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J. Biol. Chem., Vol. 279, Issue 37, 38736-38748, September 10, 2004

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Identification and Characterization of the Acidic pH Binding Sites for Growth Regulatory Ligands of Low Density Lipoprotein Receptor-related Protein-1^*

Thai-Yen Ling, Chun-Lin Chen, Yen-Hua Huang¶, I-Hua Liu, Shuan Shian Huang||, and Jung San Huang**

From the Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, the Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, and the ^¶Department of Biochemistry, Taipei Medical University, Taipei 110, Taiwan

Received for publication, September 23, 2003 , and in revised form, June 4, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The type V TGF- receptor (TR-V) plays an important role in growth inhibition by IGFBP-3 and TGF- in responsive cells. Unexpectedly, TR-V was recently found to be identical to the LRP-1/₂M receptor; this has disclosed previously unreported growth regulatory functions of LRP-1. Here we demonstrate that, in addition to expressing LRP-1, all cells examined exhibit low affinity but high density acidic pH binding sites for LRP-1 growth regulatory ligands (TGF-₁, IGFBP-3, and ₂M^*). These sites, like LRP-1, are sensitive to receptor-associated protein and calcium depletion but, unlike LRP-1, are also sensitive to chondroitin sulfate and heparin and capable of directly binding ligands, which do not bind to LRP-1. Annexin VI has been identified as a major membrane-associated protein capable of directly binding ₂M^* at acidic pH. This is evidenced by: 1) structural and Western blot analyses of the protein purified from bovine liver plasma membranes by ₂M^* affinity column chromatography at acidic pH, and 2) dot blot analysis of the interaction of annexin VI and ¹²⁵I-₂M^*. Cell surface annexin VI is involved in ¹²⁵I-TGF-₁ and ¹²⁵I-₂M^* binding to the acidic pH binding sites and ¹²⁵I-₂M^* binding to LRP-1 at neutral pH as demonstrated by the sensitivity of cells to pretreatment with anti-annexin VI IgG. Cell surface annexin VI is also capable of mediating internalization and degradation of cell surface-bound ¹²⁵I-TGF-₁ and ¹²⁵I-₂M^* at pH 6 and of forming ternary complexes with ¹²⁵I-₂M^* and LRP-1 at neutral pH as demonstrated by co-immunoprecipitation. Trifluoperazine and fluphenazine, which inhibit ligand binding to the acidic pH binding sites, block degradation after internalization of cell surface-bound ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^*. These results suggest that cell surface annexin VI may function as an acidic pH binding site or receptor and may also function as a co-receptor with LRP-1 at neutral pH.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The type V TGF- receptor (TR-V)¹ is a high molecular weight glycoprotein receptor, which co-expresses with type I, type II, and type III TGF- receptors (TR-I, TR-II, TR-III) in most cell types (1-3). Many carcinoma cells express little or no TR-V (3, 4). Their growth is not inhibited by TGF-₁, suggesting that TR-V may be involved in the growth inhibitory response to TGF-₁ and that loss of TR-V may contribute to the malignant phenotype (3-5). Identification of TR-V as the insulin-like growth factor-binding protein-3 (IGFBP-3), which mediates the IGF-independent growth inhibitory response upon IGFBP-3 stimulation of responsive cells, has highlighted the likely importance of TR-V in mediating the growth inhibitory response to TGF-₁ (5, 6). Structural and functional analysis of TR-V seems to be required for elucidating the molecular mechanisms by which TGF-₁ and IGFBP-3 induce growth inhibition in responsive cells (5-7). Unexpectedly, we recently found that the TR-V is identical to the low density lipoprotein receptor-related protein-1/activated ₂M receptor (LRP-1/₂M receptor) as determined by structural and functional analyses of purified TR-V (8). Genetic evidence and evidence of rescue experiments has revealed that TR-V/LRP-1 is required for cellular growth inhibition caused by IGFBP-3 and TGF-₁ (8).

