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Cleveland, T. E., Lax, A. R., Lee, L. S.,& Bhatnagar, D. ( 1987b) Appl. Environ. Microbiol. 53, 17 1 1-1 7 13. Cole, R. J., & Kirksey, J. W. (1979) Tetrahedron Lett. 35, 3109-31 12. Cole, R. J., & Cox, R. H. (1981) in Handbook of Toxic Fungal Metabolites, Academic Press, New York. Diener, U. L., & Davis, N. N . (1966) Phytopathology 56, 1390-1393. Dutton, M. F. (1988) Microbiol. Rev. 52, ,274-295. Dutton, M. F., & Anderson, M. S . (1982) Appl. Environ. Microbiol. 43, 548-55 1. Dutton, M. F., Ehrlich, K., & Bennett, J. W. (1985) Appl. Environ. Microbiol. 49, 1392-1 395. Elsworthy, G. E., Holker, J. S. E., McKeown, J. M., Robinson, J. B., & Mulheirn, L. J. (1970) Chem. Commun., 1069-1 070. Floyd, J. C., Mills, J., & Bennett, J. W. (1987) Exp. Mycol. 11, 109-114. Hatsuda, Y., Hamasaki, T., Tshida, M., Matsui, K., & Hara, S . (1972) Agric. Biol. Chem. 36, 521-522. Heathcote, J. G., Dutton, M. F., & Hibbert, J. R. (1976) Chem. Ind. (London), 270-274. Hsieh, D. P. H., Lin, M. T., & Yao, R. C. (1973) Biochem. Biophys. Res. Commun. 52, 992-997. Hsieh, D. P. H., Wan, C. C., & Billington, J. A. (1989) Mycopathologia 107, 121-1 30.
Leaich, L. L., & Papa, K. E. (1974) Mycopathol. Mycol. Appl. 52, 223-229. Lee, L. S . , Bennett, J. W., Cucullu, A. F., & Ory, R. L. (1976) J . Agric. Food Chem. 24, 1167-1 171. Maggon, K. K., & Vekitasubramanian, T. A. (1973) Experientia 29, 1211-1212. Maggon, K. K., Gupta, S . K., & Vekitasubramanian, T. A. (1977) Bacteriol. Rev. 41, 822-855. Malik, V. S . (1982) Adv. Appl. Microbiol. 28, 27-116. McCormick, S . P., Bhatnagar, D., & Lee, L. S . (1987) Appl. Environ. Microbiol. 53, 14-1 6. Raj, H. G., Viswanathan, L., Murthy, H. S. R., & Venkitasubramanian, T. A. (1969) Experientia 25, 1141-1 142. Schroeder, H. W., & Carlton, W. W. (1973) Appl. Microbiol. 25, 146-148. Sedmak, J. J., & Grossburg, S . E. (1977) Anal. Biochem. 79, 544-552. Steyn, P. S . , Vleggar, R., & Wessels, P. L. (1980) in The Biosynthesis of Mycotoxins (Steyn, P. S., Ed.) pp 105-155, Academic Press, New York. Thomas, R. (1 965) in Biogenesis of Antibiotic Substances, pp 155-167, Academic Press, New York. Townsend, C. A. (1986) Pure Appl. Chem. 58, 227-238. Yabe, K., Ando, Y., & Hamasaki, T. (1988) Appl. Environ. Microbiol. 54, 2 101-2 106.
