The Mannose 6-Phosphate/Insulin-like Growth Factor-II Receptor Is a Substrate of Type V Transforming Growth Factor-β Receptor

Abstract The type V transforming growth factor β (TGF-β) receptor (TβR-V) is a ligand-stimulated acidotropic Ser-specific protein kinase that recognizes a motif of SXE/S(P)/D. This motif is present in the cytoplasmic domain of the mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor. We have explored the possibility that the Man-6-P/IGF-II receptor is a substrate of TβR-V. Purified bovine Man-6-P/IGF-II receptor was phosphorylated by purified bovine TβR-V in the presence of [γ-32P]ATP and MnCl2with an apparent K m of 130 nm. TGF-β stimulated the phosphorylation of the Man-6-P/IGF-II receptor at 0 °C in mouse L cells overexpressing the Man-6-P/IGF-II receptor and in wild-type mink lung epithelial (Mv1Lu cells) metabolically labeled with [32P]orthophosphate. The in vitro andin vivo phosphorylation of the Man-6-P/IGF-II receptor occurred at the putative phosphorylation sites as revealed by phosphopeptide mapping and amino acid sequence analysis. TGF-β stimulated Man-6-P/IGF-II receptor-mediated uptake (∼2-fold after 12 h treatment) of exogenous β-glucuronidase in Mv1Lu cells and type II TGF-β receptor (TβR-II)-defective mutant cells (DR26 cells) but not in type I TGF-β receptor (TβR-I)-defective mutant cells (R-1B cells) and human colorectal carcinoma cells (RII-37 cells) expressing TβR-I and TβR-II but lacking TβR-V. These results suggest the Man-6-P/IGF-II receptor serves as an in vitroand in vivo substrate of TβR-V and that both TβR-V and TβR-I may play a role in mediating the TGF-β-stimulated uptake of exogenous β-glucuronidase.

The multiple functions of TGF-␤ are mediated by specific cell-surface receptors. A number of receptors and binding proteins for TGF-␤ have been identified in cultured cells and tissues by cross-linking 125 I-labeled TGF-␤ ( 125 I-TGF-␤) to these proteins in the presence of bifunctional reagents. These include type I (M r ϳ53,000), type II (M r ϳ66,000), type III (M r ϳ280,000 -370,000), type IV (M r ϳ60,000), type V (M r ϳ400,000), and type VI (M r ϳ180,000) TGF-␤ receptors, as well as several other membrane-binding proteins (M r ϳ38,000 -190,000) (4 -13). Among these receptors and membrane-binding proteins, type I, type II, type III, and type V TGF-␤ receptors (T␤R-I, T␤R-II, T␤R-III, and T␤R-V) co-express in most cell types (9). Other TGF-␤ receptors and binding proteins are found only in certain cell types and tissues. Recent studies have revealed that the T␤R-I, T␤R-II, and T␤R-V are membrane receptor Ser/Thr-specific protein kinases, implicating these receptors in signal transduction leading to various cellular responses following TGF-␤ stimulation (13)(14)(15). T␤R-V, first identified in our laboratory (8,9), is an acidotropic Ser/Thrspecific protein kinase that recognizes a motif of SXE/S(P)/D (15). In TGF-␤-responsive cells, T␤R-V appears to form heterocomplexes with T␤R-I in a ligand-independent manner (14).
The signal transduction leading to the cellular responses mediated by T␤R-V in response to TGF-␤ is likely to be initiated by the kinase activity of the receptor, as this is stimulated by TGF-␤ both in vitro and in vivo (14 -16). Its potential substrates are likely to be cytoplasmic proteins or the cytoplasmic domains of transmembrane proteins. To discern the substrate specificity of T␤R-V, we studied nonphysiological substrates such as caseins and insulin-like growth factor binding protein-3, and we recently identified the consensus substrate motif (SXE/S(P)/D) of T␤R-V in these substrates (14,15). The elucidation of the consensus substrate motif enabled us to search for potential physiological substrates possessing the SXE/S(P)/D motif. The many candidate proteins include the mannose 6-phosphate/insulin-like growth factor II (Man-6-P/IGF-II) receptor. This receptor appears especially interesting to examine for its substrate activity because of the following reasons. 1) The cytoplasmic domain of the Man-6-P/IGF-II receptor actually contains two consensus substrate motif sequences that are highly conserved among species (DSEDE and DSDED) (17)(18)(19)(20). This evolutionary conservation suggests the importance of these sequences in the function of the Man-6-P/IGF-II receptor molecule.
2) The Man-6-P/IGF-II receptor has already been shown to be an in vitro substrate for casein kinase II (21,22), the consensus sequence of which (SXXE) is similar to that of T␤R-V (SXE/S(P)/D) (15,23). Conceivably, T␤R-V and casein kinase II could recognize either or both of the putative phosphorylation sites (DSEDE and DSDED) in the cytoplasmic domain of the Man-6-P/IGF-II receptor (17)(18)(19)(20).
3) The Man-6-P/IGF-II receptor has been shown to be involved in the presentation of latent TGF-␤ on the cell surface for proteolytic activation (24,25). It seemed possible that T␤R-V might regulate the function of the Man-6-P/IGF-II receptor via phosphorylation of the cytoplasmic domain of the Man-6-P/IGF-II receptor.
The Man-6-P/IGF-II receptor is a multifunctional 270-kDa transmembrane receptor that binds lysosomal enzymes and mannose 6-phosphate (Man-6-P)-containing proteins and also binds IGF-II (26 -29). At steady state, the Man-6-P/IGF-II receptor is predominantly present in endosomes/prelysosomal compartments and in the trans-Golgi network (TGN). Only a small fraction of the Man-6-P/IGF-II receptor is present on the cell surface. This cell-surface Man-6-P/IGF-II receptor undergoes constitutive internalization and recycling. It is involved in the uptake of lysosomal enzymes, of IGF-II from the extracellular compartment, and also in the cell-surface activation of latent TGF-␤ (24 -29).