LRP-1 was identified by Herz et al. (9) through homologous sequence screening. It is best known as an endocytic receptor and has been shown to bind multiple ligands with distinct structures and to mediate their plasma clearance and cellular catabolism (10-12). One of the prominent ligands is the activated form of ₂-macroglobulin (₂M^*) (13). Hepatic LRP-1 appears to be responsible for plasma clearance of ₂M^*. Recently, LRP-1 has been reported to mediate signaling in several cell types upon binding by ₂M^*; ₂M^* has also been shown to regulate cell growth of certain cell types (14-17). The cytoplasmic domain of LRP-1 has also been shown to modulate the MEKK/JNK/c-Jun signaling cascade (18). The finding that TR-V is identical to LRP-1 has provided new insights into the role of LRP-1 in mediating growth regulatory signaling. Since the growth regulatory functions of IGFBP-3 and TGF-₁ are biologically important (8), we investigated the interactions of the newly identified growth regulatory ligands for LRP-1 with LRP-1 in cells. We predicted that these investigations would help to disclose mechanisms by which LRP-1 regulates cellular proliferation upon stimulation by IGFBP-3 and TGF-₁. During these investigations, we discovered that all cell types examined exhibited low affinity but high density acidic pH binding sites for LRP-1 growth regulatory ligands and that these differ from LRP-1 (which has ligand binding activity with a neutral pH optimum). Since these acidic pH binding sites are likely to modulate ligand binding to LRP-1 at neutral pH, we characterized these acidic pH binding sites in responsive cells. In this communication, we demonstrate that the acidic pH binding sites share some properties (receptor-associated protein- and calcium depletion-sensitive ligand binding) with LRP-1 but differ from LRP-1 in their sensitivity to heparin and chondroitin sulfate and in their ability to bind non-LRP-1 ligands. We also show that annexin VI is the major protein purified from bovine liver plasma membranes by ₂M^* affinity column chromatography at acidic pH and that annexin VI directly interacts with ₂M^* in a pH-dependent manner with an optimum pH of 5 as demonstrated by dot blot analysis. We further demonstrate that cell surface annexin VI is involved in the binding of ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^* at acidic pH. Cell surface annexin VI is also capable of mediating internalization and degradation of cell surface-bound ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^* at acidic pH. Additionally, it is involved in the binding of ¹²⁵I-₂M^* to LRP-1 at neutral pH and forms ternary complexes with ¹²⁵I-₂M^* and LRP-1 at neutral pH as demonstrated by co-immunoprecipitation. Finally, we show that trifluoperazine and fluphenazine both inhibit binding of ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^* to these acidic pH binding sites and to LRP-1 at neutral pH, and block cellular degradation after internalization of cell surface-bound ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^*.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Na¹²⁵I (17.4 Ci/mg), Zn²⁺ chelate-Sepharose FF and Sephacryl S-300 HR were purchased from Amersham Biosciences. TGF-₁ was obtained from Austral Biologicals (San Ramon, CA) and R & D Systems, Inc. (Minneapolis, MN). Human IGFBP-3 (expressed in Escherichia coli, molecular weight 35,000) was obtained from Upstate (Charlottesville, VA). Molecular mass protein standards (myosin, 205 kDa; -galactosidase, 116 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa; -lactoglobulin, 18 kDa), chloramine T, Triton X-100, BAPTA (ethylenedioxybis (o-phenylenenitrilo) tetraacetic acid), EGTA (tetrasodium salt), EDTA (disodium salt), Pseudomonas exotoxin A, human transferrin, human low density lipoprotein (LDL), bovine lactoferrin, human apoE, trifluoperazine, fluphenazine, promethazine, W-5, W-7, verapamil, monodansylcadaverine, and bovine serum albumin (BSA) were purchased from Sigma. GST-RAP (a fusion protein of glutathione S-transferase (GST) and receptor-associated protein (RAP)) was expressed in E. coli using pGEX-KG-RAP (6.4 kb) plasmid and purified according to the procedure of Herz et al. (19). Pure human annexin I, II, III, IV, V, and VI were obtained from BioDesign (Saco, ME). Goat anti-annexin VI IgG was prepared using the N-terminal 19 amino acid residues of human annexin VI, reacted with human, murine (e.g. in MEF/PEA-13 cells), and mink (e.g. in Mv1Lu cells) annexin VI but did not react with other members of the annexin family and was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-annexin I (N19), rabbit anti-annexin II (H-50), and control IgG were also obtained from Santa Cruz Biotechnology. Protein A-Sepharose and activated Sepharose 4B were obtained from Amersham Biosciences. ₂M^*-Sepharose 4B was prepared according to the protocol of the manufacturer. Mv1Lu cells, MEF cells, homozygous LRP-1-deficient mouse embryonic fibroblasts (PEA-13 cells) (20), and human hepatocarcinoma cells (HepG2 and H3B cells) were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum.

