-β for 16 h, in the presence or absence of MG132 or chloroquine. Cell lysates were analyzed by western blot using the indicated antibodies (left panel). Rightpanel: Densitometry of TGFBR1 (mean of n=3 independent experiments, ± SEM, *: pb0.05).

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compartmentalization/trafficking and ligand-induced receptor degra-dation. Our results demonstrating that CD109 facilitates TGF-β receptordegradation is consistent with CD109's ability to direct TGF-β receptorlocalization into the caveolae. Although TGF-β receptors have beenshown to be degraded by both lysosomal and proteasomal machineriesfollowing receptor ubiquitination [35,40], limited data are availableon factors controlling TGFBR1 proteasomal degradation. Our resultsshowing that CD109 enhancement of TGFBR1 degradation is blocked bythe proteasome inhibitor MG132 but not by the lysosome inhibitorchloroquine, suggest that CD109 may regulate TGFBR1 proteasomaldegradation.

An important finding in the present study is that CD109 inhibitsSMAD3 phosphorylation in a caveolin-dependent manner. One poten-tial explanation is that CD109, bypromoting TGF-β receptor endocytosis

into the caveolae, sequesters the receptors away from SMAD2/3.Furthermore, CD109 promotes TGF-β receptor degradation, which inturn can lead to inhibition of signaling. CD109 may thus dampen TGF-βresponses in order to avoid a deleterious effect of excess TGF-β thatoften leads to many human diseases such as tissue fibrosis and cancermetastasis. Because deregulation of CD109 expression may result inaberrant TGF-β receptor internalization and degradation, CD109 mayrepresent a novel therapeutic target for treatment of diseases in whichTGF-β is known to play a pathophysiological role.

5. Conflict of interest

The authors have no competing financial interests in relation to thework described.

Fig. 8. Schematicmodel of the potentialmechanism bywhich CD109may regulate TGF-β receptor internalization and degradation. TGF-β receptors can internalize via the clathrin-coatedpits or the caveolar pathway [13]. CD109 increases TGF-β binding to TGF-β receptors and promotes TGF-β receptor localization to the caveolae, thereby facilitating TGF-β receptorendocytosis via the caveolar pathway and/or enhancing proteasomal degradation of TGFBR1. These effects of CD109 ultimately lead to downregulation of TGF-β signaling.

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Supplementarymaterials related to this article can be found onlineat doi:10.1016/j.bbamcr.2011.01.028.

Acknowledgments

We thank Hahn Soe-Lin for generation of HaCaT stable transfectedcells, Erik Meulmeester, Peter ten Dijke and Daniel Bernard for criticalreview of the manuscript, P. Boukamp, J. Wrana, C. Hardin, S. Egan, T.Imamura and Genzyme Corporation for reagents. This work wassupported by CIHR operating grant to A.P (FRN13732).

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