To test the kinase activity of T␤R-V on the Man-6-P/IGF-II receptor, we investigated the phosphorylation of the Man-6-P/ IGF-II receptor by purified T␤R-V, and we also examined the effect of TGF-␤ on the phosphorylation and function of cellsurface Man-6-P/IGF-II receptors in cultured cells. In this communication, we demonstrate that the bovine Man-6-P/IGF-II receptor is phosphorylated at serine residues by T␤R-V purified from bovine liver plasma membranes. However, the truncated receptor that lacks the transmembrane domain and cytoplasmic tail (soluble Man-6-P/IGF-II receptor) is not phosphorylated. We also show that TGF-␤ stimulates serine-specific phosphorylation of the Man-6-P/IGF-II receptor in [ 32 P]orthophosphate metabolically labeled cells. The in vitro and in vivo phosphorylation appears to occur at the putative phosphorylation sites. Furthermore, we show that TGF-␤ stimulates the Man-6-P/IGF-II receptor-mediated uptake of exogenous ␤-glucuronidase in wild-type mink lung epithelial cells (Mv1Lu cells) and in T␤R-II-defective mutant cells (DR26 cells) but not in T␤R-I-defective mutant cells (R-1B cells) nor in human colorectal carcinoma cells (RII-37 cells) that express T␤R-I and T␤R-II but lack T␤R-V.
Recombinant human ␤-glucuronidase and rabbit antisera to human ␤-glucuronidase and bovine Man-6-P/IGF-II receptor were prepared as described previously (30,31). Antiserum to bovine Man-6-P/IGF-II receptor was found to react with the Man-6-P/IGF-II receptor from Mv1Lu cells. Disuccinimidyl suberate was obtained from Pierce. Mouse L cells stably transfected with neo vector only and with vector expressing human Man-6-P/IGF-II receptor cDNA (Lmpr Ϫ and Lmpr ϩ cells) were prepared as described previously (32). Human colorectal carcinoma cells transfected with neo vector only and with vector expressing T␤R-II cDNA (HCT-116 Neo and RII-37 cells) were provided by Dr. Michael G. Brattain, Medical College of Ohio. Wild-type mink lung epithelial cells (Mv1Lu cells) were obtained from American Type Cul-ture Collection (Rockville, MD). T␤R-Iand T␤R-II-defective mutants (R-1B and DR 26 cells) were provided by Dr. Joan Massagué, Memorial Sloan-Kettering Cancer Center, New York. All other cell types were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum.
Purification of T␤R-V from Bovine Liver Plasma Membranes-The T␤R-V was purified from bovine liver plasma membranes by sequential column chromatography on wheat germ lectin-Sepharose 4B, DEAEcellulose, and Sepharose CL-4B following Triton X-100 extraction of liver plasma membranes according to the procedure of O'Grady et al. (8). Purified T␤R-V showed the homogeneity of an ϳ400-kDa protein band on SDS-polyacrylamide gel after SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining (8). However, some preparations of purified T␤R-V were found to contain proteolytic products of molecular weights ϳ100,000 -130,000.
Phosphorylation of Bovine Man-6-P/IGF-II Receptor by T␤R-V-The reaction mixture (50 l volume) contained ϳ0.25 g of T␤R-V purified from bovine liver plasma membranes, various concentrations of bovine Man-6-P/IGF-II receptor, or 100 nM soluble bovine Man-6-P/IGF-II receptor, in 20 mM HEPES, pH 7.4, 3.3% glycerol, 0.08% Triton X-100, 2.5 mM MnCl 2 , and 0.1% ␤-mercaptoethanol. The phosphorylation reaction was started by addition of 5 Ci/5 M [␥-32 P]ATP. After 20 min at 0°C, the reaction mixture was analyzed by 5% SDS-PAGE under reducing conditions and autoradiography. The 32 P-labeled Man-6-P/IGF-II receptor band which co-migrated with its protein band was excised from the dried gels and subjected to phosphoamino acid analysis as described previously (16).
Phosphorylation of the Man-6-P/IGF-II Receptor in [ 32 P]Orthophosphate Metabolically Labeled Cells-Cells were grown on 60-mm Petri dishes in DMEM containing 5% dialyzed fetal calf serum and 3.2 M methotrexate (for Lmpr Ϫ and Lmpr ϩ cells) or 10% fetal calf serum (for Mv1Lu, R-1B, DR26, RII-37, and NIH 3T3 cells). The cells were then metabolically labeled with [ 32 P]orthophosphate as described previously (33). 32 P-Labeled Lmpr ϩ and Lmpr Ϫ cells were then incubated with 0.1 nM TGF-␤ in the presence of 16 M okadaic acid and 100 M vanadate. After 30 min at 0°C, the cells were then lysed with 1% Triton X-100 buffer and diluted to a final concentration of 0.1% Triton X-100 buffer according to the procedure of Liu et al. (14). The supernatant of the cell lysates was incubated with antiserum to Man-6-P/IGF-II receptor (1: 100 dilution). The immunocomplexes were precipitated with 20 l of protein A-Sepharose gel (50%, v/v). After washing with 0.1% Triton X-100 buffer, the immunoprecipitates were analyzed by 5% SDS-PAGE under reducing conditions and autoradiography.
[ 32 P]Orthophosphate metabolically labeled Mv1Lu cells were treated with 0.1 nM TGF-␤ in the presence of 16 M okadaic acid and 100 M vanadate at 0°C for 30 min. The cells were then incubated with antiserum to Man-6-P/IGF-II receptor (1:100 dilution) for 2 h at 0°C. After washing with serum-free DMEM, the cells were lysed with 1% Triton X-100 buffer containing 20 g of purified bovine Man-6-P/IGF-II receptor and diluted to a final concentration of 0.1% Triton X-100 as described previously (14). The immunocomplexes in the supernatant of the cell lysates were then precipitated with 20 l of protein A-Sepharose gel (50%, v/v). After washing with 0.1% Triton X-100 buffer, the immunocomplexes were dissociated in SDS sample buffer and analyzed by 5% SDS-PAGE under reducing conditions and autoradiography. 125 I-Labeled TGF-␤ ( 125 I-TGF-␤) Affinity Labeling of Cell-surface TGF-␤ Receptors-Preparation of 125 I-TGF-␤ 1 and 125 I-TGF-␤ 3 and 125 I-TGF-␤-affinity labeling of cell-surface TGF-␤ receptors were carried out as described previously (8,9). 125 I Iodination of Recombinant Human ␤-Glucuronidase-␤-Glucuronidase (33 g in 15 l of 0.5 M sodium phosphate buffer, pH 7.5, containing 0.15 M NaCl and 12 M ␤-glycerol phosphate) was mixed with 10 l of 1.5 M sodium phosphate buffer, pH 7.4, and 10 l of 125 I (1 mCi in 0.1 N NaOH). Five l of chloramine T (100 g/ml) were sequentially added into the reaction mixture at 2-, 3.5-, and 4.5-s intervals. After 1 min, the reaction was terminated by addition of 20 l of 20 mM N-acetyltyrosine and 200 l of 10% KI. The reaction mixture was then applied onto a column of Sephadex G-25 to remove free iodide. The 125 I-␤-glucuronidase had a specific radioactivity of 2-6 ϫ 10 4 cpm/ng and was stored at Ϫ20°C before use.