Preparation of Human ₂M and ₂M^*—Human ₂M was purified from pooled citrate-treated human plasma using Zn²⁺ chelate-Sepharose FF affinity chromatography followed by gel-filtration on Sephacryl S-300 HR as described previously (21, 22). ₂M activated by methylamine (₂M^*) was prepared as described previously (23, 24).

Iodination of IGFBP-3, TGF-₁, and ₂M^*—IGFBP-3 or TGF-₁ (5 µg) was iodinated with 2 mCi of Na¹²⁵I using chloramine T according to the procedure of Leal et al. (4, 5) and O'Grady et al. (2), respectively. The specific radioactivities of ¹²⁵I-labeled IGFBP-3 (¹²⁵I-IGFBP-3) and ¹²⁵I-labeled TGF-₁ (¹²⁵I-TGF-₁) were 1-4 x 10⁵ cpm/ng and 1-5 x 10⁵ cpm/ng, respectively. Iodination of ₂M^* (100 µg) was done as described previously (23-25). The specific radioactivity of ¹²⁵I-labeled ₂M^* (¹²⁵I-₂M^*) was 2 x 10⁴ cpm/ng. ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^* was mixed with unlabeled TGF-₁ or ₂M^* to yield a specific radioactivity of 2-5 x 10³ cpm/ng in specific experiments.

Specific Binding of ¹²⁵I-IGFBP-3, ¹²⁵I-TGF-₁, and ¹²⁵I-₂M^* to Cells—Mv1Lu, MEF, and PEA-13 cells were plated at a cell density of 8 x 10⁴ cells/well in 48-well clustered dishes and grown at 37 °C overnight in DMEM/50 mM HEPES, pH 7.4 containing 10% fetal calf serum. The cells were then washed and incubated with 6 nM ¹²⁵I-¹²⁵I-TGF-₁, or 10 nM ¹²⁵I-₂M^* in the presence or absence of EGTA (tetrasodium salt) or BAPTA (5 mM), GST-RAP (15 µg/ml), or 200-fold excess of unlabeled TGF-₁ or ₂M^* in DMEM/50 mM HEPES/acetate at pH 4.0, 5.0, 6.0, 7.4 (or 7.0), and 8.0, all containing BSA (1 mg/ml). GST-RAP or 200-fold excess of unlabeled TGF-₁ or ₂M^* was used to estimate nonspecific binding. After 2.5 h at 0 °C, the specific binding of ¹²⁵I-IGFBP-3, ¹²⁵I-TGF-₁, or ¹²⁵I-₂M^* was determined. BAPTA and the tetrasodium salt (but not the free acid form) of EGTA appeared to function well as chelators of Ca²⁺ at acidic pH (26). The experiments were performed in quadruplicate.

Internalization and Degradation of Cell Surface-bound ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^*—Cells (8 x 10⁴ cells/well) in 48-well clustered dishes were incubated with ¹²⁵I-TGF-₁ (100 pM) or ¹²⁵I-₂M^* (2 nM) with or without 10 µM trifluoperazine, fluphenazine, or promethazine in the presence and absence of 200-fold excess of unlabeled TGF-₁ or ₂M^* (to estimate nonspecific binding) in DMEM/25 mM HEPES, pH 7.4 containing BSA (1 mg/ml). After 2 h at 0 °C, the cells were washed and incubated with DMEM/25 mM HEPES, pH 7.4 containing BSA (1 mg/ml) with or without 10 µM trifluoperazine, fluphenazine or promethazine. After 1 h at 37 °C, the medium was collected and precipitated with 10% trichloroacetic acid. The trichloroacetic acid-soluble radioactive material in the medium represented the cellular degradation products of ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^*. The cells were then treated with trypsin (5 mg/ml), maintained for 20 min at 0 °C, and centrifuged. The radioactivity in the supernatant and cell pellets represented cell surface-bound and internalized ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^*, respectively. The experiments were performed in quadruplicate.

