Communicated by Elwood V. Jensen, January 7, 1991. ABSTRACT. Bone morphogenetic protein 2B (BMP 2B), is a heparin-binding bone differentiation factor ...
Proc. Nati. Acad. Sci. USA Vol. 88, pp. 3397-3401, April 1991 Cell Biology
Identification and characterization of cellular binding proteins (receptors) for recombinant human bone morphogenetic protein 2B, an initiator of bone differentiation cascade (bone cells/morphogenesis/cartflage/development/transfonning growth factor f superfamily)
VISHWAS M. PARALKAR*, R. GLENN HAMMONDSt, AND A. H. REDDI*f *Bone Cell Biology Section, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892; and the tDepartment of Developmental
Biology, Genentech, Inc., South San Francisco, CA 94080
Communicated by Elwood V. Jensen, January 7, 1991
ABSTRACT Bone morphogenetic protein 2B (BMP 2B), is a heparin-binding bone differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously. The molecular mechanism of action of this differentiation factor is not known, and as a first step we have examined BMP 2B-responsive cells for the presence of specific cellular binding proteins. Using 125I-labeled BMP 2B, specific high-affinity binding sites for recombinant human BMP 2B on MC3T3 El osteoblast-like cells as well as on NIH 3T3 fibroblasts were identified. Platelet-derived growth factor, insulinlike growth factor 1, basic fibroblast growth factor, epidermal growth factor, and transforming growth factor ,B did not compete for the binding of radiolabeled BMP 2B. The binding of BMP 2B is a time- and temperature-dependent process. Chemical crosslinking of radiolabeled BMP showed two components (apparent size, 200 and 70 kDa in MC3T3 El cells and 200 and 90 kDa in NIH 3T3 cells). A minor component at 60 kDa was also detected in both cell lines. Scatchard analysis of the binding data showed a high-affinity receptor with an apparent dissociation constant of 128 ± 40 pM in MC3T3 El cells. These data demonstrate specific, high-affinity cellsurface binding proteins for BMP 2B.
The occurrence of growth and differentiation factors in bone has been demonstrated by subcutaneous implantation of demineralized collagenous bone matrix, which induces local bone differentiation (1, 2). The bone differentiation activity of bone matrix can be dissociatively extracted and reconstituted with inactive residual collagenous matrix to restore full bone-inductive activity (3). Several bone-inductive molecules have been purified to homogeneity and variously named as osteogenin and bone morphogenetic proteins (BMPs) (4, 5). Several genes have been cloned and designated BMP 1, BMP 2A, BMP 2B, BMP 3/osteogenin, and OP-1 (5, 6). BMPs are members of the transforming growth factor ,B (TGF-,B) superfamily based on sequence similarity (5). Recombinant human BMP 2A, BMP 2B, and osteogenin induce endochondral bone differentiation in ectopic sites in the rat (7, 8). As a first step toward elucidation of the mechanism of action of BMPs, we have investigated specific binding in BMP 2B-responsive cells. Polypeptide growth factors, in general, initially interact with the cell through membranebound receptors (9). Membrane receptors for BMPs have not been previously demonstrated due to the lack of sufficient quantities of homogenous factors. The recent expression of biologically active recombinant BMPs (8) permitted us to address this question. We report here the identification of specific cellular binding proteins for BMP 2B.
