©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distribution and Characterization of Specific Cellular Binding Proteins for Bone Morphogenetic Protein-2 (*)

(Received for publication, September 22, 1994; and in revised form, November 28, 1994)

Shoji Iwasaki (1) Nobuo Tsuruoka (2) Akira Hattori (1)(§) Masahiro Sato (1) Masafumi Tsujimoto (2) Michiaki Kohno (1)(¶)

From the  (1)Department of Biology, Gifu Pharmaceutical University, 5-6-1, Mitahora-higashi, Gifu 502, Japan and the (2)Suntory Institute for Biomedical Research, Mishima, Osaka 618, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Bone morphogenetic proteins (BMPs), which were originally identified by their novel ability to induce de novo cartilage and bone formation in vivo, are multifunctional proteins structurally related to transforming growth factor-betas, activins, and inhibins. As a first step to elucidate the precise physiological function as well as the action mechanism of BMPs, we have examined the distribution of the specific cellular binding proteins for BMP-2 on a wide variety of cell types. A single class of high affinity-specific binding sites for BMP-2 were identified not only on osteoblastic cells but also on major types of non-hematopoietic cells in a rather ubiquitous fashion (1,20060,000 receptors/cell, K = 35230 pM); these cells included fibroblasts, keratinocytes, astrocytes, kidney epithelial cells, and tumor cells of bone, muscle, lung, liver, kidney, stomach, colon, prostate, and neuronal tissue. Other growth factors including transforming growth factor-beta(1), activin A, and inhibin A did not compete for the binding of I-labeled BMP-2 to the cells. Affinity cross-linking of radiolabeled BMP showed five components with apparent molecular masses of 170, 105, 90, 80, and 70 kDa common to all three fibroblast cell lines analyzed. On the other hand, no specific binding sites for BMP-2 were identified on vascular endothelial cells or on hematopoietic cells including RPMI 1788 and RPMI 8226 (B-lymphocyte lineage), MOLT-3 and MOLT-4 (T-lymphocyte lineage), HL-60 (myeloid lineage), and K-562 (erythroid lineage). These results suggest that major types of cells other than hematopoietic cells and vascular endothelial cells may be potential targets for BMP-2 action.


INTRODUCTION

Bone morphogenetic proteins (BMPs) (^1)are a family of proteins, which were originally identified and characterized by their novel ability to induce cartilage and bone formation in ectopic extraskeletal sites in vivo (Refs. 1 and 2 and reviewed in (3) ). Recently, several members of this protein family have been isolated, cloned, and expressed as recombinant proteins; these include BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, and BMP-7 (also referred to as osteogenic protein-1)(4, 5, 6, 7, 8) . The BMPs are 30-kDa glycosylated proteins with disulfide-linked dimeric structures(5, 6, 9) . Comparative amino acid sequence analysis of the BMP family of proteins has suggested that these molecules can be further divided into three groups. The members of the first group, BMP-2 and BMP-4, are very closely related to one another (90% amino acid identity), but their sequences differ significantly from those of BMP-3, -5, -6, and -7. BMP-5, BMP-6, and BMP-7 exhibit 80% identity to each other, thereby defining the second group. Of all the BMPs, BMP-3 is the most distinct and by itself forms the third group(7) .

All the BMPs contain the characteristic 7 highly conserved cysteines in their carboxyl-terminal portions and thus belong to the transforming growth factor-beta (TGF-beta) superfamily, which includes TGF-betas, activins, inhibins, and Müllerian inhibiting substance (reviewed in (10) ). The members of TGF-beta superfamily are multifunctional, e.g. TGF-betas are known to be implicated in the regulation of a wide range of biological phenomena such as cell proliferation, cell differentiation, tissue repair, inflammation, angiogenesis, immunosuppression, and embryogenesis. In this respect, expression of the BMP transcripts and presence of the BMP proteins in various tissues including kidney, brain, and skin have been reported(11, 12, 13) . Increasing evidence suggests a regulatory role for BMPs in embryonic development(14, 15, 16, 17) , indicating that the BMP family of proteins also has much broader biological effects, unrelated to bone induction, on different cell types. However, the precise physiological function as well as the mechanism of action of BMPs are largely unknown.