Immunoprecipitation of Cell Surface-bound ¹²⁵I-₂M^*—MEF and PEA-13 cells (1 x 10⁵ cells/well) grown in 24-well clustered dishes were incubated with 1 nM ¹²⁵I-₂M^* in the presence and absence of 200-fold excess of unlabeled ₂M^* in DMEM/25 mM HEPES, pH 7.4 containing BSA (1 mg/ml). After 2 h at 0 °C, the cells were lysed with 50 mM HEPES/HCl buffer, pH 7.4 containing 0.1% Triton X-100, 0.15 M NaCl, and 2 mM Ca²⁺, and the cell lysates were immunoprecipitated with anti-annexin VI IgG or control IgG in the same HEPES/HCl buffer as described previously (4, 5, 7). The immunoprecipitates were analyzed by 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. ¹²⁵I-₂M^* appeared as a 180-kDa (monomer) band on the autoradiogram.

Cell Surface Localization of Annexin VI—Cells grown on coverslips in DMEM/25 mM HEPES, pH 7.4 containing 10% fetal calf serum were fixed with 3.7% formaldehyde in DMEM/25 mM HEPES, pH 7.4 (ice cold). After 1 h, the fixed cells were washed with DMEM/25 mM HEPES, pH 7.4 (ice cold). The coverslips were then blocked with BSA (5 mg/ml) in DMEM/25 mM HEPES, pH 7.4 on ice overnight. After washing with DMEM/25 mM HEPES, pH 7.4, the fixed cells were treated with anti-annexin VI IgG or control IgG (1:75 dilution) in DMEM/25 mM HEPES, pH 7.4 containing BSA (5 mg/ml) at room temperature for 2 h. After washing, fixed cells were incubated with anti-rabbit IgG-fluorescein isothiocyanate conjugate (1:50 dilution) at room temperature for 1.5 h and then washed twice with ice-cold phosphate-buffered saline prior to visualize with a confocal fluorescent microscope.

Affinity Column Chromatography on ₂M^*-Sepharose 4B—Bovine liver plasma membranes were subjected to Triton X-100 extraction according to published procedures (2), except that 50 mM HEPES/acetate buffer, pH 6 (or pH 5), containing 0.15 M NaCl and 4 mM CaCl₂ was used. The Triton X-100 extracts were applied onto a column of ₂M^*-Sepharose 4B (1.6 x 20 cm) in 50 mM HEPES/acetate buffer, pH 6.0 (or pH 5.0), 0.15 M NaCl, and 0.1% Triton X-100 (HEPES/acetate buffer) containing 4 mM Ca²⁺. After extensive washing with HEPES/acetate buffer containing 4 mM CaCl₂, the column was eluted with 10 mM EDTA in HEPES/acetate buffer. The fractional volume of the eluents was 1 ml. An aliquot of fractions (EDTA eluents) was subjected to 7.5% SDS-PAGE under non-reducing and reducing conditions and silver staining. The concentrated flow-through fractions and peak fraction were analyzed by Western blot analysis using anti-annexin VI IgG (8).

MALDI-TOF Analysis—A 68-kDa protein purified from ₂M^*-Sepharose 4B affinity column chromatography was subjected to 7.5% SDS-PAGE under reducing conditions, stained with Coomassie Blue, and digested with trypsin. MALDI-TOF analysis of the tryptic digests was carried out at Applied Biosystems (Foster City, CA).

Dot Blot Analysis—1 µl of annexin I, II, III, IV, V, and VI (0.1 mg/ml in H₂O) was applied onto nitrocellulose membranes and air-dried. The membranes were blocked with 5% BSA in phosphate-buffered saline. After 2 h at room temperature, the membranes were incubated overnight with ¹²⁵I-₂M^* (10 nM) in 50 mM HEPES/acetate at pH 4, 5, 6 and 7 containing BSA (2 mg/ml), 2 mM Ca²⁺, and 0.15 M NaCl in the presence and absence of RAP (100 µg/ml) or BAPTA (5 mM). The membranes were extensively washed with the respective binding buffers containing BSA (5 mg/ml) and analyzed by autoradiography.