MATERIALS AND METHODS BMP 2B Expression and Purification. Recombinant human BMP 2B was expressed and purified as described (8). In brief, cDNAs for BMP 2A and BMP 2B were isolated from a human placental cDNA library constructed in AgtlO by using oligonucleotide probes based on the human nucleotide sequence by standard cloning techniques. The coding regions were subcloned into the expression vector pRK5 to create expression plasmids. Mammalian 293 cells were grown to 70% confluence and were transfected with expression plasmids by the calcium phosphate method. BMP 2B was purified from the conditioned medium by heparin-Sepharose affinity chromatography and Mono Q column and reverse-phase HPLC. Iodination of BMP 2B. Recombinant BMP 2B was iodinated by a modified chloramine-T method (10, 11). Briefly, 0.5 pug of BMP 2B in 10 Al of 4 mM HCl was diluted with 10 pl of 1.5 M sodium phosphate (pH 7.5). Then, 150 ACi of Na125I (1 Ci = 37 GBq; Amersham) was added to the reaction mixture and the reaction was started by adding 5-A1 aliquots of chloramine T (100 mg/ml) (Sigma). After 2 min, 5 A.l was added followed by the addition of the last aliquot of S Al for 1 min. The reaction was terminated by the addition of 20 ,ul of N-acetyltyrosine (50 mM) followed by the addition of 200 ,ul of 60 mM KI and 200 ,ul of 8 M urea. Radiolabeled BMP 2B was separated from unincorporated Nal by passing the reaction mixture over a PD-10 column (Pharmacia) equilibrated with 4 mM HCI/75 mM NaCl/0.1% bovine serum albumin (BSA). The tube with peak radioactivity was stored in 4 mM HCI containing 10 mg of BSA per ml at -70°C in smaller aliquots. Specific activity of BMP 2B ranged from 50 to 66 mCi/mg after correcting for trichloroacetic acid-precipitable activity, which was >95%. A sample (100,000 cpm) of each iodinated preparation was analyzed by SDS/PAGE on a 15% separating gel followed by autoradiography. '2SI-Labeled BMP 2B (12'I-BMP 2B) Binding Assay. MC3T3 El osteoblasts or NIH 3T3 fibroblasts were seeded in a 24-well plate at 5 x 104 cells per ml in M-199 or Dulbecco's modified Eagle's medium (DMEM) containing 10% serum and gentamicin (50 ,ug/ml). After 2 days, the cell monolayer (2 x 105 cells for MC3T3 and 4 x 105 cells for NIH 3T3) was washed twice with binding buffer [128 mM NaCl/5 mM KCI/5 mM MgSO4 containing 5 mg of BSA per ml, 1.3 mM CaC12, and 50 mM Hepes (pH 7.4)] and incubated with 1 ml of binding buffer for 30 min at 37°C. The cell monolayer was then incubated with 200 ,ul of binding buffer containing BMP 2B on a rotator at either 4°C or room temperature. After incubation, the cells were washed three times with Hanks' balanced salt solution containing 0.1% BSA (12). The cells Abbreviations: BMP, bone morphogenetic protein; BSA, bovine serum albumin; TGF-p1, transforming growth factor /81. fTo whom reprint requests should be addressed at: Building 30, Room 211, National Institutes of Health, Bethesda, MD 20892.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3397
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were then solubilized with 750 ,ul of Triton solution [20 mM Hepes/1% Triton X-100/10%6 (vol/vol) glycerol/0.01% BSA, pH 7.4] at 370C for 20 min. Radioactivity was determined in aliquots (600 ILI) in a Beckman gamma counter. Nonspecific binding was determined by using a 100-fold excess of unlabeled BMP 2B. More than 95% of BMP 2B was found to be trichloroacetic acid precipitable at the end of the incubation. In Vitro Activity of BMP 2B. Twenty-four-well tissue culture plates were coated with fibronectin (2 ,pg/ml) for 30 min at 370C. NIH 3T3 or MC3T3 cells were then seeded into these wells at a density of 5 x 104 cells per well in medium containing 0.2% serum. After 1 hr, either radiolabeled or unlabeled BMP 2B was added to the wells and the cells were incubated for 22 hr at 370C in 5% C02/95% air. After 22 hr, 2.5 p.Ci of [3H]thymidine (2.5 A.l of 1-mCi/ml stock; NEN) was added to each well and the cells were labeled for 2 hr. The reaction was stopped by the addition of methanol/acetic acid (3:1; 1 ml per well) directly to the wells (13). The cells were then washed two times with 80% (vol/vol) methanol and released by incubation with 0.05% trypsin (0.5 ml) for 1 hr at 37°C, followed by 0.5 ml of 1% SDS, and then counted in a 'y counter (13). The radioactivity