As a first step to elucidate the biological action of BMPs, we have examined the distribution of specific cellular binding proteins (receptors) for BMP-2 on a wide variety of cell types and tissues; all the hormonally active polypeptides are believed to act on target cells by binding to specific cell surface receptors that are coupled to cytoplasmic signal transducers. In the present study, we have shown that the high affinity receptors for BMP-2 are present not only on osteoblastic cells but also on other types of cells in a rather ubiquitous fashion; these include fibroblasts, keratinocytes, astrocytes, kidney epithelial cells, and tumor cells of lung, liver, kidney, stomach, and neuronal tissue. On the other hand, cells of hematopoietic origin and vascular endothelial cells are shown not to express specific binding sites for BMP-2. In addition, we have characterized the specific cellular binding proteins for BMP-2 in several fibroblastic cell lines.


EXPERIMENTAL PROCEDURES

Materials

Recombinant human BMP-2, expressed in silkworm larvae, was purified to homogeneity as previously described (18) . Purified human activin A and purified bovine inhibin A were kindly provided by Dr. K. Miyamoto (National Cardiovascular Center, Research Institute, Osaka, Japan). Recombinant human TGF-beta(1) was purchased from Wako Chemical Co. (Osaka, Japan); recombinant human fibroblast growth factor (acidic and basic), recombinant human insulin-like growth factor-II, and epidermal growth factor purified from mouse submaxillary glands were from Toyobo Co. (Osaka, Japan); and recombinant human insulin was from Sigma. Other chemicals and reagents were of the purest grade available.

Cell Culture

Mouse osteoblast-like cells (MC3T3-E1) (19) were kindly provided by Dr. M. Kumegawa (Meikai University); Swiss albino mouse fibroblasts (Swiss 3T3), Balb/c mouse fibroblasts (Balb 3T3, clone A31-1-1), C3H mouse fibroblasts (C3H/10T1/2, clone 8), human skin fibroblasts (SF-TY), human embryo lung fibroblasts (TIG-3-20), Madin-Darby canine kidney epithelial cells (MDCK (NBL-2)), rat kidney epithelial cells (NRK-52E), human osteosarcoma cells (HuO-3N1), human neuroblastoma cells (NB-1), rat pheochromocytoma cells (PC12), human lung adenocarcinoma cells (ABC-1), human colon adenocarcinoma (COLO201), human peripheral blood-derived B lymphocytes (RPMI 1788), human myeloma-derived B lymphocytes (RPMI 8226), human acute lymphoblastic leukemia cells (MOLT-3 and MOLT-4), human chronic myelogenous leukemia cells (K-562), and human promyelocyte leukemia cells (HL-60) were obtained through the Japanese Cancer Research Resources Bank; mouse keratinocytes (PAM-212) and human hepatocellular carcinoma cells (HepG2) were kindly provided by Dr. T. Nakamura (Osaka University); human rhabdomyosarcoma cells (KT006) were kindly provided by Dr. S. Toyama (Kyoto University); human renal adenocarcinoma cells (NC65) and human prostate adenocarcinoma cells (PC 3) were kindly provided by Dr. H. Oka (Kyoto University); human stomach adenocarcinoma cells (TMK-1) were kindly provided by Dr. E. Tahara (Hiroshima University); human umbilical vein endothelial cells (HUVEC) were purchased from Kurabou Co. (Osaka, Japan); and astrocytes were prepared from the whole brains of 8-day-old ICR mice as previously described(20) . Each cell line was maintained under the recommended and standard condition; most of the non-hematopoietic cells were cultured in DMEM supplemented with 10% fetal bovine serum, while the cells of hematopoietic origin were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum.