Anti-annexin VI IgG Treatment of Cells—Mv1Lu, MEF, and PEA-13 cells (8 x 10⁴ cells/well) grown on 48-well clustered dishes were treated with various concentrations (0, 7.5, 15, and 30 µg/ml) of anti-annexin VI IgG or control IgG in DMEM/25 mM HEPES/acetate buffer at pH 6.4 or 7.4 containing BSA (1 mg/ml) at 37 °C for 2 h. The treated cells were then kept on ice and additional anti-annexin VI IgG or control IgG in the same DMEM/HEPES/acetate buffer was added to wells (0, 7.5, 15, and 30 µg/ml). The binding assay at pH 6.4 or 7.4 (at 0 °C) was started by adding ¹²⁵I-TGF-₁ (100 pM) or ¹²⁵I-₂M^* (1 nM) with or without 200-fold excess of unlabeled TGF-₁ or ₂M^* (to estimate nonspecific binding) to wells. After 2 h at 0 °C, the cell-associated ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^* was determined. The specific binding of ¹²⁵I-TGF-₁ or ¹²⁵I-₂M^* was estimated by subtracting nonspecific binding from total binding. The experiments were performed in duplicate.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
LRP-1 Ligands Exhibit High Capacity Binding to Cells at Acidic pH—LRP-1 is a 600-kDa type I membrane glycoprotein which has been shown to bind more than 35 ligands (10-12). These ligands have different structures and functions but share two receptor binding properties: 1) Ca²⁺ dependence and 2) inhibition by RAP. Since IGFBP-3 and TGF-₁ are the newly identified ligands for LRP-1, we determined whether they share these two receptor binding properties with ₂M^* in Mv1Lu cells. Mv1Lu cells are a standard model cell system for investigating TGF-₁ and IGFBP-3 receptors and activities (4, 5, 27-29). These cells were incubated with 6 nM ¹²⁵I-IGFBP-3, 1 nM ¹²⁵I-TGF-₁, or 10 nM ¹²⁵I-₂M^* in the presence or absence of GST-RAP (15 µg/ml), EGTA or BAPTA (5 mM), or 200-fold excess of unlabeled IGFBP-3, TGF-₁ or ₂M^* at pH 4, 5, 6, 7.4 (or 7.0), and 8.0. After 2.5 h at 0 °C, the specific binding of ¹²⁵I-IGFBP-3, ¹²⁵I-TGF-₁, and ¹²⁵I-₂M^* was determined. GST-RAP is a fusion protein of glutathione S-transferase and RAP, which inhibits binding of all known ligands to LRP-1 (9, 10, 19, 30, 31). The tetrasodium salt (but not the free acid form) of EGTA functions well as a chelator of Ca²⁺ at acidic pH. BAPTA is a Ca²⁺ chelator independent of pH (26). As shown in Fig. 1, A-C, ¹²⁵I-IGFBP-3, ¹²⁵I-TGF-₁, and ¹²⁵I-₂M^* bound to Mv1Lu cells in a pH-dependent manner. The specific binding (GST-RAP-sensitive) of ¹²⁵I-IGFBP-3, ¹²⁵I-TGF-₁, and ¹²⁵I-₂M^* exhibited a maximum at pH 5. The specific binding (GST-RAP-sensitive) of ¹²⁵I-IGFBP-3, ¹²⁵I-TGF-₁, and ¹²⁵I-₂M^* at pH 7.4 was much less than at pH 5 (Fig. 1, A-C). The pH profiles of the EDTA- or BAPTA-sensitive binding for these radioactive ligands were similar to those of the GST-RAP sensitive binding (data not shown). The apparent K_d values for binding of IGFBP-3, ¹²⁵I-TGF-₁, and ¹²⁵I-₂M^* to TR-V/LRP-1 at pH 7.4 are known to be 6 nM, 50-400 pM, and 75 pM, respectively (7, 8, 32). These results suggest that Mv1Lu cells may possess low affinity, high density binding sites (which are GST-RAP- and Ca²⁺ depletion-sensitive) for ¹²⁵I-IGFBP-3, ¹²⁵I-TGF-₁, and ¹²⁵I-₂M^* with acidic pH optima. This suggestion is supported by Scatchard plot analysis of ¹²⁵I-₂M^* 1nM binding to Mv1Lu cells at pH 5. As shown in Fig. 2A, ¹²⁵I-₂M^* bound to Mv1Lu cells in a concentration-dependent manner at pH 5 with a saturating concentration of 120 nM. Scatchard plot analysis of the binding data revealed a single class of low affinity binding sites with an apparent K_d of 54 nM and 1.5 x 10⁶ sites/cell (Fig. 2B). The K_d values of the low affinity acidic pH binding sites for ¹²⁵I-IGFBP-3 and ¹²⁵I-TGF-₁ were not determined. However, it is very possible that the K_d values of the low affinity acidic pH binding sites for ¹²⁵I-IGFBP-3 and ¹²⁵I-TGF-₁ are similar to the apparent K_d of the acidic pH binding sites for ¹²⁵I-₂M^*. We therefore focused on characterizing ¹²⁵I-TGF-₁ and ¹²⁵I-₂M^* binding to the acidic pH binding sites in all of the following experiments.