Man-6-P/IGF-II Receptor-mediated Binding of 125 I-Glucuronidase in Mv1Lu Cells-Mv1Lu cells (0.6 -1.0 ϫ 10 5 cells/well) were grown on 24-well clustered dishes in 10% fetal calf serum in DMEM. After treatment with 0.1 nM TGF-␤ in 0.1% fetal calf serum at 37°C for 12 h, the monolayers were incubated with various concentrations (0, 0.2, 0.4, 0.8, 1.6, 2.0, and 2.4 nM) of 125 I-␤-glucuronidase in the presence and absence of 10 mM Man-6-P in binding buffer (50 mM HEPES, pH 7.5, 128 mM NaCl, 5 mM KCl, 5 mM MgSO 4 , and 1.2 mM CaCl 2 ) containing 0.2% bovine serum albumin. After the binding of 125 I-␤-glucuronidase reached equilibrium (2.5 h at 0°C), the cells were washed with 1 ml of binding buffer three times. The cells were then dissolved in 0.2 ml of 0.2 N NaOH and counted. The Man-6-P/IGF-II receptor-mediated binding of 125 I-␤-glucuronidase was estimated by subtracting the binding in the presence of 10 mM Man-6-P from the binding in the absence of Man-6-P. The apparent K d values and receptor number of the Man-6-P/IGF-II receptor-mediated binding of 125 I-␤-glucuronidase were determined by Scatchard plot analysis. The experiments for Scatchard plot analysis were repeated three times.
Man-6-P/IGF-II Receptor-mediated Uptake of 125 I ␤-Glucuronidase in Cultured Cells-Approximately 0.6 -1.0 ϫ 10 5 cells per well of Mv1Lu, R-1B, DR26, and RII-37 cells were plated on 24-well clustered dishes in 0.1% fetal calf serum in DMEM. The cells were then incubated with 0.1 nM TGF-␤ for various periods (0, 2, 6, 12, and 18 h). At each period, the cells were incubated with 2 nM 125 I-␤-glucuronidase in the presence or absence of 2 mM Man-6-P in 0.1% fetal calf serum in DMEM. After 2 h at 37°C, the cells were washed three times with binding buffer and dissolved in 0.4 ml of 0.2 N NaOH for radioactivity counting. The Man-6-P/IGF-II receptor-mediated uptake of 125 I-␤-glucuronidase was calculated by subtracting the uptake in the presence of 2 mM Man-6-P from the uptake in the absence of Man-6-P. The uptake assays were carried out in triplicate.
Indirect Immunofluorescence Staining-Mv1Lu cells grown on cover glass slides were treated with 0.1 nM TGF-␤ for 12 h. The uptake of ␤-glucuronidase in these cells was carried out at 37°C for 2 h with 2 nM ␤-glucuronidase in the presence or absence of 10 mM Man-6-P. After incubation, the cells were fixed with 100% methanol (4°C). The fixed cells were incubated with 10% bovine serum albumin in phosphatebuffered saline for 30 min or longer. The cells were then incubated with non-immune serum or antiserum to ␤-glucuronidase (1:20 dilution in phosphate-buffered saline) at 37°C for 1 h. After being washed with 0.1% bovine serum albumin in phosphate-buffered saline five times, the cells were incubated with fluorescein-labeled goat anti-rabbit IgG at room temperature for 1 h. The cells were then washed with 0.1% bovine serum albumin in phosphate-buffered saline and visualized under a fluorescence microscope.
Phosphopeptide Mapping-After phosphorylation and SDS-PAGE, the 32 P-labeled Man-6-P/IGF-II receptor was located by Coomassie Blue staining. The 32 P-Man-6-P/IGF-II receptor band was cut out and washed extensively with 50% ethanol and vacuum-dried. The dried gels were rehydrated using 0.5 ml of 50 mM ammonium bicarbonate, pH 8.5, containing 2 g of L-1-tosylamido-2-phenylethyl chloromethyl ketonetreated trypsin. After incubation at 37°C for 18 h, the supernatant was dried by Speed Vac, dissolved in 10 l of H 2 O, and subjected to twodimensional thin layer cellulose electrophoresis and chromatography. The solvent for the first-dimensional electrophoresis was acetic acid: pyridine:H 2 O (20:1:379, by volume). The solvent for the second-dimensional chromatography was n-butyl alcohol:pyridine:acetic acid:H 2 O (37.5:25:7.5:30, by volume). The thin layer cellulose plates were dried in air and exposed to Kodak X-Omat films.
Automated Amino Acid Sequencing of 32 P-Labeled Tryptic Peptides Derived from 32 P-Labeled Man-6-P/IGF-II Receptors-32 P-Labeled bovine Man-6-P/IGF-II eluted from the SDS-polyacrylamide gel was subjected to trypsin digestion as described above. The tryptic digests were separated on reverse phase high pressure liquid chromatography (C8 column) with a linear gradient of acetonitrile from 0 to 70% in 0.1% trifluoroacetic acid using an Applied Biosystems model 130A separation system. The peptide containing radioactivity was eluted at ϳ20% acetonitrile and subjected to automated amino acid sequencing on an Applied Biosystems model 477 gas/liquid phase protein sequenator with an on-line Applied Biosystems model 120A phenylthiohydantoin amino acid analyzer. The average repetitive yield was estimated to be 93%. The identification of phosphorylated amino acid residues in 32 P-labeled tryptic peptides was carried out as described previously (34). 32 P-Labeled human Man-6-P/IGF-II was eluted from SDS-polyacrylamide gel and subjected to trypsin digestion and two-dimensional phosphopeptide mapping as described above. The 32 P-labeled tryptic peptides were extracted from the cellulose particles scraped from the thin layer plates with 0.1% trifluoroacetic acid. The 32 P-labeled tryptic peptides were then analyzed by automated Edman amino acid sequencing.