Iodination of BMP-2

Recombinant human BMP-2 was radioiodinated by using the Bolton and Hunter reagent (Amersham International). Briefly, 10 µg of BMP-2 in 20 µl of 20 mM HCl was diluted with 10 µl of 0.3 M sodium borate (pH 8.5) and then transferred to a polypropylene tube containing 500 µCi (1 Ci = 37 GBq) of dried I-labeled Bolton and Hunter reagent. The reaction was allowed to proceed for 30 min at 0 °C with occasional agitation. To terminate the reaction, 200 µl of 0.4 M glycine in 0.1 M sodium borate (pH 8.5) was added, mixed well, and incubated for 10 min at 0 °C. Then, 4 mg of bovine serum albumin in 400 µl of 4 M urea was added, and this mixture was chromatographed on a 10-ml Sephadex G-25 column that had been equilibrated with 5 mM HCl, 0.1 M NaCl, 0.2% bovine serum albumin. I-Labeled BMP-2 collected in the void volume of the column was stored at -70 °C in small aliquots up to 4 weeks. BMP-2 did not show any significant loss of biological activity after radioiodination, which was determined by its alkaline phosphatase-inducing activity in MC3T3-E1 cells(18) . The specific radioactivities of the I-labeled BMP-2 ranged from 18.9 to 29.0 µCi/µg after correcting for trichloroacetic acid-precipitable activity, which was >95%. Each of I-labeled BMP-2 preparations was analyzed by SDS-PAGE on a 12.5% acrylamide gel followed by autoradiography, which always showed a single band of 30 kDa under non-reducing conditions or a 16-kDa band under reducing conditions.

Binding Assay

For cells that attached to culture plates, a solid-phase monolayer assay was used, while for hematopoietic cells a suspension binding assay was used. The monolayer assay was performed as described (20, 21) with slight modification. Briefly, cells were seeded in their normal growth medium at 515 times 10^4 cells/well in 24-well culture plates (collagen (type I)-coated plates were used for PC12 cells) and allowed to grow for 24 h, such that at the time of assay they were somewhat subconfluent and still actively growing. The monolayers were then washed twice and incubated in binding buffer (DMEM containing 10% fetal bovine serum and 25 mM Hepes, pH 7.4) for 1 h at 37 °C. After washing, cells were incubated in 0.3 ml of binding buffer containing 1500 pMI-labeled BMP-2 for 2 h at room temperature on a rocker platform. At the end of the incubation, the cells were washed five times with binding buffer and then solubilized with 750 µl of solubilization buffer (1% Triton X-100, 10% glycerol, 20 mM Hepes, pH 7.5, 0.05% bovine serum albumin) for 20 min at 37 °C. Radioactivity was determined in aliquots (600 µl) in a Packard counter. Nonspecific binding was determined by using a 100-fold excess of unlabeled BMP-2. The number of cells in replicate wells was determined by counting in a hematocytometer. The decrease in trichloroacetic acid precipitability of I-labeled BMP-2 at the end of the incubation was less than 3%. Each data point was determined in triplicate, and binding data were analyzed according to the method of Scatchard(23) .

Binding of I-labeled BMP-2 to hematopoietic cells in suspension (10^6 cells in 0.2 ml) was performed under essentially the identical conditions as for the monolayer cells described above. After the binding incubation, cells were pelleted, resuspended in 0.2 ml of ice-cold binding buffer, and centrifuged through a silicone oil/paraffin oil layer (0.2 ml); the radioactivity associated with the resulting pellet was then determined(24, 25) . Although all the hematopoietic cells assayed in suspension lacked any specific binding sites for BMP-2 (see Fig. 5), this binding assay could be successfully used to detect BMP binding proteins in PC12 and NB-1 cells and gave essentially identical results as with the solid-phase monolayer assay described above (data not shown).


Figure 5: Binding of I-labeled BMP-2 to various lines of hematopoietic cells and human umbilical vein endothelial cells. 83.3 (L) or 500 pM (H) I-labeled BMP-2 was incubated with various types of cells for 2 h at room temperature. Total binding (&cjs2110;) and nonspecific binding (&cjs2112;) for each line of cells are shown; cell-associated I-labeled BMP-2 radioactivities are normalized by respective cell numbers and are expressed as cpm/10^4 cells. The data represent mean ± S.D. of triplicate determinations. Three independent experiments for each line of cells yielded similar results.