View larger version (22K): [in this window] [in a new window]   FIG. 1. IGFBP-3, TGF-1, and 2M* exhibit high capacity binding to Mv1Lu (A-C), MEF (D), and PEA-13 (E) cells at acidic pH. Mv1Lu (A-C), MEF (D), and PEA-13 (E) cells were incubated with 6 nM 125I-IGFBP-3 ± 15 µg GST-RAP (A), 125I-TGF-1± 15 µg/ml GST-RAP (B), or 10 nM 125I-2M* ± 15 µg/ml GST-RAP (C-E) in DMEM/HEPES/acetate at pH 4.0, 5.0, 6.0, 7.4 (or 7.0), and 8.0. After 2.5 h at 0 °C, the cell-associated 125I-labeled ligand was determined. The specific binding (GST-RAP-sensitive binding) is estimated by subtracting nonspecific binding (GST-RAP-insensitive binding which was determined in the presence of 15 µg/ml GST-RAP) from total binding (which was determined in the absence of GST-RAP). The nonspecific binding (GST-RAP-insensitive binding) was comparable to the nonspecific binding which was determined in the presence of 200-fold excess of unlabeled 2M* and 15 µg/ml GST-RAP. Each data point is the mean of quadruplicate determinations.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Acidic pH binding of 125I-2M* to Mv1Lu cells. A, concentration dependence of binding of 125I-2M* to Mv1Lu cells at pH 5.0 (DMEM/50 mM HEPES/acetate buffer). The nonspecific binding was determined in the presence of GST-RAP (15 µg/ml). Each data point represents the mean of triplicate determinations. B, Scatchard plot analysis of the specific binding data from A. The line shows the fitted curve with GraphPad Prism analysis program and indicates a single class of low affinity binding sites (B). The data are representative of eight independent experiments

The Acidic pH Binding Sites in Cells Have Broad Ligand Specificity—₂M is a plasma protease inhibitor that inhibits all four classes of proteases. ₂M is cleaved by the protease in the bait region (which is close to the thioester bond in the ₂M three-dimensional structure) and undergoes conformational changes, resulting in trapping of the protease (21-25). The protease-activated ₂M is termed ₂M^*. ₂M^* can be mimicked by methylamine-treated ₂M since methylamine also cleaves the same thioester bond, thus inducing the same conformational changes in ₂M (21-25). LRP-1 has been shown to bind ₂M^* and native ₂M with different affinities (K_d: 40-75 pM and 2 nM, respectively) (10-14). To see if, like LRP-1, the acidic pH binding sites have different affinities for ₂M^* and native ₂M, we determined the effects of increasing concentrations of unlabeled ₂M^* and native ₂Mon ¹²⁵I-₂M^* binding at pH 5.5 in MEF cells. As shown in Fig. 3A, increasing concentrations of ₂M^* and native ₂M correspondingly inhibited ¹²⁵I-₂M^* binding to the acidic pH binding sites with IC₅₀ values of 50 nM and 150 nM, respectively. The IC₅₀ of unlabeled ₂M^* appeared to be similar to the apparent K_d for ¹²⁵I-₂M^* binding to the acidic pH binding sites in Mv1Lu, MEF, and PEA-13 cells as determined by Scatchard plot analysis (Fig. 2 and Table I). We also determined the effects of increasing concentrations of unlabeled ₂M^* and native ₂M on ¹²⁵I-₂M^* binding to PEA-13 cells which are known to be deficient in LRP-1. As shown in Fig. 3B, unlabeled ₂M^* and native ₂M also inhibited ¹²⁵I-₂M^* binding to the acidic pH binding sites in a concentration-dependent manner with IC₅₀ values of 120 nM and > 400 nM, respectively. These IC₅₀ values were higher than those found in wild-type cells (MEF cells). This result suggests that in PEA-13 cells, the absence of LRP-1 may decrease the binding affinity of native ₂M or ₂M^* for acidic pH binding sites. Alternatively, LRP-1 may collaborate with the acidic pH binding sites for ligand interactions at acidic pH.