was corrected for contributions from 1251 by subtracting the cpm due to 1251 in the 3H channel from the total cpm. The correction was 0.1-4% of total cpm. Other Procedures. MC3T3 El osteoblast-like cells (14, 15) were grown in M-199 containing 10% fetal calf serum, 1% insulin/transferrin/selenium, and 50 mg of gentamicin and fungizone per ml. NIH 3T3 cells were cultured in DMEM containing 10%o calf serum and 50 mg of gentamicin and fungizone per ml. Cells were passaged by first washing the cell monolayer with phosphate-buffered saline (PBS) and then exposing to 5 ml of 0.05% trypsin-EDTA (GIBCO). The trypsin solution was removed after 1 min and the cultures were observed under the microscope for cell detachment. The reaction was stopped by the addition of medium containing 10%o serum. A cell pellet was then obtained by centrifuging the detached cells in a Sorvall centrifuge for 5 min at 800 rpm. Crosslinking of 125I-BMP 2B to Cell-Surface Receptors. Binding of radiolabeled BMP 2B to cells was carried out as described (16). After the cell monolayer was washed with Hanks' balanced salt solution, 1 ml of disuccinimidyl suberate (Pierce) [prepared fresh by first dissolving in dimethyl sulfoxide (20 mM) and then diluting 1:100 in PBS] was added to each well and the reaction was carried out at room temperature for 20 min. The reaction was stopped by the addition of quenching buffer (10 mM Tris-HCI, pH 7.5/200 mM glycine/2 mM EDTA). Each plate then was incubated for 1 min at room temperature and the incubation medium was removed. Each well was then washed three times with cold PBS (1 ml) and subsequently 0.5 ml of PBS containing 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA was added to each well. The cells were detached with a rubber policeman and the cell suspension was centrifuged for 30 sec at 12,000 x g in a microcentrifuge. The supernatant was aspirated and the pellet was solubilized in lysis buffer [10 mM Tris HCl (pH 7.5) containing 0.5% Nonidet P-40, 0.1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride] for 10 min. SDS/PAGE sample buffer (5 x) was then added to each tube and the tubes were centrifuged. The supernatant fraction was then analyzed by gradient (4-12%) SDS/PAGE (17).
RESULTS AND DISCUSSION Structural and Biological Integrity of Iodinated BMP 2B. The integrity of the iodinated BMP 2B was confirmed by SDS/PAGE. The autoradiogram of a 15% SDS gel of radiolabeled BMP 2B under reducing and nonreducing conditions showed a 36-kDa band under nonreducing conditions and a
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FIG. 1. SDS/PAGE of
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125I-BMP 2B were dissolved in SDS sample buffer in the presence
(lane b) and absence (lane a) of 10 mM dithiothreitol and 0.1% 2-mercaptoethanol. Samples containing the reducing agents were heated at 370C for 2 hr followed by 3 min at 1000C before application to a 15% SDS gel with a 4% stacking gel. After electrophoresis, the gel was dried and subjected to autoradiography. Note a single broad band at 30-38 kDa before reduction and at 20-22 kDa after reduction. Molecular mass markers are shown in kDa.
20- to 22-kDa band under reducing conditions (Fig. 1). It was essential that the iodinated derivative of BMP 2B be tested for physiological activity. As shown in Fig. 2, both native and radiolabeled BMP 2B had a similar biological activity profile when tested on MC3T3 El cells. The radiolabeled BMP 2B displayed good stability for periods up to 1 month when
stored at -70°C in 4 mM HCl/1% BSA. Demonstation of Specific '25I-BMP 2B B g to NIH 3T3 and MC3T3 El Cels. Radiolabeled BMP 2B bound to both MC3T3 El and NIH 3T3 cells in a dose-dependent manner 25
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[3H]thymidine incorporation into DNA. MC3T3 El cells were seeded
in a 24-well plate at 1 x 105 cells per ml using 0.5 ml per well followed by the addition of either native (o) or radiolabeled (e) BMP 2B at the concentrations indicated. [3H]Thymidine was added to the cells after 22 hr and the amount of incorporation of [3H]thymidine in DNA was measured. Each point represents the mean of three observations. The assay was repeated with two different iodinated preparations. Note the inhibition of [3H]thymidine incorporation in MC3T3 El cells.