Cross-linking ofI-Labeled BMP-2 to Cell Surface Receptors

Binding of radiolabeled BMP-2 to cells was carried out as described above, except that cells were in 35-mm culture dishes, 250 pMI-labeled BMP-2 was used, and binding incubation was for 4 h at 4 °C. After the cell monolayer was washed three times each with the binding buffer and then with DMEM containing 25 mM Hepes, pH 7.4, 2 ml of 0.5 mM disuccinimidyl suberate (Pierce) (prepared fresh by first dissolving in dimethyl sulfoxide (50 mM) and then diluted 1:100 in DMEM containing 25 mM Hepes, pH 7.4) was added to each dish, and cross-linking reaction was carried out for 20 min at room temperature (26) . The reaction was terminated by adding quenching buffer (20 mM Tris-HCl, pH 7.4, 200 mM glycine, 2 mM EDTA). After washing the cell monolayer three times with ice-cold phosphate-buffered saline containing 0.5 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and 0.5% aprotinin (Sigma), the cells were detached with a rubber policeman and collected by centrifugation for 1 min at 8,000 times g. The resulting cell pellet was solubilized in lysis buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 10 µg/ml antipain (Peptide Institute, Inc., Osaka, Japan), 10 µg/ml leupeptin (Peptide Institute, Inc.), 10 µg/ml pepstatin (Peptide Institute, Inc.), 100 µg/ml benzamidine-HCl) for 20 min on ice and then centrifuged for 20 min at 12,000 times g to obtain the supernatant. Concentrated SDS-PAGE sample buffer (5times) was added and heated for 5 min at 95 °C, and the sample was analyzed by SDS-PAGE on a 7% acrylamide gel. The gels were fixed, dried, and then subjected to autoradiography or analysis using a FUJIX Bioimaging analyzer BAS 2000 (Fuji Photo Film Co., Tokyo, Japan).


RESULTS

Specific Binding of BMP-2 to a Wide Variety of Non-hematopoietic Cells

The time course of specific binding (total binding minus nonspecific binding) of I-labeled BMP-2 to MC3T3-E1 at 4 °C and at room temperature (2025 °C) was determined. MC3T3-E1 is a well characterized mouse osteoblastic cell line, which responds to BMP-2 by the induction of alkaline phosphatase activity(18, 19) . As shown in Fig. 1, maximal binding was reached after incubation of 4 h at 4 °C. At room temperature, binding of I-labeled BMP-2 to the cells was faster and reached the same maximal level as at 4 °C after a 2-h incubation; binding assays performed in the presence of 0.1% sodium azide (a metabolic poison that inhibits endocytosis by lowering cellular ATP level) (27) gave essentially identical results, indicating that there was little, if any, ligand-induced receptor down-regulation at this temperature. Essentially the same time course profiles of specific binding of I-labeled BMP-2 to human and mouse fibroblasts (TIG-3-20 and Swiss 3T3, respectively) were obtained (data not shown). All BMP-2 binding assays hereafter were performed at room temperature for 2 h, since several of the cell types analyzed detached from the culture plates during prolonged incubation at 4 °C.


Figure 1: Time course of binding of I-labeled BMP-2 to MC3T3-E1 cells. 333 pMI-labeled BMP-2 was added to 30 times 10^4 MC3T3-E1 cells in 0.3 ml of binding buffer, and at the indicated time intervals, cell-associated I-labeled BMP-2 was determined. At each time point, the binding of I-labeled BMP-2 was corrected for nonspecific binding, and the specifically bound I-labeled BMP-2 radioactivity was plotted. box, binding at 4 °C; circle, binding at room temperature; bullet, binding at room temperature in the presence of 0.1% NaN(3). Results are mean ± S.D. of triplicate determinations and are representative of two independent experiments.



Radiolabeled BMP-2 specifically bound to MC3T3-E1 cells in a dose-dependent manner (Fig. 2). A typical set of binding curves and corresponding Scatchard analysis are shown in Fig. 2. These gave a linear plot characteristic of a single high affinity binding site with 5,400 receptors/cell and a dissociation constant (K(d)) of 150 pM.