View larger version (16K): [in this window] [in a new window]   FIG. 3. 2M* exhibits higher affinity than native 2M to the acidic pH binding sites in MEF (A) and PEA-13 (B) cells. MEF (A) and PEA-13 (B) cells were incubated with 2 nM 125I-2M* in the presence of increasing concentrations (as indicated) of unlabeled 2M* or native 2M (2M) at pH 5.5 (DMEM/HEPES/acetate). After 2.5 h at 0 °C, the cell-associated specific binding of 125I-2M* was determined. The 125I-2M* binding obtained in the absence of unlabeled 2M* and 2M was taken as 100% binding. Each data point is the mean ± S.D. of quadruplicate determinations.

View larger version (18K): [in this window] [in a new window]   FIG. 4. Transferrin, lactoferrin, and LDL inhibit 125I-2M* binding to Mv1Lu cells at acidic pH. Cells were incubated with 2 nM 125I-2M* with or without 15 µg/ml GST-RAP (to estimate nonspecific binding) in the presence of increasing concentrations of transferrin, lactoferrin (A), or LDL (B). After 2 h at 0 °C, the specific binding (at pH 5 for transferrin and lactoferrin and at pH 5 and 6 for LDL) of 125I-2M* was determined. The specific binding of 125I-2M* obtained in the absence of competitors was taken as 100% binding. Each data point is the mean ± S.D. of quadruplicate determinations.

Cell Surface Annexin VI Is Involved in the Acidic pH Binding of ¹²⁵I-TGF-₁ and ¹²⁵I-₂M^* in Cells

View larger version (47K): [in this window] [in a new window]   FIG. 5. Annexin VI binds to 2M* Sepharose 4B in a calcium-dependent manner. The Triton X-100 extracts of bovine liver plasma membranes were applied onto a column of 2M*-Sepharose 4B (1.6 x 20 cm) in HEPES/acetate, pH 6.0, 0.15 M NaCl, 0.1% Triton X-100 (HEPES/acetate buffer) containing 4 mM Ca2+. After washing with HEPES/acetate buffer extensively, the column was eluted with 10 mM EDTA in HEPES/acetate buffer. The fractional volume was 1 ml. An aliquot of fractions (EDTA eluents) was subjected to 7.5% SDS-PAGE under non-reducing conditions and silver stained (A). M indicates the protein molecular mass standards (175, 62, 47, and 33 kDa). The peak fraction (fraction 33) containing a 68-kDa protein (lane 2) and the concentrated column flow-through fraction (lane 1) were analyzed by Western blot analysis using anti-annexin VI IgG (B). The arrowhead indicates the location of annexin VI.

View larger version (34K): [in this window] [in a new window]   FIG. 6. Annexin VI and other annexins directly interact with 125I-2M*. Dot blot analysis was performed to analyze the direct interactions of pure annexin I, II, III, IV, and VI with 125I-2M* at pH 4, 5, 6, and 7. Dots of annexins immobilized on nitrocellulose membranes were probed with 125I-2M* as described in the text and analyzed by autoradiography. The relative intensities of 125I-2M* bound to the annexins were quantitated by a PhosphorImager and estimated to be 1.0, 0.5, 0.4, 0.3, and 0.1 for annexin VI, annexin IV, annexin III, annexin I, and annexin II, respectively.

View larger version (15K): [in this window] [in a new window]   FIG. 7. Chondroitin sulfate (A) and heparin (B) inhibit 125I-2M* binding (at acidic pH) to Mv1Lu cells. Cells were incubated with 2 nM 125I-2M* with or without 15 µg/ml GST-RAP (to estimate nonspecific binding) in the presence of various concentrations (as indicated) of chondroitin sulfate A, B, and C (A) or heparin (B). After 2 h at 0 °C, the specific binding of 125I-2M* was determined. The specific binding of 125I-2M* obtained in the absence of chondroitin sulfate and heparin was taken as 100% binding. Each data point is the mean ± S.D. of quadruplicate determinations.

View larger version (26K): [in this window] [in a new window]   FIG. 8. Western blot analysis of annexins using anti-annexin VI IgG. 100 ng or 400 ng of annexin I, II, III, IV, and VI were subjected to 10% SDS-PAGE under reducing conditions, electrophoretically transferred to Immobilon-P membranes, and analyzed by Western blotting using anti-annexin VI IgG (1:1000 dilution) (A) or by Coomassie Blue staining (B). The arrow indicates the location of annexin VI (A).