A Transforming Growth Factor /3 (TGF-0) Receptor from Human Placenta Exhibits a Greater Affinity for TGF-P2 than for TGF-/31+*$ E. Jane Mitchell* and Maureen D. O'Connor-McCourt Receptor Group, National Research Council of Canada, Biotechnology Research Institute, 61 00 Royalmount Avenue, Montreal, Quebec H4P 2R2. Canada Received September 14, 1990; Revised Manuscript Received January 8, 1991 ABSTRACT: Affinity-labeling techniques have been used to identify three types of high-affinity receptors
for transforming growth factor P (TGF-P) on the surface of many cells in culture. Here we demonstrate that membrane preparations from tissue sources may also be used as an alternative system for studying the binding properties of TGF-/3 receptors. Using a chemical cross-linking technique with '251-TGF-P1 and '251-TGF-P2 and bis(su1fosuccinimidyI)suberate (BS3),we have identified and characterized two high-affinity binding components in membrane preparations derived from human term placenta. The larger species, which migrates as a diffuse band of molecular mass 250-350 kDa on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, is characteristic of the TGF-P receptor type 111, a proteoglycan containing glycosaminoglycan (GAG) chains of chondroitin and heparan sulfate. The smaller species of molecular mass 140 kDa was identified as the core glycoprotein of this type I11 receptor by using the techniques of enzymatic deglycosylation and peptide mapping. Competition experiments, using IZ5I-TGF-P1or lZSI-TGF-P2and varying amounts of competing unlabeled TGF-P1 or TGF-62, revealed that both the placental type I11 proteoglycan and its core glycoprotein belong to a novel class of type I11 receptors that exhibit a greater affinity for TGF-P2 than for TGF-PI. This preferential binding of TGF-P2 to placental type I11 receptors suggests differential roles for TGF-P2 and TGF-P1 in placental function. T a n s forming growth factor P (TGF-P)' represents a family of multifunctional regulatory proteins involved in cellular differentiation and proliferation. TGF-PI and TGF-P2 have been isolated from tissue sources, whereas the amino acid sequences of TGF-P3, TGF-04, and TGF-P5 have been de'This article is NRCC Publication 32411. $These results have been presented in preliminary form at the Annual Meeting of the American Society for Cell Biology in Houston, TX, November 1989 (Mitchell & OConnor-McCourt, 1989). * Author to whom correspondence should be addressed.
0006-296019 110430-4350$02.50/0
duced from cDNA clones. TGF-Ps, as with other polypeptide hormones, appear to act by binding to specific cell surface I Abbreviations: TGF-6, transforming growth factor p; GAG, glycosaminoglycan; BS3, bis(sulfosuccinimidy1) suberate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, DulbecGO'S phosphate-buffered saline; BSA, bovine serum albumin; EGTA, ethylene glycol bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid; EDTA, disodium ethylenediaminetetraacetate;STI, soybean trypsin inhibitor; TCA, trichloroacetic acid; PMSF, phenylmethanesulfonyl fluoride; hCG, human chorionic gonadotropin; hPL, human placental lactogen.
0 1991 American Chemical Society
TGF-/3 Affinity Labeling of Placental Membranes receptors that transduce the regulatory signal [for reviews, see Roberts and Sporn (1989) and Massagui (1990)l. The nature of the TGF-P signaling pathway(s) remains uncertain; however, some mechanism(s) may be linked to the phosphorylation state of the retinoblastoma protein (Laiho et al. 1990a) or to a GTP-binding protein (Howe et al., 1990). Although the signaling activity of TGF-/3 receptors remains obscure, it has been possible to identify putative TGF-/3 receptors by chemical cross-linking of '2SI-TGF-Pto specific, high-affinity binding components on cell surfaces (Massagub, 1985; Massagui & Like, 1985; Fanger et al., 1985; Segarini, 1989). This affinity-labeling technique has revealed three types of high-affinity binding components, operationally termed TGF-@receptors, on the cell surface of most established cell lines examined (Massagub & Like, 1985; Cheifetz et al., 1986; Wakefield et al., 1987). Types I and XI receptors are glycoproteins identified as affinity-labeled complexes having molecular masses of 65 kDa and 85-95 kDa, respectively, and both bind TGF-PI with a higher affinity than TGF-P2 (Cheifetz et al., 1987, 1988a). The type 111 component is a membrane proteoglycan of approximately 250-350 kDa containing glycosaminoglycan (GAG) chains of chondroitin and heparan sulfate. Its core glycoprotein is identified as an affinity-labeled complex of molecular mass 100-140 kDa that contains the functional binding site for TGF-P (Segarini & Seyedin, 1988; Cheifetz et al., 1988b). In general, type 111 receptors bind TGF-Dl and TGF-02 with