Man-6-P/IGF-II Receptor-mediated Uptake of ␤-Glucuronidase in Mv1Lu Cells-Mv1Lu cells were plated on 35-mm Petri dishes in DMEM containing 0.1% fetal calf serum. The cells were incubated with 0.1 nM TGF-␤ or insulin-like growth factor binding protein-3 (0.5 and 1 g/ml) for 12 or 18 h. The cells were incubated with 2 nM ␤-glucuronidase in the presence or absence of 10 mM Man-6-P in 0.1% fetal calf serum in DMEM. After 2 h at 37°C, the cells were washed. The cell-associated ␤-glucuronidase was assayed using 4-methylumbelliferyl ␤-D-glucuronide as substrate according to the procedure of Brot et al. (35). One enzyme unit was defined as the nanomoles of 4-methylumbelliferone product released per ml of enzyme solution per h. The Man-6-P/IGF-II receptor-mediated uptake of ␤-glucuronidase was calculated by subtracting the uptake in the presence of 10 mM Man-6-P from the uptake in the absence of Man-6-P. The cellular proteins were determined with the Lowry method.
TGF-␤-stimulated Secretion of Lysosomal Enzymes in Mv1Lu Cells-Mv1Lu cells grown on 35-mm Petri dishes were treated with 5 mM Man-6-P Ϯ 0.1 nM TGF-␤ for 12 h. The medium was then assayed for ␤-glucuronidase, ␤-hexosaminidase, ␣-galactosidase, and ␣-mannosidase. The enzyme assays were carried out as described previously (35). To exclude the possibility that the increased secretion of lysosomal enzymes might result from their increased biosynthesis, the Tran 35 Slabel-metabolic labeling of ␤-glucuronidase in Mv1Lu cells treated with and without 0.1 nM TGF-␤ was carried out as described previously (33). Mv1Lu cells were treated with and without 0.1 nM TGF-␤ in DMEM containing 0.1% fetal calf serum at 37°C for 12 h. After Tran 35 S-labelmetabolic labeling (33), 35 S-metabolically labeled ␤-glucuronidase was immunoprecipitated with antiserum to ␤-glucuronidase. The immunoprecipitates were analyzed by 7.5% SDS-PAGE under reducing conditions and fluorography. The 35 S-labeled ␤-glucuronidase identified as an ϳ80-kDa protein band on the autoradiogram was quantitated using a PhosphorImager. TGF-␤ appeared to have no effect on the level of 35 S-labeled ␤-glucuronidase in Mv1Lu cells.

RESULTS
The Man-6-P/IGF-II Receptor Is an in Vitro Substrate for T␤R-V-The Man-6-P/IGF-II receptor is a transmembrane protein composed of three structural domains as follows: a cellsurface ligand binding domain, a transmembrane domain, and a cytoplasmic domain (26 -29). The putative phosphorylation sites (DSEDE and DSDED) on the Man-6-P/IGF-II receptor for T␤R-V are located in its cytoplasmic domain (17)(18)(19)(20). To examine the substrate activity of the Man-6-P/IGF-II receptor, purified bovine receptor and its cytoplasmic tail-and transmembrane-truncated form (soluble Man-6-P/IGF-II receptor) were incubated with purified T␤R-V in HEPES, 0.08% Triton X-100 buffer containing [␥-32 P]ATP and MnCl 2 . After 20 min at 0°C, the reaction mixture was analyzed by 5% SDS-PAGE under reducing conditions and autoradiography. As shown in Fig. 1A, bovine Man-6-P/IGF-II receptor monomers and dimers were phosphorylated (lane 2), and the truncated receptor was not phosphorylated (lane 5). Purified bovine Man-6-P/IGF-II receptor contained monomers (ϳ85%) and covalent dimers (ϳ15%) that were stable at 100°C in 0.1% SDS (5 min) and migrated slightly more slowly than T␤R-V on SDS-PAGE (Fig. 1B). Phosphoamino acid analysis of 32 P-labeled bovine Man-6-P/IGF-II receptor monomers revealed that the phosphorylation occurred at serine residues (Fig. 1C). The phosphorylation of bovine Man-6-P/IGF-II receptor monomers by T␤R-V appeared to be dose-dependent with an apparent K m of 130 nM (Fig. 2). At 200 nM bovine Man-6-P/IGF-II receptor monomers, the 32 P incorporation was estimated to be ϳ0.5 mol of [ 32 P]phosphate per mol of the receptor. These results demonstrate that the Man-6-P/IGF-II receptor is a substrate for T␤R-V in vitro.
TGF-␤ Stimulates the Phosphorylation of the Man-6-P/ IGF-II Receptor in Mouse L Cells Stably Transfected with Human Man-6-P/IGF-II Receptor cDNA-We previously demonstrated that TGF-␤ stimulates the kinase activity of T␤R-V in vitro and in vivo (14 -16). If the Man-6-P/IGF-II receptor is a substrate for T␤R-V in vivo, TGF-␤ should stimulate the phos-phorylation of the Man-6-P/IGF-II receptor in cultured cells at 0°C. To test this possibility, we investigated the effect of TGF-␤ on the phosphorylation of the Man-6-P/IGF-II receptor in mpr Ϫ mouse L cells stably transfected with human Man-6-P/IGF-II receptor cDNA (Lmpr ϩ cells). The parental mpr Ϫ mouse L cells are deficient in endogenous mouse Man-6-P/ IGF-II receptor (36). In preparation for investigation of the phosphorylation of the human Man-6-P/IGF-II receptor, we first performed 125 I-labeled TGF-␤ ( 125 I-TGF-␤) affinity labeling to ensure that Lmpr ϩ cells expressed T␤R-V and other TGF-␤ receptor types. As shown in Fig. 3, mouse L cells transfected with neo vector only and with vector expressing human Man-6-P/IGF-II receptor cDNA (Lmpr Ϫ and Lmpr ϩ cells, respectively) expressed all major TGF-␤ receptors including T␤R-I, T␤R-II, T␤R-III, and T␤R-V as demonstrated in Mv1Lu cells (14,30).

