Mapping signal transduction pathways by phage display.

Rapid identification of proteins that interact with a novel gene product is an important element of functional genomics. Here we describe a phage display-based technique for interaction screening of complex cDNA libraries using proteins or synthetic peptides as baits. Starting with the epidermal growth factor receptor (EGFR) cytoplasmic tail, we identified known protein interactions that link EGFR to the Ras/MAP kinase signal transduction cascade and several novel interactions. This approach can be used as a rapid and efficient tool for elucidating protein networks and mapping intracellular signal transduction pathways.

Neuregulins signaling via a glial erbB-2-erbB-4 receptor complex contribute to the neuroendocrine control of mammalian sexual development.

Activation of erbB-1 receptors by glial TGFalpha has been shown to be a component of the developmental program by which the neuroendocrine brain controls mammalian sexual development. The participation of other members of the erbB family may be required, however, for full signaling capacity. Here, we show that activation of astrocytic erbB-2/erbB-4 receptors plays a significant role in the process by which the hypothalamus controls the advent of mammalian sexual maturation. Hypothalamic astrocytes express both the erbB-2 and erbB-4 genes, but no erbB-3, and respond to neuregulins (NRGs) by releasing prostaglandin E(2) (PGE(2)), which acts on neurosecretory neurons to stimulate secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual development. The actions of TGFalpha and NRGs in glia are synergistic and involve recruitment of erbB-2 as a coreceptor, via erbB-1 and erbB-4, respectively. Hypothalamic expression of both erbB-2 and erbB-4 increases first in a gonad-independent manner before the onset of puberty, and then, at the time of puberty, in a sex steroid-dependent manner. Disruption of erbB-2 synthesis in hypothalamic astrocytes by treatment with an antisense oligodeoxynucleotide inhibited the astrocytic response to NRGs and, to a lesser extent, that to TGFalpha and blocked the erbB-dependent, glia-mediated, stimulation of LHRH release. Intracerebral administration of the oligodeoxynucleotide to developing animals delayed the initiation of puberty. Thus, activation of the erbB-2-erbB-4 receptor complex appears to be a critical component of the signaling process by which astrocytes facilitate the acquisition of female reproductive capacity in mammals.

Negative signaling pathways of the killer cell inhibitory receptor and Fc gamma RIIb1 require distinct phosphatases.

Inhibition of natural killer (NK) cells by the killer cell inhibitory receptor (KIR) involves recruitment of the tyrosine phosphatase SHP-1 by KIR and is prevented by expression of a dominant negative SHP-1 mutant. Another inhibitory receptor, the low affinity Fc receptor for immunoglobulin G (IgG) (Fc gamma RIIb1), has been shown to bind SHP-1 when cocross-linked with the antigen receptor on B cells (BCR). However, coligation of Fc gamma RIIb1 with BCR and with Fc epsilon RI on mast cells leads to recruitment of the inositol 5' phosphatase SHIP and to inhibition of mast cells from SHP-1-deficient mice. In this study, we evaluated the ability of these two inhibitory receptors to block target cell lysis by NK cells, and the contribution of SHP-1 and SHIP to inhibition. Recombinant vaccinia viruses encoding chimeric receptors and dominant negative mutants of SHP-1 and SHIP were used for expression in mouse and human NK cells. When the KIR cytoplasmic tail was replaced by that of Fc gamma RIIb1, recognition of HLA class I on target cells by the extracellular domain resulted in inhibition. A dominant negative mutant of SHP-1 reverted the inhibition mediated by the KIR cytoplasmic tail but not that mediated by Fc gamma RIIb1. In contrast, a dominant negative mutant of SHIP reverted only the inhibition mediated by the Fc gamma RIIb1 tail, providing functional evidence that SHIP plays a role in the Fc gamma RIIb1-mediated negative signal. These data demonstrate that inhibition of NK cells by KIR involves primarily the tyrosine phosphatase SHP-1, whereas inhibition mediated by Fc gamma RIIb1 requires the inositol phosphatase SHIP.

Co-ligation of the antigen and Fc receptors gives rise to the selective modulation of intracellular signaling in B cells. Regulation of the association of phosphatidylinositol 3-kinase and inositol 5'-phosphatase with the antigen receptor complex.