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FIG. 3. Binding of radiolabeled BMP 2B to MC3T3 El cells. Increasing concentrations of radiolabeled BMP 2B were incubated with MC3T3 El cells at room temperature for 1 hr and the cell-associated radioactivity was determined. Points represent specifically bound radioactivity determined by subtracting nonspecific binding (10-40%o of total binding) from total radioactivity bound. (Inset) Scatchard analysis of the binding data. B, bound; F, free.
(Figs. 3 and 4) (Inset, Scatchard plot) (18). The data do not fit the usual single straight line but instead show a downward "hook" at lower concentrations of bound BMP 2B. It is possible that this may be due to positive cooperative behavior in the binding of radiolabeled BMP 2B to its receptor. Alternatively, low concentrations of growth factors have
been shown to approach equilibrium slowly, which could also result in the formation of a hook (19). MC3T3 El cells show the presence of a high-affinity site with a Kd of 128 ± 40 pM with 5600 ± 500 receptors per cell. NIH 3T3 cells have high levels of nonsaturable binding, making valid estimates of binding parameters difficult. BMP 2B is a heparin binding
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FIG. 4. Binding of radiolabeled BMP 2B to NIH 3T3 cells. Increasing concentrations of radiolabeled BMP 2B were incubated with NIH 3T3 cells at room temperature for 1 hr and the cell-associated radioactivity was determined. Nonspecific binding was determined by using a 100-fold excess of native BMP 2B. Each point represents the mean of triplicate observations for total binding and duplicate observations for nonspecific binding. (Inset) Scatchard analysis of the binding data. B, bound; F, free.
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FIG. 5. Time course of 1251-BMP 2B binding to NIH 3T3 cells.
125I-BMP 2B (final concentration, 2.5 ng/ml) was added to each cell in binding buffer (200,ul). At the indicated time intervals, the reaction was stopped and the cell-bound radioactivity was determined. *, Binding at 40C; o, binding at room temperature.
differentiation factor. The existence of a similar small number of high-affinity sites and a high number of low-affinity sites has been demonstrated for other matrix binding growth factors (20). It is likely that the low-affinity sites could be due to interaction of 125I-BMP 2B with matrix macromolecules such as heparan sulfate proteoglycans. These sites have a high capacity and are difficult to saturate. The low-affinity sites may also be cell-surface proteoglycans as has been shown to be the case for TGF-.8 (21, 22). Time and Temperature Dependence of Binding. The time course of binding of 125I-BMP 2B to NIH 3T3 fibroblasts at 4TC and at room temperature was determined (Fig. 5). Maximal binding was reached after incubations of 40-45 min at room temperature (250C) and of 2 hr at 40C. Higher binding was observed at room temperature than at 40C. Cell detachment precluded longer incubation at 40C. Specificity of Binding. As shown in Table 1 480% of the 125I-BMP 2B bound to NIH 3T3 and MC3T3 El cells was displaced by a 100-fold (250 ng/ml) excess of nonradioactive BMP 2B. In contrast, none of the other polypeptide growth factors tested (platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor 1, epidermal growth factor, or TGF-,B1) at a concentration of 250 ng/ml had any effect on binding of radiolabeled BMP 2B to the cells. Preliminary experiments with purified natural osteogenin/ BMP 3 showed that a 100-fold excess of osteogenin could compete 30% of the bound BMP 2B. Among the polypeptide Table 1. Competition with growth factors for binding of iodinated BMP 2B to MC3T3 El and NIH 3T3 cells cpm bound NIH 3T3 MC3T3 El Growth factor ± 800 1251-BMP 2B 10,480 ± 680 9,600 ± 230 BMP 2B 2,300 ± 265 4,000 PDGF 10,050 ± 202 11,720 ± 1000 FGF 9,510 ± 230 8,210 ± 217 IGF-l 9,670 ± 275 8,930 ± 110 EGF 9,070 ± 660 9,806 600 9,570 ± 330 9,260 ± 460 TGF-,B1 Each factor was in a 100-fold excess of the iodinated BMP 2B. Values shown are means ± SD of four observations. PDGF, plateletderived growth factor; FGF, basic fibroblast growth factor; IGF-1, insulin-like growth factor 1; EGF, epidermal growth factor.