In Vitro and in Vivo Phosphorylation of the Man-6-P/IGF-II
Receptor Occurs at the Putative Phosphorylation Sites-To define the in vitro phosphorylation sites of the Man-6-P/IGF-II receptor, the 32 P-labeled bovine Man-6-P/IGF-II receptor band was cut from the gel after in vitro phosphorylation by T␤R-V and 5% SDS-PAGE and then subjected to trypsin digestion and two-dimensional peptide mapping. As shown in Fig. 5A, a 32 Plabeled tryptic peptide was detected on the peptide map. The amino acid sequence analysis of this 32 P-labeled peptide revealed that this peptide contained the amino-terminal amino acid sequence A A D T L S A L H G D E Q D S E D E . During automated Edman amino acid sequencing, the 32 P radioactivity started to appear at the 15th phenylthiohydantoin-derivative from the amino terminus of the peptide which corresponds to the 15th residue (Ser). This result suggested that the 15th residue (Ser) was phosphorylated (34). The cytoplasmic domain of the Man-6-P/IGF-II receptor contains two putative phosphorylation sites, DSEDE and DSDED. Finding a 32 P-labeled peptide corresponding to only one of the two phosphorylation sites may be due to the fact that the extreme carboxyl terminus of bovine Man-6-P/IGF-II containing the other phosphorylation site was removed proteolytically during its purification (22).
On the other hand, when the human Man-6-P/IGF-II receptor was phosphorylated in Lmpr ϩ cells, the phosphopeptide map showed two 32 P-labeled tryptic peptides (Fig. 5B). TGF-␤ treatment did not generate new 32 P-labeled peptides but enhanced the 32 P labeling of both peptides (Fig. 5, C versus B). The smear near the origin of the peptide map and the U shape near the 32 P-labeled peptides were artifacts. The amounts of these two 32 P-labeled peptides were so small that we were unable to identify the phosphorylated residues. However, these two 32 P-labeled tryptic peptides (slow and fast migrating peptides on the second-dimensional thin layer chromatography) from human Man-6-P/IGF-II receptor showed amino-terminal amino acid sequences of A L S and L V S F H D that corresponded to those of the tryptic peptides containing putative phosphorylation sites (ALSSLHGDDQDSEDEVLTIPEVK and LVSFH- The arrows indicate the locations of 32 P-labeled Man-6-P/IGF-II receptor ( 32 P-M6P/IGF-IIR) monomers and dimers. One and two asterisks indicate the locations of the 32 P-labeled autophosphorylated T␤R-V and its proteolytic products, respectively. The bar indicates the location of soluble Man-6-P/IGF-II receptor that was located by Coomassie Blue staining. B, purified bovine Man-6-P/IGF-II receptor (M6P/IGF-II R) (ϳ5 g) was subjected to 5% SDS-PAGE under reducing conditions and Coomassie Blue staining. The apparent molecular weights of Man-6-P/IGF-II receptor monomers and dimers were estimated to be ϳ230,000 and ϳ460,000, respectively. C, phosphoamino acid analysis of the acid hydrolysates of 32 P-Man-6-P/IGF-II receptor monomers eluted from the SDS-polyacrylamide gel was carried out by thin layer cellulose electrophoresis at pH 3.5. The standard phosphoamino acids, P-Ser, P-Thr, and P-Tyr were co-electrophoresed with the acid hydrolysates of 32 P-Man-6-P/IGF-II receptor monomers. DDSDEDLLHI) (19). These results suggest that the in vitro and in vivo phosphorylations of the Man-6-P/IGF-II receptors occur at the putative phosphorylation sites.

TGF-␤ Stimulates the Man-6-P/IGF-II Receptor-mediated Uptake of Exogenous ␤-Glucuronidase in Mv1Lu Cells-Cell-
surface Man-6-P/IGF-II receptors undergo constitutive internalization and recycling and are involved in the uptake of lysosomal enzymes and IGF-II in the extracellular compartment (26 -29). To test the possibility that the TGF-␤-stimulated phosphorylation of cell-surface Man-6-P/IGF-II receptors may affect the function of the cell-surface Man-6-P/IGF-II receptor, we investigated the effect of TGF-␤ on the internalization and recycling of cell-surface Man-6-P/IGF-II receptors in Mv1Lu cells. Mv1Lu cells are the standard cell system for investigating TGF-␤-induced cellular responses (1). The serinespecific phosphorylation of cell-surface Man-6-P/IGF-II receptors in response to TGF-␤ stimulation (in the presence of okadaic acid and vanadate) at 0°C was also found in Mv1Lu cells. Fig. 6 shows a 1.6-fold TGF-␤ stimulation (1.6 Ϯ 0.2; mean Ϯ S.D., n ϭ 3) of 32 P labeling of cell-surface Man-6-P/IGF-II receptors in Mv1Lu cells metabolically labeled with [ 32 P]orthophosphate. Phosphoamino acid analysis also revealed that the phosphorylation occurred at serine residues (data not shown).