similar affinities (Cheifetz et al., 1987, 1988a; Cheifetz & Massagui, 1989), although there is evidence for a type 111 subset, termed class B, found on NRK and Swiss 3T3 fibroblasts that preferentially binds TGF-P2 (Segarini et al., 1987). Until recently, the only criteria that could be used to define TGF-P signaling receptors was the correlation between receptor affinities for TGF-@I and TGF-P2 and the potencies of TGF-@I and TGF-@2in biological assays. On this basis, Massagui and co-workers had originally proposed that the type I11 (250-350 kDa) receptor mediates the effect of TGF-P on regulation of extracellular matrix production and inhibition of epithelial cell proliferation (Cheifetz et al., 1987, 1988a), whereas the type 1 (65-kDa) receptor mediates the effect of TGF-/3 on the growth of mouse hematopoietic progenitor cells (Ohta et al., 1987). Recently, however, mutants of epithelial cells have been isolated that selectively lose either the type I or both the types I and TI receptors as they become resistant to growth inhibition by TGF-/3 (Boyd & Massagu6, 1989; Laiho et al., 1990b). In addition, certain cells that do not display detectable type 111 receptors are still able to respond to TGF-/3 (Segarini et al., 1989). Therefore, it has now been proposed that the type 111 receptor may not be a true, signaling receptor for many TGF-@activities including regulation of DNA synthesis and extracellular matrix production (Segarini et al., 1989). The recent discovery of both membrane-anchored and soluble forms of the type 111 proteoglycan supports the notion of novel nonsignaling roles, such as high-affinity storage sites for TGF-@s,activators of latent TGF-@,or clearance receptors, for these molecules (Andres et al., 1989). With a few exceptions (Kyprianou & Isaacs, 1988; MacKay et al., 1989, 1990; Gruppuso et al., 1990), most research on the characterization of TGF-/3 receptors has been done with established or primary cells in culture. We were particularly interested in determining whether TGF-P-binding components in membrane preparations from tissue sources could be characterized. Human term placental membrane preparations were chosen because of their ready availability and their broad usage for receptor assays and receptor purification [e.g., the
Biochemistry, Vol. 30, No. 17, 1991 4351 epidermal growth factor receptor (Downward et al., 1984)]. Moreover, since the factors involved in placental growth, development, and immunosuppression are poorly understood, it is important to investigate the role(s) of TGF-P during pregnancy. In the present study, we have identified and characterized novel type 111 TGF-(3 receptors in human placental membrane preparations that exhibit a greater affinity for TGF-02 than TGF-P1. EXPERIMENTAL PROCEDURES Placental Membranes. Human placentas obtained at term after Caesarean section from nonlabored mothers were held on ice for approximately 30 min during transport to the laboratory. Membranes were prepared as described (Valentine et al., 1987). Briefly, fresh spongy placental tissue was dissected away from the cord and large vessels and thoroughly washed with buffer 1 (100 mM NaCl, 5 mM EGTA, 5 mM EDTA, 25 mM Tris-HCI, pH 7.4) containing the following protease inhibitors: 2 pg/mL aprotinin, 2 pg/mL STI, 2 pg/mL leupeptin, 25 mM benzamidine, 1 mM PMSF. Throughout the procedure, the tissue was maintained at 0-4 "C. The tissue was first homogenized in a Waring blender for 90 s with buffer 2 (250 mM sucrose, 5 mM EGTA, 5 mM EDTA, 25 mM Tris-HC1, pH 7.4) containing the above protease inhibitors in a final volume of 1 L. An additional 1-L volume of buffer 2 was then added, followed by further homogenization with a Brinkman Polytron with use of a single burst for 90 s at setting 7. The homogenate was centrifuged at 7500g for 30 min at 4 OC, and the supernatant was made to 100 mM NaCl and 0.2 mM MgS04 by addition of salts. This material was centrifuged at 30000g for 60 min at 4 OC, and the creamy white membrane pellets were resuspended in a total volume of 500 mL of buffer 3 ( 5 mM EGTA, 5 mM EDTA, 50 mM Tris-HC1, pH 7.4, containing the above protease inhibitors) with use of a glass/Teflon Dounce homogenizer. The membranes were washed two times, and the final pellet was resuspended in approximately 50 mL of buffer 3 and frozen in aliquots at -80 "C. Protein content was estimated by the method of Lowry et al. (1951). Iodination of TGF-@. TGF-PI and TGF-P2 (both purified from porcine platelets) were purchased from R & D Systems (Minneapolis, MN). 12SI-LabeledTGF-01 and 'ZSI-labeled TGF-@2were prepared by the chloramine T method as described (Ruff & Rizzino, 1986) with use of l pg of carrier-free TGF-0 (redissolved into 15 p L of 30% acetonitrile/O.l% trifluoroacetic acid) and 1 mCi of Na[I2