Figure 2: Binding of I-labeled BMP-2 to MC3T3-E1 cells. Increasing concentrations of I-labeled BMP-2 were incubated with 31.5 times 10^4 MC3T3-E1 cells for 2 h at room temperature, and the cell-associated radioactivity was determined. The data are presented in a Scatchard plot, and the inset shows BMP-2 binding as a function of I-labeled BMP-2 concentration where the BMP-2 specifically bound () is the difference between the total () and nonspecific (bullet) binding. The data represent mean ± S.D. of triplicate determinations. Three independent experiments yielded similar results.



The binding of I-labeled BMP-2 to other types of cells (non-hematopoietic origin) was then analyzed. Representative Scatchard plots for cells with low (1,000), intermediate (20,000), and high (60,000) numbers of high affinity receptors per cell are shown in Fig. 3. The results of binding assays for other cell lines using two different concentrations (83.3 and 500 pM) of I-labeled BMP-2 are shown in Fig. 4. The number and affinity of the receptors for BMP-2 on a variety of different cell types are summarized in Table 1. Presence of the specific binding sites for BMP-2 (with high affinity) was demonstrated on all the cell lines assayed; these cells included fibroblasts, keratinocytes, astrocytes, kidney epithelial cells, and tumor cells of bone, muscle, lung, liver, kidney, stomach, colon, prostate, and neuronal tissue, derived from different species (human, rodent, and dog) and from adult and embryonic tissues. The relative high background of nonspecific binding was observed for all cell types analyzed ( Fig. 2and Fig. 4), making it difficult to exclude the possible existence of additional lower affinity sites in some cases since data in which the nonspecific binding exceeded 50% of the total binding could not be analyzed reliably. A similar high background of nonspecific binding was reported for TGF-beta(22) .


Figure 3: Representative Scatchard plots of BMP-2 binding to cells expressing low, intermediate, and high numbers of BMP-2 binding sites per cell. Increasing concentrations of I-labeled BMP-2 were incubated with 52 times 10^4 PAM212 cells (A), 10 times 10^4 TIG-3-20 cells (B), or 5 times 10^4 SF-TY cells (C) for 2 h at room temperature, and the cell-associated radioactivity was determined. The data are presented in Scatchard plots. Results shown are representative of two (A), two (B), or three (C) independent experiments.




Figure 4: Binding of I-labeled BMP-2 to various types of non-hematopoietic cells. 83.3 (L) or 500 pM (H) I-labeled BMP-2 was incubated with various types of cells for 2 h at room temperature. Total binding (&cjs2110;) and nonspecific binding (&cjs2112;) for each line of cells are shown; cell-associated I-labeled BMP-2 radioactivities are normalized by respective cell numbers and are expressed as cpm/10^4 cells. The data represent mean ± S.D. of triplicate determinations. Two or three independent experiments for each line of cells yielded similar results.





No Specific Binding Sites for BMP-2 on Major Types of Hematopoietic Cells and on Vascular Endothelial Cells

Binding of I-labeled BMP-2 to various types of hematopoietic cells was analyzed; these included cells of B-lymphocyte lineage (RPMI 1788 and RPMI 8226), T-lymphocyte lineage (MOLT-3 and MOLT-4), myeloid lineage (HL-60), and erythroid lineage (K-562). The results of binding assays for these cell lines using two concentrations (83.3 and 500 pM) of radiolabeled BMP-2 are shown in Fig. 5. As clearly shown in the figure, none of these hematopoietic cells expressed any specific binding sites for BMP-2. Neither could specific binding sites for BMP-2 be detected on human umbilical vein endothelial cells.