growth factors tested, the BMP 2B binding protein is specific for BMP 2B. Affinity Crosslnking of '25I-BMP 2B to Its Binding Proteins. The binding proteins in both NIH 3T3 and MC3T3 El cells were further characterized by affinity crosslinking techniques. Radiolabeled BMP 2B specifically bound to cells and was covalently crosslinked to its binding protein by the homobifunctional crosslinking agent disuccinimidyl suberate. Analysis by SDS gel under nonreducing conditions revealed two crosslinked macromolecular components in both NIH 3T3 and MC3T3 El cells (Fig. 6). The apparent molecular mass of these species was 200 kDa in both cell types and the second species showed a molecular mass of 90 kDa in NIH 3T3 cells and '=70 kDa in MC3T3 El cells. Since these molecular masses include that of BMP 2B (36 kDa), the corresponding putative receptors prior to crosslinking would have apparent molecular masses of 164, 54, and 34 kDa, respectively. The crosslinking to both macromolecular species was inhibited by a 100-fold excess of native BMP 2B (lanes B and D). No crosslinking of '25I-BMP 2B to larger molecular mass was observed in the absence of cells (data not shown). Many of the characteristics of binding of 1251I-BMP 2B described here are similar to those of other hormones and growth factors (22-26). BMP 2B is able to bind with high affinity to specific, saturable, binding sites in a time- and temperature-dependent manner. None of the growth factors tested had any effect on the ability of BMP 2B to bind to its receptor. Both NIH 3T3 cells (unpublished observations) and MC3T3 El cells are responsive to BMP 2B and it is not surprising to find specific high-affinity binding proteins for BMP 2B on the cell surface/membrane. Since BMP 2B is a member of the TGF-,B superfamily, we also examined the occurrence of binding proteins in MvlLu epithelial cells (CCL-64), which have receptors for TGF-/3 (22, 27). We could not detect any binding of BMP 2B to these cells. The apparent molecular sizes of receptor species obtained by crosslinking studies are 164 and 54 kDa in NIH 3T3 cells. On the other hand, in MC3T3 El cells binding species were around 164 and 34 kDa. The variations in size could be due to differences in cells or to differential glycosylation, as has been shown to be the case for TGF-/ (22). Additional experiments are needed to demonstrate that the BMP 2B binding proteins are indeed bona fide functional receptors involved in the signal transduction pathway. The recent autoradiographic demonstration (28) of binding sites for natural osteogenin (BMP 3) and recombinant BMP 2B (S. Vukicevic, personal communication) in rat embryos during
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FIG. 6. Affinity labeling of NIH 3T3 and MC3T3 El cells with radiolabeled BMP 2B. Lanes A and C, confluent cultures of NIH 3T3 and MC3T3 El cells were incubated with 125I-BMP 2B (2.5 ng/ml); lanes B and D, cells also received native BMP 2B (250 ng/ml). Cultures were incubated at room temperature for 1 hr. They were then washed and crosslinked. Aliquots were then subjected to SDS/PAGE on a 1.0-mm-thick 4-12% gradient SDS gel followed by autoradiography. The bands at the bottom of the gel represent free 125I-BMP 2B. Molecular mass markers are shown in kDa.