Cross-linking of the Fc receptor (FcR) to surface immunoglobulin (sIg) on B cells inhibits the influx of extracellular calcium and abrogates the proliferative signal. The mechanism by which this occurs is not well understood. In this report we show that co-cross-linking the FcR to the antigen receptor gives rise to very selective modulation of signal transduction in B cells. Co-cross-linking sIg and the FcR enhanced the phosphorylation of the FcR, the adapter protein, Shc, and the inositol 5'-phosphatase Ship. Furthermore, phosphorylation of the FcR induced its association with Ship. Cross-linking of the FcR and sIg decreased the tyrosine phosphorylation of CD19, which led to a reduction in the association of phosphatidylinositol 3-kinase. In addition, the phosphorylation of several other proteins of 73, 39, and 34 kDa was reduced. Activation of the cells with either F(ab')2 or intact anti-IgG induced very similar changes in levels of tyrosine phosphorylation of most other proteins, and no differences in the activation of several protein kinases were observed. These results indicate that the inhibitory signal that is transmitted through the FcR is not mediated by a global shutdown of tyrosine phosphorylation but is, rather, a selective mechanism involving localized changes in the interactions of adapter proteins and the enzymes Ship and phosphatidylinositol 3-kinase with the antigen receptor complex.

Growth and differentiation signals regulated by the M-CSF receptor.

The normal proto-oncogene c-fms encodes the macrophage growth factor (M-CSF) receptor involved in growth, survival, and differentiation along the monocyte-macrophage lineage of hematopoietic cell development. A major portion of our research concerns unraveling the temporal, molecular, and structural features that determine and regulate these events. Previous results indicated that c-fms can transmit a growth signal as well as a signal for differentiation in the appropriate cells. To investigate the role of the Fms tyrosine autophosphorylation sites in proliferation vs. differentiation signaling, four of these sites were disrupted and the mutant receptors expressed in a clone derived from the myeloid FDC-P1 cell line. These analyses revealed that: (1) none of the four autophosphorylation sites studied (Y697, Y706, Y721, and Y807) are essential for M-CSF-dependent proliferation of the FDC-P1 clone; (2) Y697, Y706, and Y721 sites, located in the kinase insert region of Fms, are not necessary for differentiation but their presence augments this process; and (3) the Y807 site is essential for the Fms differentiation signal: its mutation totally abrogates the differentiation of the FDC-P1 clone and conversely increases the rate of M-CSF-dependent proliferation. This suggests that the Y807 site may control a switch between growth and differentiation. The assignment of Y807 as a critical site for the reciprocal regulation of growth and differentiation may provide a paradigm for Fms involvement in leukemogenesis, and we are currently investigating the downstream signals transmitted by the tyrosine-phosphorylated 807 site. In Fms-expressing FDC-P1 cells, M-CSF stimulation results in the rapid (30 sec) tyrosine phosphorylation of Fms on the five cytoplasmic tyrosine autophosphorylation sites, and subsequent tyrosine phosphorylation of several host cell proteins occurs within 1-2 min. Complexes are formed between Fms and other signal transduction proteins such as Grb2, Shc, Sos1, and p85. In addition, a new signal transduction protein of 150 kDa is detectable in the FDC-P1 cells. The p150 is phosphorylated on tyrosine, and forms a complex with Shc and Grb2. The interaction with Shc occurs via a protein tyrosine binding (PTB) domain at the N-terminus of Shc. The p150 is not detectable in Fms signaling within fibroblasts, yet the PDGF receptor induces the tyrosine phosphorylation of a similarly sized protein. In hematopoietic cells, this protein is involved in signaling by receptors for GM-CSF, IL-3, KL, MPO, and EPO. We have now cloned a cDNA for this protein and found at least one related family member. The related family member is a Fanconia Anemia gene product, and this suggests potential ways the p150 protein may function in Fms signaling.

p150Ship, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity.

The production, survival, and function of monocytes and macrophages is regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms. Binding of M-CSF to Fms induces the tyrosine phosphorylation and association of a 150-kD protein with the phosphotyrosine-binding (PTB) domain of Shc. We have cloned p150 using a modified yeast two-hybrid screen. p150 contains one SH2 domain, two potential PTB-binding sites, an ATP/GTP-binding domain, several potential SH3-binding sites, and a domain with homology to inositol polyphosphate-5-phosphatases. p150 antibodies detect this protein in FDC-P1 myeloid cells, but the same protein is not detectable in fibroblasts. The antibodies immunoprecipitate a 150-kD protein from quiescent or M-CSF-stimulated FDC-P1 cells that hydrolyzes PtdIns(3,4,5)P3, to PtdIns(3,4)P2. This activity is observed in Shc immunoprecipitates only after M-CSF stimulation. Retroviral expression of p15O in FD-Fms cells results in strong inhibition of cell growth in M-CSF and a lesser inhibition in IL-3. Ectopic expression of p150 in fibroblasts does not inhibit growth. This novel protein, p150(ship) (SH2-containing inositol phosphatase), identifies a component of a new growth factor-receptor signaling pathway in hematopoietic cells.