Specificity of Binding

As shown in Fig. 6, more than 70% of the I-labeled BMP-2 bound to Swiss 3T3 cells was displaced by a 100-fold excess of unlabeled BMP-2, indicating the specificity of the BMP-2 binding sites on the cells. In contrast, some of the other TGF-beta superfamily peptides (TGF-beta activin A, inhibin A) or growth factors (epidermal growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, insulin-like growth factor-II, insulin), when present at a 100-fold excess, did not show any significant effect on the binding of radiolabeled BMP-2 to the cells. The identical specificity of I-labeled BMP-2 binding was observed on PC12 and MC3T3-E1 cells (data not shown). Thus, although BMP-2 belongs to the TGF-beta superfamily, these related family members appear to have distinct cellular specific binding sites.


Figure 6: Competition with growth factors for binding of I-labeled BMP-2 to Swiss 3T3. 333 pMI-labeled BMP-2 was incubated with 5 times 10^4 Swiss 3T3 cells in either the absence or presence of a 100-fold excess (33.3 nM) of the appropriate growth factor for 2 h at room temperature, and the cell-associated radioactivity (total binding) was determined. Results are mean ± S.D. of triplicate determinations and are representative of two independent experiments. EGF, epidermal growth factor; IGF-II, insulin-like growth factor-II; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor.



Affinity Cross-linking ofI-Labeled BMP-2 to Its Binding Proteins

The specific cellular binding proteins for BMP-2 were further characterized by affinity cross-linking techniques; for their analysis we used human and mouse fibroblast cell lines (TIG-3-20, SF-TY, and Swiss 3T3) because these cells express especially high numbers of high affinity binding sites for BMP-2 per cells (Table 1). I-Labeled BMP-2 specifically bound to cells was covalently cross-linked to its binding proteins by the homobifunctional cross-linking agent, disuccinimidyl suberate. Analysis by SDS-PAGE repeatedly revealed five cross-linked macromolecular components common in all three cell lines analyzed (Fig. 7). The apparent molecular mass of these species were 170, 105, 90, 80, and 70 kDa. The cross-linking to all of these macromolecules was inhibited by a 100-fold excess of unlabeled BMP-2.


Figure 7: Affinity labeling of SF-TY, TIG-3-20, and Swiss 3T3 cells with I-labeled BMP-2. SF-TY, TIG-3-20, and Swiss 3T3 cells were incubated with I-labeled BMP-2 (50 or 250 pM) for 4 h at 4 °C in either the absence or presence of a 100-fold excess of unlabeled BMP-2. Free ligand was removed, and bound I-labeled BMP-2 was affinity cross-linked with disuccinimidyl suberate. Samples were analyzed by SDS-PAGE followed by analysis using a FUJIX Bioimaging analyzer BAS 2000. Arrowheads indicate positions of the cross-linked macromolecular components. Results shown are representative of three independent experiments.




DISCUSSION

In this paper, we have examined the distribution of the cellular specific binding sites for BMP-2 on a large variety of cell types. A complete Scatchard analysis was carried out for most cell lines, but due to limitations in BMP-2 protein availability, some cell lines were tested using only two different concentrations (83.3 and 500 pM) of I-labeled BMP-2. All of these results are summarized in Table 1. The data show rather ubiquitous expression of a single class of high affinity receptors for BMP-2 on the major types of non-hematopoietic cells analyzed to date, which include bone, muscle, skin, lung, liver, kidney, stomach, colon, prostate, and neuronal cells, and suggest that all of these cell types may be potential targets for BMP-2 action. In this respect, expression of the BMP transcripts and presence of the BMP proteins in a wide variety of tissues besides bone has been reported(11, 12, 13) , and BMPs are suggested to play important roles in the morphogenesis of the embryo(14, 15, 16, 17) . Thus, it seems very likely that BMP-2 and probably other BMP family members of proteins have much broader biological effects on various cell types, some of which are unrelated to bone formation. The precise physiological function of BMPs on each of these cell types remains to be elucidated. The presence of specific binding sites for BMP-4 on MC3T3-E1 cells and NIH 3T3 fibroblasts(26) , PC12 cells(28) , and articular chondrocytes (29) has also been recently reported.