To quantitate the internalization and recycling of cell-surface Man-6-P/IGF-II receptors, we determined the Man-6-P/ IGF-II receptor-mediated uptake of exogenous human ␤-glucuronidase (32). Mv1Lu cells were treated with TGF-␤ for several periods during which the Man-6-P/IGF-II receptor-mediated uptake of 125 I-␤-glucuronidase was measured in 2-h incubations. During this incubation period, the uptake of 125 I-␤-glucuronidase was linear. As shown in Fig. 7, TGF-␤ stimulated the Man-6-P/IGF-II receptor-mediated uptake of 125 I-␤-glucuronidase in a time-dependent manner. A maximal 125 I-␤-glucuronidase uptake rate (ϳ28,000 cpm/well/hr) was generated by treatment of Mv1Lu cells with TGF-␤ for 12 h. This 125 I-␤glucuronidase uptake rate was nearly twice that of the cells treated without TGF-␤ for the same period (ϳ15,000 cpm/well/ h). During the 12-h period of incubation with TGF-␤, the ligand binding activity (4100 Ϯ 801 cpm/well; mean Ϯ S.D., n ϭ 3) or receptor number (ϳ7000 Ϯ 1200/cell; mean Ϯ S.D., n ϭ 3) of cell-surface Man-6-P/IGF-II receptors did not show a significant change compared with that estimated from the cells treated without TGF-␤ (8500 Ϯ 2000/cell; mean Ϯ S.D., n ϭ 3). The apparent K d of 125 I-␤-glucuronidase binding to the receptor was determined to be 1.0 Ϯ 0.1 and 0.9 Ϯ 0.05 nM in Mv1Lu cells treated with and without TGF-␤, respectively. These results suggest that TGF-␤ treatment of cells increases the uptake of 125 I-␤-glucuronidase without altering the receptor number and ligand affinity of cell-surface Man-6-P/IGF-II receptors. Assuming a constant number of cell-surface Man-6-P/ IGF-II receptors, we estimate that cell-surface Man-6-P/IGF-II receptors are replaced approximately every 8 min after treatment with TGF-␤ for 12 h. In TGF-␤-unstimulated cells, we estimate that cell-surface Man-6-P/IGF-II receptors are replaced every 16 min during uptake of the 125 I-␤-glucuronidase ligand. It is important to note that the time of 16 min for replacing cell-surface Man-6-P/IGF-II receptors in Mv1Lu cells without TGF-␤ stimulation is longer than that reported for human fibroblasts during uptake of ␤-glucuronidase (ϳ5 min) (37). TGF-␤ was also able to increase the uptake of 125 I-␤glucuronidase ϳ2-fold in NIH 3T3 cells (Table I). Thus, TGF-␤ stimulates the internalization and recycling of cell-surface Man-6-P/IGF-II receptors in a time-dependent manner in more than one cell type.
In the previous experiments, we found that the treatment of the cultured cells with okadaic acid was required for demonstration of TGF-␤-stimulated phosphorylation of the Man-6-P/ IGF-II receptor, suggesting that the Man-6-P/IGF-II receptor undergoes okadaic acid-sensitive dephosphorylation following TGF-␤-stimulated phosphorylation. The okadaic acid-sensitive dephosphorylation and TGF-␤-stimulated phosphorylation may be required for TGF-␤-stimulated internalization and recycling of cell-surface Man-6-P/IGF-II receptors. To test this possibility, we investigated the effect of okadaic acid on the Man-6-P/IGF-II receptor-mediated uptake of 125 I-␤-glucuronidase in Mv1Lu cells. As shown in Table I, okadaic acid inhibited the Man-6-P/IGF-II receptor-mediated uptake of 125 I-␤glucuronidase in Mv1Lu cells treated with and without TGF-␤ (ϳ90 and ϳ70% inhibition, respectively). The inhibition by okadaic acid of the Man-6-P/IGF-II receptor-mediated uptake of 125 I-␤-glucuronidase appears to result at least partially from the depletion of cell-surface Man-6-P/IGF-II receptors. The analysis of cell-surface Man-6-P/IGF-II receptors indicates that, following treatment with okadaic acid, the number of cell-surface receptors decreases by ϳ70 and ϳ55% in Mv1Lu cells incubated with and without TGF-␤, respectively. The oka-daic acid treatment did not alter the apparent K d of 125 I-␤glucuronidase binding to cell-surface Man-6-P/IGF-II receptors. Similar okadaic acid-induced depletion of cell-surface P-labeled cells were treated with TGF-␤ at 0°C for 30 min. 32 P-Man-6-P/IGF-II receptor was then immunoprecipitated with antiserum to Man-6-P/IGF-II receptor. The immunoprecipitates were analyzed by 5% SDS-PAGE under reducing conditions and autoradiography. The arrow indicates the location of 32 P-Man-6-P/IGF-II receptor ( 32 P-M6P/IGF-II R). The intensity of 32 P-Man-6-P/IGF-II receptor on the autoradiogram was quantitated by a PhosphorImager. B, phosphoamino acid analysis of the acid hydrolysates of 32 P-labeled Man-6-P/IGF-II receptor eluted from the SDS-polyacrylamide gel was performed by thin layer cellulose electrophoresis at pH 3.5. The standard phosphoamino acids, P-Ser, P-Thr, and P-Tyr were co-electrophoresed with the acid hydrolysates of 32 P-Man-6-P/IGF-II receptor. 32 P-Bovine Man-6-P/IGF-II receptor (b M6P/IGF-II R) and 32 P-human Man-6-P/IGF-II receptor (h M6P/ IGF-II R) were prepared as shown in Figs. 1 and 3, eluted from Coomassie Blue-stained gels, and subjected to phosphopeptide mapping following trypsin digestion. The arrows indicate the locations of 32 Plabeled tryptic peptides. The smear spot near the origin (C) and U shape near the 32 P-labeled peptides (A, B, and C) are artifacts. The 32 P-labeled tryptic peptides on the phosphopeptide maps were extracted from the cellulose particles scraped from the thin layer plates and then subjected to automated Edman amino acid sequencing.

FIG. 6. TGF-␤-stimulated phosphorylation of cell-surface Man-6-P/IGF-II receptors in Mv1Lu cells.
Cells metabolically labeled with [ 32 P]orthophosphate were treated with (ϩ) and without (Ϫ) TGF-␤ in the presence of okadaic acid and vanadate. 32 P-Labeled cell-surface Man-6-P/IGF-II receptors ( 32 P-M6P/IGF-II R) was then immunoprecipitated by incubation of the 32 P-labeled cells with antiserum to the Man-6-P/IGF-II receptor followed by washing to remove unbound antibodies. The immunoprecipitates were then analyzed by 5% SDS-PAGE under reducing conditions and autoradiography. The arrow indicates the location of 32 P-Man-6-P/IGF-II receptor ( 32 P-M6P/IGF-II R). The intensity of 32 P-Man-6-P/IGF-II receptor on the autoradiogram was quantitated by a PhosphorImager.