Shc, Grb2, Sos1, and a 150-kilodalton tyrosine-phosphorylated protein form complexes with Fms in hematopoietic cells.

Fms, the macrophage colony-stimulating factor (M-CSF) receptor, is normally expressed in myeloid cells and initiates signals for both growth and development along the monocyte/macrophage lineage. We have examined Fms signal transduction pathways in the murine myeloid progenitor cell line FDC-P1. M-CSF stimulation of FDC-P1 cells expressing exogenous Fms resulted in tyrosine phosphorylation of a variety of cellular proteins in addition to Fms. M-CSF stimulation also resulted in Fms association with two of these tyrosine-phosphorylated proteins, one of which was identified as the 55-kDa Shc, which is shown in other systems to be involved in growth stimulation, and the other was a previously uncharacterized 150-kDa protein (p150). Fms also formed complexes with Grb2 and Sos1, and neither contained phosphotyrosine. Whereas both Grb2 and Sos1 complexed with Fms only after M-CSF stimulation, the amount of Sos1 complexed with Grb2 was not M-CSF dependent. Shc coimmunoprecipitated Sos1, Grb2, and tyrosine-phosphorylated p150, while Grb2 immunoprecipitates contained mainly phosphorylated p150, Fms, Shc, and Sos1. Shc interacted with tyrosine-phosphorylated p150 via its SH2 domain, and the Grb2 SH2 domain likewise bound tyrosine-phosphorylated Fms and p150. Analysis of Fms mutated at each of four tyrosine autophosphorylation sites indicated that none of these sites dramatically affected p150 phosphorylation or its association with Shc and Grb2. M-CSF stimulation of fibroblast cell lines expressing exogenous murine Fms did not phosphorylate p150, and this protein was not detected either in cell lysates or in Grb2 or Shc immunoprecipitates. The p150 protein is not related to known signal transduction molecules and may be myeloid cell specific. These results suggest that M-CSF stimulation of myeloid cells could activate Ras through the nucleotide exchange factor Sos1 by Grb2 binding to either Fms, Shc, or p150 and that Fms signal transduction in myeloid cells differs from that in fibroblasts.

Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain.

The c-fms gene encodes the receptor for the macrophage colony-stimulating factor (M-CSF), and its extracellular domain consists of five immunoglobulin-like subdomains. To identify which of the five immunoglobulin-like regions are involved in ligand binding, we polymerase chain reaction-cloned five segments of the extracellular domain of the murine c-fms gene, each starting with the normal initiation codon and containing successive additions of the immunoglobulin-like subdomains. These protein segments are designated A, B, C, D, and E and contain, from the N-terminal end, either one, two, three, four, or all five immunoglobulin-like subdomains, respectively. Each segment was expressed as a secreted soluble protein from a baculovirus expression vector in Sf9 insect cells. In addition, segments A, B, C, and E were produced as soluble alkaline phosphatase fusion proteins, as was a segment containing only the fourth and fifth immunoglobulin domains. These segments of the Fms extracellular domain were used to assess M-CSF binding by competition radioimmunoassays, plate binding immunoassays, and immunoprecipitation analyses. The results indicated that the first two N-terminal immunoglobulin-like domains did not interact with M-CSF but, in combination with the third immunoglobulin-like domain, provided high-affinity M-CSF binding. The fourth and fifth immunoglobulin-like domains near the cell membrane did not exhibit M-CSF binding and may inhibit interaction of M-CSF with the first three immunoglobulin domains. These results suggest that the three N-terminal immunoglobulin-like domains constitute the high-affinity M-CSF binding region and that the fourth and fifth immunoglobulin-like domains may perform functions other than ligand binding.

Site-directed mutagenesis of glycosylation sites in the transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 (414) precursors and of cysteine residues within mature TGF beta 1: effects on secretion and bioactivity.

The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.

Characterization of latent transforming growth factor-beta 2 from monkey kidney cells.