View larger version (15K): [in this window] [in a new window]   FIG. 9. Treatment of cells with anti-annexin VI IgG prevents the binding of 125I-TGF-1 or 125I-2M* to Mv1Lu (A, B, E), MEF (C, F), and PEA-13 (D) cells at pH 6.4 (A-D) or pH 7.4 (E, F). Cells were treated with increasing concentrations (as indicated) of anti-annexin VI IgG or control IgG at pH 6.4 or pH 7.4 at 37 °C for 2 h. The specific binding of 125I-TGF-1 (0.1 nM) or 125I-2M* (1 nM) in these cells treated with anti-annexin VI IgG or control IgG was then determined after incubation of cells with 125I-TGF-1 (A) or 125I-2M* (B-F) at pH 6.4 (A-D) or 7.4 (E, F) at 0 °C for 2 h. The specific binding 125I-TGF-1 or 125I-2M* at pH 6.4 or 7.4 in cells treated without anti-annexin VI IgG and control IgG was taken as 100% binding. Each data point is the average of triplicate determinations.

View larger version (69K): [in this window] [in a new window]   FIG. 10. Annexin VI is localized at the cell surface of Mv1Lu, MEF, PEA-13, and Hep3B cells. Mv1Lu, MEF, PEA-13, and Hep3B cells were fixed with paraformaldehyde and reacted with anti-annexin IgG. The primary antibody was visualized by reaction with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (A, E, C, G). The phase contrast microscopy is also shown (B, D, F, H). Hep3B cells are human hepatoma cells. The control IgG did not show any significant immunofluorescent staining in these cells (data not shown).

View larger version (37K): [in this window] [in a new window]   FIG. 11. Anti-annexin VI IgG immunoprecipitates cell surface-bound 125I-2M* in MEF cells but not in PEA cells. MEF and PEA-13 cells were incubated with 1 nM 125I-2M* with or without 200-fold excess of unlabeled 2M* at pH 7.4. After 2.5 h at 0 °C, the cells were washed, and the cell lysates were immunoprecipitated with anti-annexin VI IgG (lanes 1 and 3) or control IgG (lanes 2 and 4). The immunoprecipitates were analyzed by 7.5% SDS-PAGE under reducing conditions and autoradiography. The bar indicates the locations of protein molecular mass markers (83 and 175 kDa). The arrow indicates the location of 125I-2M* (monomer, 180,000).

View this table: [in this window] [in a new window]   TABLE II Internalization and degradation of cell surface-bound 125I- 2M* at 37° C in DMEM (pH 6.0 or pH 7.4) in PEA-13 and MEF cells Cells were incubated with 2 nM 125I- 2M* in the presence and absence of 200-fold excess of unlabeled 2M* or 15 µ g/ml of GST-RAP (for estimating nonspecific binding) in DMEM, pH 6.0 or pH 7.4 at 0° C for 2.5 h. The cells were then washed and warmed to 37° C. After 1 h, the cell surface-bound, internalized, and degraded fractions of 125I- 2M* were determined. The experiments were performed in quadruplicate. Data are represented as mean ± S.D.

View larger version (19K): [in this window] [in a new window]   FIG. 12. Trifluoperazine and fluphenazine inhibits 125I-2M* binding to MEF cells at pH 5.5 and 7.4 (A, B) and 125I-TGF-1 binding to Mv1Lu cells at pH 5 (C) in a concentration-dependent manner. MEF cells were incubated with 1 nM 125I-2M* (A-C) or 100 pM 125I-TGF-1 (C) in the presence of various concentrations (as indicated) of trifluoperazine (TFP), fluphenazine (FL), promethazine (PM), W-7, and monodansylcadaverine (MD) as indicated at pH 5.5 (A), 5 (C), or 7.4 (B). After 2.5 h at 0 °C, the specific binding of 125I-2M* or 125I-TGF-1 was determined. The specific binding of 125I-2M* or 125I-TGF-1 in the absence of trifluoperazine and other compounds was taken as 100% binding. Each data point is the average of quadruplicate determinations.

Trifluoperazine and Fluphenazine Inhibit Cellular Degradation of ¹²⁵I-TGF-₁ and ¹²⁵I-₂M^* in Mv1Lu and MEF Cells