Man-6-P/IGF-II receptors was shown previously in human skin fibroblasts (38). These results suggest that both phosphorylation and dephosphorylation of the cell-surface Man-6-P/IGF-II receptor are required for its internalization and recycling and are involved in the uptake of 125 I-␤-glucuronidase.
To confirm that TGF-␤ stimulated the Man-6-P/IGF-II receptor-mediated uptake of ␤-glucuronidase, we performed two additional sets of experiments. First, we determined the effect of TGF-␤ on the cumulative uptake of ␤-glucuronidase enzyme activity into Mv1Lu cells over an 18-h period (Table II). TGF-␤ stimulated the Man-6-P/IGF-II receptor-mediated uptake of ␤-glucuronidase approximately 1.5-fold (1.6 Ϯ 0.2; mean Ϯ S.D., n ϭ 3). In the second set of experiments, we used indirect immunofluorescence staining to compare exogenous ␤-glucuronidase taken up by Mv1Lu cells that have been treated with and without TGF-␤ for 12 h at 37°C. By this technique also, there was more ␤-glucuronidase in intracellular compartments of Mv1Lu cells treated with TGF-␤ than in cells treated with-out TGF-␤ (Fig. 8, A and B). The ␤-glucuronidase accumulation was completely blocked by Man-6-P (Fig. 8, C and D).
TGF-␤ Does Not Stimulate the Man-6-P/IGF-II Receptormediated Uptake of Exogenous ␤-Glucuronidase in Cells Lacking Either T␤R-V or T␤R-I-Mv1Lu cells express all major TGF-␤ receptor types (8,9). The TGF-␤-stimulated internalization and recycling of cell-surface Man-6-P/IGF-II receptors could be mediated by T␤R-V in concert with other TGF-␤ receptors. The T␤R-V as well as T␤R-I and T␤R-II are Ser/Thrspecific protein kinases and are believed to be involved in signaling. To define the roles of T␤R-I, T␤R-II, and T␤R-V in the TGF-␤-stimulated uptake of exogenous ␤-glucuronidase, we examined the effect of TGF-␤ on the uptake of exogenous ␤-glucuronidase in T␤R-I and T␤R-II-defective mutants (R-1B and DR26 cells) of Mv1Lu cells (39,40) and human colorectal carcinoma cells (which lack T␤R-V and T␤R-II but express T␤R-I) stably transfected with neo vector only (HCT 116 Neo cells) and with vector expressing T␤R-II cDNA (RII-37 cells) (41). As shown in Table III, TGF-␤ stimulated the Man-6-P/ IGF-II receptor-mediated uptake of 125 I-␤-glucuronidase in Mv1Lu cells and DR26 cells but not in R-1B cells and RII-37 cells. These results also are consistent with the hypothesis that T␤R-V plays a role in mediating the TGF-␤-stimulated uptake of exogenous ␤-glucuronidase but suggest that T␤R-I is also required.
TGF-␤ Stimulates the Secretion of Lysosomal Enzymes in Mv1Lu Cells-TGF-␤ stimulates the Man-6-P/IGF-II receptormediated uptake of exogenous ␤-glucuronidase, but the Man-6-P/IGF-II receptor is involved not only in the uptake of extracellular lysosomal enzymes but also in the delivery of newly synthesized lysosomal enzymes from TGN to endosomes/prelysosomal compartments and to the extracellular compartment.   It was therefore of interest to see whether TGF-␤ influenced the rate of the secretion of endogenous lysosomal enzymes. To address this question, we examined the effect of TGF-␤ on the secretion of lysosomal enzymes in Mv1Lu cells. As shown in Table IV, TGF-␤ stimulated the secretion of all four lysosomal enzymes measured (␤-glucuronidase, ␤-hexosaminidase, ␣-galactosidase, and ␣-mannosidase) under conditions in which TGF-␤ has no significant effect on cell number. The TGF-␤ stimulation of lysosomal enzyme secretion ranged from ϳ1.4to 1.8-fold. The Tran 35 S-label metabolic labeling experiments demonstrated that TGF-␤ does not affect the biosynthetic rate of ␤-glucuronidase in Mv1Lu cells under conditions where TGF-␤ stimulates the secretion of lysosomal enzymes (data not shown). Together with the observations described above, these results suggest that TGF-␤ stimulates the overall cellular trafficking of the Man-6-P/IGF-II receptor, including internalization and recycling of the cell-surface receptor and the transport of the Man-6-P/IGF-II receptor from TGN to the cell surface, resulting in the secretion of lysosomal enzymes.

DISCUSSION
Several lines of evidence presented herein support the suggestion that the Man-6-P/IGF-II receptor is a substrate for T␤R-V in vitro and in vivo. These include the following. 1) Purified bovine Man-6-P/IGF-II receptor but not its cytoplasmic tail-and transmembrane-truncated form (soluble Man-6-P/IGF-II receptor) is phosphorylated with a low K m by T␤R-V purified from bovine liver plasma membranes. 2) When incubated at 0°C, which minimizes downstream phosphorylation, TGF-␤ stimulates the phosphorylation of the Man-6-P/IG-II receptor in Lmpr ϩ cells and Mv1Lu cells.
3) The in vitro and in vivo phosphorylation of the Man-6-P/IGF-II receptor occurs at the putative phosphorylation sites in the cytoplasmic domain of the receptor. 4) TGF-␤ is unable to stimulate the phosphorylation of cell-surface Man-6-P/IGF-II receptors in cells lacking the expression of T␤R-V (e.g. RII-37 and HCT 116 Neo cells).
The putative phosphorylation sites of the Man-6-P/IGF-II receptor are not only highly conserved among species but also homologous to the putative phosphorylation sites of several TGN-trafficking proteins including cation-dependent Man-6-P receptor and furin (42)(43)(44)(45). It is possible that both cation-dependent Man-6-P receptor and furin can also be phosphorylated by T␤R-V. The phosphorylation at the SXE/D motifs in the cytoplasmic domains of these two proteins might also affect the function of these molecules at the cell surface.