Serum-free medium conditioned by BSC-40 cells was analyzed for the presence of transforming growth factor-beta 2 (TGF beta 2)-related proteins. Western blot analysis was performed using site-specific antipeptide antibodies directed against the pro- and mature regions of the TGF beta 2 precursor. When conditioned medium was analyzed by polyacrylamide gel electrophoresis under reducing conditions, proteins with mol wt of 53 kDa (containing both mature and proregion sequences), 34-38 kDa (containing proregion sequences only), and 12 kDa (containing mature sequences) were detected. Under nonreducing conditions, complexes of 60- to 80-kDa, 160- to 200-kDa, as well as 24-kDa mature dimers were seen. Cleavage of mature TGF beta 2 from its precursor was inhibited by monensin and chloroquin, but not by ammonium chloride or methylamine. Two peaks of bioactivity were detected after fractionation on a TSK column corresponding to mol wt of 130 and 400 kDa. These peaks contained TGF beta 2 and pro-TGF beta 2 proteins. Partial purification of the 130-kDa complex followed by N-glyconase digestion indicated that the pro-TGF beta 2 proteins were glycosylated. These data demonstrate that BSC-40 cells secrete mature TGF beta 2 complexed with proregion-containing proteins and suggest that this association may contribute to the latency phenomena observed with respect to this growth regulator.

Expression of recombinant TGF-beta 2(442) precursor and detection in BSC-40 cells.

Analysis of cDNA clones encoding transforming growth factor (TGF)-beta 2 predicts two different precursor proteins derived by alternative mRNA splicing; a 414 amino acid precursor [TGF-beta 2(414)] and a 442 amino acid precursor [TGF-beta 2(442)]. The two proteins differ by a 28 amino acid insertion within the pro-region of TGF-beta 2(442). In order to characterize the TGF-beta 2-related proteins encoded by the TGF-beta 2(442) cDNA and determine whether it could, in fact, direct the synthesis of active growth factor, we have expressed this gene in Chinese hamster ovary (CHO) cells and, after amplification with methotrexate, obtained stable clones secreting TGF-beta 2(442). The TGF-beta 2 secreted by these cells was latent as acidification was necessary to detect optimal biological activity. In addition to mature TGF-beta 2, high molecular weight pro-region containing proteins were also secreted as analyzed by immunoblotting using site-specific anti-peptide antibodies. These proteins migrated differently than those secreted by CHO cells transfected with cDNA encoding TGF-beta 2(414), indicating that structural differences exist between the two complexes. An anti-peptide antiserum was produced in rabbits against the 28 amino acid insert region of TGF-beta 2(442). This sera was then used to detect the presence of TGF-beta 2(442) in serum-free media conditioned by BSC-40 cells. Since the TGF-beta 2(442) precursor is produced and secreted by a non-recombinant cell line, this suggests that it may play a physiological role in regulating the activity of TGF-beta 2.

Characterization of latent recombinant TGF-beta 2 produced by Chinese hamster ovary cells.

Latent recombinant transforming growth factor-beta 2 (LrTGF-beta 2) complex has been purified from serum-free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF-beta 2 (414) precursor. Under neutral conditions, LrTGF-beta 2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro-region sequences. Acidification of LrTGF-beta 2 resulted in the release of mature 24 kDa TGF-beta 2 from the high molecular weight pro-region-containing complex, suggesting that TGF-beta 2 was non-covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF-beta 2. Protein sequence analysis of the purified TGF-beta 2 pro-region indicated that signal peptide cleavage occurred between ser(20) and leu(21). The pro-region, which previously was found to contain mannose-6-phosphate, bound to the mannose-6-phosphate receptor. Proteolytic cleavage of mature TGF-beta 2 from pro-TGF-beta 2 was inhibited by monensin and chloroquine suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF-beta 2 precursor.