On the contrary, hematopoietic cells (B-lymphocyte lineage, T-lymphocyte lineage, myeloid lineage, and erythroid lineage) and vascular endothelial cells were found not to express any specific binding sites for BMP-2. All of the hematopoietic cells we have analyzed in the present study are transformed cells; although they are quite randomly selected, the observed absence of the specific binding sites for BMP-2 on those cells might be the result of malignant transformation. In this respect, however, expression of the TGF-beta receptors on any given cell type has been reported to be relatively unaffected by cellular transformation(22) . We have obtained similar results with 3Y1 cells (Fischer rat embryo fibroblasts) and various derived transformed cells such as SR-3Y1 (transformed by Rous sarcoma virus infection), HR-3Y1 (v-Ha-Ras transfection), Py-3Y1 (mouse polyoma virus infection), SV-3Y1 (SV-40 infection), and NG-3Y1 (nitrosoguanidine treatment). Specific binding of I-labeled BMP-2 was demonstrated on all of these cells in a one-point binding assay using 167 pMI-labeled BMP-2, although degrees of specific binding varied to some extent. We have also analyzed other cell lines, such as Raji and RPMI 8866 for the B-lymphocyte lineage, Jurkat and HUT-102 for the T-lymphocyte lineage, and U-937 for the myeloid lineage, or phorbol 12-myristate 13-acetate-pretreated HL-60 and U-937 (which induces the differentiation of these cells into the monocyte/macrophage lineage) (30) in the one-point binding assay as described above. None of these cells shows any significant expression of the specific binding sites for BMP-2. Furthermore, our recent study on normal leukocytes prepared from the peripheral blood of healthy human donors by Ficoll-Hypaque centrifugation does not reveal any significant specific binding of BMP-2 to these cells. (^2)In the meantime, Cunningham et al.(25) recently have reported that highly purified populations of normal human monocytes possess specific binding sites for BMP-4. Although all our results suggest that the major, if not all, types of hematopoietic cells do not express any specific binding sites for BMP-2, further detailed analysis may be necessary to draw a final conclusion in this matter.

However, our finding that BMP-2 receptors are absent from vascular endothelial cells and probably from hematopoietic cells in adult animals seems interesting because all of these cells are of splanchnic mesodermal origin. On the other hand, all of the other cell types we have analyzed in the present study possess specific binding sites for BMP-2, which include those developed from ectodermal origin, endodermal origin, and mesodermal origin except for the splanchnic lineage. The observed difference in the distribution of specific binding sites for BMP-2 could be the result of diverged expression of the BMP receptor gene(s) during embryonic development. One speculation is that populations of cells that develop along the splanchnic mesodermal lineage might be determined not to express gene(s) coding for the BMP receptors. Such a distribution of BMP-2 receptors is apparently different from that of the TGF-beta receptors, which are universally expressed on all cells of epithelial, mesenchymal, and hematopoietic origin(22) . Thus, although BMP-2 is suggested to have a wide spectrum of target tissues and cell types for its action as discussed above, it is rather restricted as compared with that of TGF-beta.

Affinity cross-linking experiments have revealed five macromolecular components that bind specifically to BMP-2 in human and mouse fibroblasts. The apparent molecular masses of these affinity-labeled complexes are 170, 105, 90, 80, and 70 kDa, which are common to all the fibroblast cell lines analyzed and also to PC12 cells. (^3)All of these cells have a single high affinity receptor class, and thus it remains to be determined how the binding sites identified by the equilibrium binding studies ( Fig. 2and Fig. 3and Table 1) relate to these five structural classes of binding proteins identified by the affinity cross-linking studies (Fig. 7). Specific cellular binding proteins for BMP-4 with apparent molecular masses of 164 and 54 kDa (in NIH 3T3 fibroblasts), 164 and 34 kDa (in MC3T3-E1 osteoblasts), and 165, 55, and 35 kDa (in articular chondrocytes) have been recently reported(26, 29) .