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cartilage and bone development implies a major role for this family of bone morphogenetic proteins in morphogenesis. The patterns ofexpression of murine BMP 2A RNA by in situ hybridization during embryonic development of mouse lends further support for the role of bone morphogenetic proteins in skeletal morphogenesis and pattern formation (29). We thank Dr. David Saloman for many helpful discussions and Dr. H. Kodama for providing MC3T3 El cells. 1. Urist, M. R. (1%5) Science 150, 893-899. 2. Reddi, A. H. & Huggins, C. B. (1972) Proc. Nail. Acad. Sci. USA 69, 1601-1605. 3. Sampath, T. K. & Reddi, A. H. (1981) Proc. Nail. Acad. Sci. USA 78, 7599-7603. 4. Luyten, F. P., Cunningham, N. S., Ma, S., Muthukumaran, N., Hammonds, R. G., Nevins, W. B., Wood, W. I. & Reddi, A. H. (1989) J. Biol. Chem. 264, 13377-13380. 5. Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Kriz, R. W., Hewick, R. M. & Wang, E. A. (1988) Science 242, 1528-1534. 6. Ozkaynak, E., Rueger, D. C., Drier, E. A., Corbett, C., Ridge, R. J., Sampath, T. K. & Oppermann, H. (1990) EMBO J. 9, 2085-2093. 7. Wang, E. A., Rosen, V., D'Alessandro, J. S., Bauduy, M., Cordes, P., Harada, T., Israel, D. I., Hewick, R. M., Kerns, K. M., LaPan, P., Luxenberg, D. P., McQuaid, D., Moutsatsos, I. K., Nove, J. & Wozney, J. M. (1990) Proc. Nail. Acad.
Sci. USA 87, 2220-2224. 8. Hammonds, R. G., Schwall, R., Dudley, A., Lai, C., Berkemeier, L., Cunningham, N. S., Reddi, A. H., Wood, W. I. & Mason, A. J. (1991) Mol. Endocrinol., 5, 149-155. 9. James, R. & Bradshaw, R. A. (1984) Annu. Rev. Biochem. 53, 259-292.
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10. Frolick, C. A., Wakefield, L. M., Smith, D. M. & Sporn, M. B. (1984) J. Biol. Chem. 259, 10995-11000. 11. Greenwood, F. C., Hunter, W. M. & Glover, J. S. (1963) Biochem. J. 89, 114-123. 12. Heldin, C.-H., Westermark, B. & Wasteson, A. (1981) Proc. Nail. Acad. Sci. USA 78, 3664-3668. 13. Danielpour, D., Dart, L. L., Flanders K. C., Roberts, A. B. & Sporn, M. B. (1989) J. Cell. Physiol. 138, 79-86. 14. Sudo, H., Kodama, H., Amagai, Y., Yamamoto, S. & Kasai, S. (1983) J. Cell Biol. 96, 191-198. 15. Vukicevic, S., Luyten, F. P. & Reddi, A. H. (1990) Biochem. Biophys. Res. Commun. 166, 750-756. 16. Cheifetz, S., Andres, J. L. & Massague, J. (1988) J. Biol. Chem. 263, 16984-16991. 17. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 18. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. 19. Bowen-Pope, D. F. & Ross, R. (1985) Methods Enzymol. 109, 69-100. 20. Moscatelli, D. (1987) J. Cell. Physiol. 131, 123-130. 21. Segarini, P. R. & Seyedin, S. M. (1988) J. Biol. Chem. 263, 8366-8370. 22. Massague, J., Cheifetz, S., Boyd, F. T. & Andres, J. L. (1990) Ann. N. Y. Acad. Sci. 593, 59-72. 23. Carpenter, G., Lembach, K. J., Morrison, M. M. & Cohen, S. (1975) J. Biol. Chem. 250, 4297-4304. 24. Carpenter, G. & Cohen, S. (1976) J. Cell. Biol. 71, 159-171. 25. Huang, J. S., Huang, S. S., Kennedy, B. & Deuel, T. F. (1982) J. Biol. Chem. 257, 8130-8136. 26. Bowen-Pope, D. F. & Ross, R. (1982) J. Biol. Chem. 257, 5161-5171. 27. Segarini, P. R., Roberts, A. B., Rosen, D. M. & Seyedin, S. M. (1987) J. Biol. Chem. 262, 14655-14662. 28. Vukicevic, S., Paralkar, V. M., Cunningham, N. S., Gutkind, J. S. & Reddi, A. H. (1990) Dev. Biol. 140, 209-214. 29. Lyons, K. M., Pelton, R. W. & Hogan, B. L. M. (1990) Development 109, 833-844.