Méresse et al. (22) isolated a casein kinase II-like kinase from bovine brain that phosphorylated the cytoplasmic domain of bovine Man-6-P/IGF-II receptor at the putative phosphorylation sites for T␤R-V. This kinase associates with hydroxylapatite-group I assembly proteins of Golgi-derived clathrin-coated vesicles (22). The T␤R-V and this casein kinase II-like kinase share some properties such as polylysine stimulation and heparin inhibition of their enzyme activities but then differ in molecular weight and cellular localization. The T␤R-V is a 400-kDa plasma membrane glycoprotein. In contrast, the casein kinase II-like kinase is a cytoplasmic vesicle-associated protein containing a catalytic subunit of molecular weight ϳ46,000. The exact functional role of this casein kinase II-like kinase in modulation of the intracellular trafficking of the Man-6-P/IGF-II receptor is unknown. It is likely that T␤R-V and the casein kinase II-like kinase have distinct roles in modulation of intracellular trafficking of the Man-6-P/IGF-II receptor because of their different subcellular localizations (T␤R-V is a plasma membrane protein and casein kinase II-like kinase is a vesicle-associated protein). Casein kinase II-like kinase may be involved in the phosphorylation of the newly synthesized or dephosphorylated Man-6-P/IGF-II receptor at TGN (46).
In this study, we demonstrate that TGF-␤ stimulates the Man-6-P/IGF-II receptor-mediated uptake of exogenous ␤-glucuronidase. The TGF-␤ stimulation appears to be mediated by T␤R-V and T␤R-I. The T␤R-II is not required for TGF-␤-stimulated uptake of exogenous ␤-glucuronidase. The mechanism by which T␤R-V and T␤R-I mediate the TGF-␤-stimulated uptake of exogenous ␤-glucuronidase is unknown. We previously reported that T␤R-V forms a heterocomplex with T␤R-I in a ligand-independent manner (14). Since T␤R-I lacks ligand binding activity in the absence of T␤R-II (46), it is possible that T␤R-V and T␤R-I form heterocomplexes in T␤R-II-defective mutant cells (DR26 cells) and mediate the TGF-␤-stimulated uptake of exogenous ␤-glucuronidase.
Recently, site-directed mutagenesis has been employed to investigate the functions of the putative phosphorylation sites of TGN-trafficking proteins including the Man-6-P/IGF-II re-  a Monolayers of cells grown on 24-well clustered dishes were incubated with 0.1 nM TGF-␤ at 37°C for 12 h. The Man-6-P/IGF-II receptormediated uptake/h rate of 125 I-␤-glucuronidase was determined.
b The presence (ϩ) and absence (Ϫ) of the TGF-␤ receptor/kinase types in these cells were previously reported by several laboratories (8, 9, 40 -42) and verified by cell-surface 125 I-TGF-␤ affinity labeling and Western blot analysis (for T␤R-V). All of these cell types expressed T␤R-III which is a non-kinase proteoglycan membrane glycoprotein. ceptor, the cation-dependent Man-6-P receptor, and furin (42)(43)(44)(45)(46)(47). The functional importance of the putative phosphorylation sites in the trafficking of these proteins has remained controversial, possibly due to the use of transfection or overexpression systems for these studies (42)(43)(44)(45)(46)(47). Here, we show that TGF-␤ stimulated phosphorylation of the Man-6-P/IGF-II receptor and the Man-6-P/IGF-II receptor mediated uptake of exogenous ␤-glucuronidase in Mv1Lu cells under non-overexpression conditions. Empirically, it appears to require 12 h of TGF-␤ treatment to reach the maximally increased rate of uptake of exogenous ␤-glucuronidase. The reason for this time lag to reach the maximum stimulation is unclear. Only a small fraction (ϳ5%) of the Man-6-P/IGF-II receptor is present on the cell surface at any particular time, and the cell-surface receptor number is not altered by the ligand. Méresse and Hoflack (48) previously reported that in Chinese hamster ovary cells, cell-surface Man-6-P/IGF-II receptors were weakly phosphorylated and did not acquire additional phosphate groups during internalization. Based on this result, they inferred that cell-surface Man-6-P/IGF-II receptors were not phosphorylated during internalization. Their observations are consistent with ours. Although we believe that the Man-6-P/IGF-II receptor undergoes continuous phosphorylation and dephosphorylation, only in the presence of phosphatase inhibitors such as okadaic acid and vanadate can a significant accumulation of phosphorylated cell-surface Man-6-P/IGF-II receptors be detected. TGF-␤ appears to stimulate this phosphorylation of cell-surface Man-6-P/IGF-II receptors. Méresse and Hoflack (48) emphasized that the phosphorylation of the Man-6-P/IGF-II receptor occurs mainly at TGN or in clathrincoated vesicles. At the TGN, other kinases such as casein kinase II-like kinase (22) may be involved in maintaining the level of the Man-6-P/IGF-II receptor at TGN by phosphorylation of newly synthesized or dephosphorylated Man-6-P/IGF-II receptors.
The Man-6-P/IGF-II receptor has been implicated in the cell-surface activation of latent TGF-␤ (TGF-␤ precursor) that possesses Man-6-P moieties (24,25,49). The cell-surface Man-6-P/IGF-II receptor is believed to bind latent TGF-␤ and present it to plasmin or other proteases associated with the cell surface of the same cell or other cells for proteolytic activation (24,25). The TGF-␤-stimulated internalization and recycling of cell-surface Man-6-P/IGF-II receptors as shown by the increased uptake of exogenous ␤-glucuronidase may play an autocrine or paracrine feedback negative regulatory role in the generation of active TGF-␤. The stimulation of cell-surface Man-6-P/IGF-II receptors may lead to faster internalization and subsequent degradation (in lysosomes) of latent TGF-␤ that binds to cell-surface Man-6-P/IGF-II receptors, resulting in less production of active TGF-␤ on the cell surface.
TGF-␤ is a potent growth inhibitor for epithelial cells both in vitro and in vivo (1)(2)(3). TGF-␤ inhibits growth of epithelial cells mainly through interaction with T␤R-I, T␤R-II, and T␤R-V (1-3, 14). The TGF-␤-stimulated cellular trafficking of the Man-6-P/IGF-II receptor may also contribute to the growth inhibition under certain conditions in which IGF-II is responsible for the cell growth. The increased uptake and subsequent degradation (in lysosomes) of IGF-II by the TGF-␤-stimulated cell-surface Man-6-P/IGF-II receptor could contribute to this growth inhibitory effect.