Although the receptors for BMPs have not been well characterized, substantial progress has been recently made on the characterization of receptors for other members of TGF-beta superfamily such as TGF-betas and activins. For example, a number of different size receptors and binding proteins for TGF-betas have been identified in cultured cells and tissues by chemical cross-linking experiments; these include type I (53 kDa), type II (75 kDa), type III (280 kDa), type IV (60 kDa), type V (400 kDa), and type VI (180 kDa) receptors, as well as several other membrane binding proteins of 40, 60, and 140 kDa (reviewed in (31, 32, 33) ). Among these receptors and membrane binding proteins, the most widely distributed are the type I, II, and III receptors. The roles of these different types of receptors in the multiple functions of TGF-betas are unclear. However, several lines of evidence suggest that the type I and type II receptors, both of which have transmembrane serine/threonine kinase structures(34, 35, 36) , are implicated in the signal transduction of many effects of TGF-betas, while type III receptors are involved in the presentation of the ligand to the signaling receptors(31, 32, 33) .

The presence of multiple cellular binding proteins for BMP-2 that we have identified in the present study suggests that the functional receptors for BMPs also are composed of several components. In this context, the Daf-4 protein, a transmembrane serine/threonine kinase obtained from Caenorhabditis elegans, has been recently shown to bind specifically to BMP-2 and BMP-4 and to form a 100-kDa cross-linked complex(37) . The structure of Daf-4 is similar to that of the activin type II receptor and TGF-beta type II receptor, and thus Daf-4 is suggested to be a type II receptor for BMPs. Very recently, when this manuscript was in the final stage of preparation, ALK-3 and ALK-6 obtained from human and mouse, respectively(38) , and Brk25D (the product of tkv gene) and Brk43E (the product of sax gene) from Drosophila(39, 40, 41) were suggested to be the type I receptors for BMPs; these molecules bind specifically to BMP-4 or BMP-2 forming 80-kDa cross-linked complexes.

After submission of this manuscript, isolation of a gene (brk-1) encoding a putative type I receptor for BMPs from mouse fibroblasts was reported(42) . Affinity labeling of brk-1 transfected COS-7 cells with I-labeled BMP-2/BMP-4 was found to yield two labeled products of 80 and 93 kDa; the 80-kDa band is suggested to represent the product of brk-1 cross-linked to the ligand monomer, while the 93-kDa band represents BRK-1 cross-linked to the ligand dimer. In this respect, some of our cross-linking experiments with I-labeled BMP-2 have revealed an additional 155-kDa band other than the five components described above in fibroblasts and PC12 cells.^3 Taken together, these results might suggest that I-labeled BMP-2 has specifically bound to cellular proteins with molecular masses of 140, 75 (this value corresponds to that of type II receptor for TGF-beta, and thus it could be a mammalian homologue of Daf-4), and 52 kDa (this value corresponds to that of type I receptor for TGF-beta and thus it could be related to BRK-1/ALK-3 or ALK-6); cross-linking of the ligand monomer (16 kDa) or dimer (30 kDa) to each of these proteins would then generate the observed 170/155, 105/90, and 80/70-kDa species, respectively. Additional experiments are needed to demonstrate whether the cellular specific binding proteins for BMP-2 we have identified indeed constitute the functional BMP receptor and how each of these molecules actually relates to Daf-4, BRK-1/ALK-3, or ALK-6.


FOOTNOTES

*
This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of the Fellowship of the Japan Society for the Promotion of Science for Japanese Junior Scientists.

To whom all correspondence should be addressed. Tel.: 81-58-237-3931 (ext. 207); Fax: 81-58-237-5979.

(^1)
The abbreviations used are: BMP, bone morphogenetic protein; TGF-beta, transforming growth factor-beta; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; MDCK, Madin-Darby canine kidney epithelial cells; HUVEC, human umbilical vein endothelial cells.

(^2)
S. Iwasaki, M. Tsujimoto, and M. Kohno, unpublished observation.

(^3)
A. Hattori, S. Iwasaki, M. Sato, M. Tsujimoto, and M. Kohno, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. M. Kumegawa, T. Nakamura, S. Toyama, H. Oka, and K. Miyamoto for supplying cells or growth factors. We also thank Drs. K. Shiota, M. Maeda, and P. Hughes for critical reading of the manuscript and helpful advice.


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