High-level expression of TGF-beta 2 and the TGF-beta 2(414) precursor in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) clones secreting high levels of transforming growth factor-beta 2 (TGF-beta 2) were obtained after transfection with a cDNA clone coding for the 414-amino acid TGF-beta 2 precursor and subsequent amplification with methotrexate. The TGF-beta 2 was secreted in a latent form since acidification was necessary for detection of maximal levels of bioactivity. Amino- and carboxy-terminal sequencing of purified recombinant TGF-beta 2 indicated that correct processing of mature TGF-beta 2 had occurred. In addition to mature TGF-beta 2, the recombinant CHO clones secreted larger proteins having molecular weights of 85, 105, and 130 kD, which consisted of both mature and pro-region sequences when analyzed by immunoblotting using site-specific anti-peptide antibodies. Analysis of serum- and cell-free media from recombinant CHO cells metabolically labeled with [3H]glucosamine and [32P]orthophosphate indicated that pro-TGF-beta 2 was glycosylated and phosphorylated. Two-dimensional electrophoretic analysis of acid hydrolysates showed that the 32P was incorporated into mannose-6-phosphate.

Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins.

Three cysteine residues are located in the pro region of the transforming growth factor beta 1 (TGF-beta 1) precursor at amino acid positions 33, 223, and 225. Previous studies (Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988) Mol. Cell. Biol. 8, 4162-4168) with purified recombinant TGF-beta 1 (rTGF-beta 1) precursor produced by Chinese hamster ovary (CHO) cells revealed that Cys-33 can form a disulfide bond with at least 1 cysteine residue in mature TGF-beta 1, contributing to the formation of a 90-110-kDa protein. We now show that Cys-223 and Cys-225 form interchain disulfide bonds. Site-directed mutagenesis was used to change these Cys codons to Ser codons, and mutant constructs were transfected into COS cells. Analysis of recombinant proteins by immunoblotting showed that by substituting Cys-33 the 90-110-kDa protein is not formed, and thus, more mature dimer (24 kDa) is obtained, corresponding to a 3- to 5-fold increase in biological activity. Substitution of Cys-223 and/or Cys-225 resulted in near wild-type levels of mature TGF-beta 1. Furthermore, cells transfected with plasmid coding for Ser at positions 223 and 225 expressed only monomeric precursor proteins and released bioactive TGF-beta 1 that did not require acid activation, suggesting that dimerization of the precursor pro region may be necessary for latency.

Interactions of recombinant and platelet transforming growth factor-beta 1 precursor with the insulin-like growth factor II/mannose 6-phosphate receptor.

Recombinant transforming growth factor (TGF)-beta 1 precursor was recently found to contain mannose 6-phosphate (Purchio et al., 1988, J. Biol. Chem. 263, 14211-14215). In the present study, recombinant TGF-beta 1 precursor was shown to bind to the insulin-like growth factor (IGF)-II/mannose 6-phosphate (man6P) receptor on the plasma membrane of cells since: 1) Insulin, which induces an increase in cell surface IGF-II/man6P receptors on adipocytes, caused a 2.7-fold increase in TGF-beta 1 precursor binding to adipocytes; 2) Chinese hamster ovary cells selected for overexpression of the IGF-II/man6P receptor exhibited an increased binding of TGF-beta 1 precursor in comparison to the parental cells; and 3) the binding of 125I-TGF-beta 1 precursor to these transfected cells and adipocytes was largely inhibited by man6P. After 15 minutes at 37 degrees C, 75% of the recombinant TGF-beta 1 precursor was found to be internalized in the transfected cells. Additional studies with latent TGF-beta 1 isolated from platelets indicated that this material could also bind to the isolated IGF-II/man6P receptor.

Expression and characterization of recombinant TGF-beta 2 proteins produced in mammalian cells.

Recombinant DNA plasmids coding for transforming growth factor beta 2 (TGF-beta 2) precursor and a hybrid TGF-beta 1(NH2)/beta 2(COOH) molecule consisting of the amino-terminal precursor portion of transforming growth factor-beta 1 (TGF-beta 1) linked in phase to the carboxyl terminus of mature TGF-beta 2 were constructed and transfected into COS cells. Both plasmids directed the synthesis of active TGF-beta 2 which was secreted into the supernatants of transfected cells. The TGF-beta 2 was secreted in a latent form, as an acidification step was required to demonstrate optimal biological activity. Using site-specific anti-peptide antibodies, we show that precursor and mature forms of TGF-beta 2 are produced. A stable Chinese hamster ovary (CHO) cell line expressing the hybrid TGF-beta 1(NH2)/beta 2(COOH) protein was isolated. This cell line secreted both precursor and mature forms of TGF-beta 1(NH2)/beta 2(COOH); acidification was required to demonstrate biological activity. Protein sequence analysis of recombinant TGF-beta 2 produced by this CHO clone demonstrated that correct proteolytic cleavage had occurred, suggesting that the processing signals contained within the TGF-beta 1 amino portion can function in producing mature TGF-beta 2. Receptor binding studies showed that TGF-beta 2 specifically bound predominantly to type III receptors on the surface of human palatal mesenchymal cells. The availability of active TGF-beta 2 should aid in determining its potential therapeutic use as a growth modulator.

Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-beta 1 precursor.

Recombinant transforming growth factor-beta 1 (TGF-beta 1) precursor produced and secreted by a clone of Chinese hamster ovary cells was found to be glycosylated and phosphorylated. Treatment of 32P-labeled precursor protein with N-glycanase indicated that phosphate was incorporated into asparagine-linked complex carbohydrate moieties. Fractionation of 32P-labeled glycopeptides followed by amino acid sequence analysis indicated that greater than 95% of the label was incorporated into two out of three glycosylation sites at Asn-82 and Asn-136 of the TGF-beta 1 precursor. Two-dimensional electrophoretic analysis of acid hydrolyzed precursor protein and precursor protein-derived glycopeptides indicated that 32P was incorporated as mannose 6-phosphate. Binding studies with the purified receptor for mannose 6-phosphate indicated that the TGF-beta 1 precursor could bind to this receptor and the binding was specifically inhibited with mannose 6-phosphate.

Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide.

Recently, the simian type 1 transforming growth factor beta (TGF-beta 1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene amplification (L.E. Gentry, N.R. Webb, G.J. Lim, A.M. Brunner, J.E. Ranchalis, D.R. Twardzik, M.N. Lioubin, H. Marquardt, and A.F. Purchio, Mol. Cell. Biol. 7:3418-3427, 1987). We have now purified and characterized the recombinant proteins released by these cells. Analyses of the precursor proteins by amino acid sequencing identified potentially important proteolytic processing sites. Signal peptide cleavage occurs at the Gly-29-Leu-30 peptide bond of pre-pro-TGF-beta 1, yielding pro-TGF-beta 1 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-beta 1 occurs at the dibasic cleavage site immediately preceding Ala-279, indicating that CHO cells possess the appropriate processing enzyme. Greater than 95% of the biological activity detected in the conditioned medium of the CHO transfectant was due to mature, properly processed growth factor. Highly purified recombinant TGF-beta 1 had the same specific biological activity as natural TGF-beta 1. The concentration of TGF-beta 1 required for half-maximal inhibition of Mv1Lu mink lung epithelial cell growth was approximately 1 to 2 pM. Purified precursor inhibited mink lung cell proliferation at 50 to 60 pM concentrations. The purified precursor preparation was shown to consist of pro-TGF-beta 1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-beta 1 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-beta1 should prove useful for further structural and functional studies.

Purification and characterization of cytostatic lymphokines produced by activated human T lymphocytes. Synergistic antiproliferative activity of transforming growth factor beta 1, interferon-gamma, and oncostatin M for human melanoma cells.

Supernatants from activated human T lymphocytes were highly growth inhibitory for A375 human melanoma cells. Three growth inhibiting polypeptides, transforming growth factor beta 1 (TGF-beta 1), interferon-gamma (IFN-gamma), and oncostatin M, were isolated from the acid-soluble fraction of serum-free T cell-conditioned medium and purified by gel permeation chromatography and reverse-phase high performance liquid chromatography in volatile solvents at acid pH. The purification was monitored in a growth inhibition assay. The release of TGF-beta 1 biologic activity by and the purification of IFN-gamma from the medium of activated human peripheral blood T lymphocytes have been reported. We now describe the isolation of oncostatin M from the conditioned medium of activated human T cells. The concentration of oncostatin M required for half-maximal inhibition of A375 melanoma cells was approximately 4 pM when assayed in the presence of 10% fetal bovine serum. The purified oncostatin M had an apparent m.w. 28,000 and an amino-terminal sequence that was identical with the sequence of oncostatin M isolated from supernatants of macrophage-like cells. Suboptimal concentrations of TGF-beta 1 in combination with suboptimal concentrations of IFN-gamma or oncostatin M resulted in synergistic antiproliferative responses for A375 cells (1.9 and 3.1 times the expected additive responses, respectively). Combinations of oncostatin M and IFN-gamma added simultaneously to A375 cells caused an additive growth inhibitory response. These results demonstrate that oncostatin M is a novel lymphokine, and its interaction with other cytostatic polypeptide growth inhibitors may play a role in the immune regulation of tumor cell growth.