Cell, Vol. 56, 691-700, February 24, 1989, Copyright 0 1989 by Cell Press

Transmembrane TGF-a Precursors Activate EGFITGF-a Receptors

Rainer Brachmann, Patricia B. Lindquist, Mariko Nagashima, William Kohr, Terry Lipari, Mary Napier, and Rik Derynck Department of Developmental Biology and Department of Medicinal and Biomolecular Chemistry Genentech, Inc. 460 Point San Bruno Boulevard South San Francisco, California 94080

Summary

TGF-a and EGF are structurally related factors that bind to and induce tyrosine autophosphorylation of a com- mon receptor. Proteolytic cleavage of the transmem- brane TGFa precursor’s external domain releases sev- eral TGF-a species. However, membrane-bound TGF-a forms remain on the surface of TGF-a-expressing cell lines. To evaluate the biological activity of these forms, we modified two cleavage sites in the TGF-a precur- sor coding sequence, making processing into the 50 amino acid TGF-a impossible. Overexpression of this cDNA in a receptor-negative cell line, partial purifica- tion, and N-terminal sequence analysis indicate the ex- istence of two transmembrane TGF-a forms. These solubilized precursors induce tyrosine autophosphory- lation of the EGF/TGFa receptor in intact receptor- ovemxpmssing cells, and anchorage-independent growth of NRK fibroblasts. Ceil-cell contact between TGF-a precursor-overexpressing cells and cells expressing high numbers of receptors also resulted in receptor activation. These findings suggest a role for trans- membrane TGF-a forms in intercellular interactions in proliferating tissues.

Introduction

Transforming growth factor-a (TGF-a) is a secreted poly- peptide that interacts with the same receptor as epidermal growth factor (EGF) and induces a mitogenic response in a variety of cells (for review see Derynck, 1988). While TGF-a was first identified in the medium of several solid- tumor cells (De Larco and Todaro, 1978), it has more re- cently been established that normal cell types also syn- thesize TGF-a. An abundant cell source for TGF-a is the skin keratinocytes. We have previously shown that TGF-a mRNA and protein can be detected in the skin and that TGF-a can induce its own synthesis (Coffey et al., 1987). In addition, activation of the protein kinase C pathway results in a strong induction of TGF-a synthesis in these cells (Pittelkow et al., 1989), and the proliferative keratino- cytes in psoriatic skin have significantly enhanced levels of TGF-a expression (Elder et al., 1989). These results and the mitogenic effect of TGF-a on keratinocytes (Barrandon and Green, 1987) indicate that TGF-a has to be considered a normal physiological ligand for EGF receptors in the skin. Furthermore, TGF-a synthesis has also been de-

tected in other types of epithelial cells (Valverius et al., 1989) and in the brain (Wilcox and Derynck, 1988a), pitu- itary (Kobrin et al., 1986), activated macrophages (Madtes et al., 1988) and various embryonic tissues (Lee et al., 1985a; Wilcox and Derynck, 1988b), implying a more general importance of TGF-a in various processes.

cDNA analysis of TGF-a suggested that the 50 amino acid, fully processed form of TGF-a is synthesized as an internal part of a 160 amino acid precursor (Derynck et al., 1984; Lee et al., 1985b). This precursor contains an ex- tracellular domain of about 100 amino acids that includes the N-terminal signal sequence and the 50 amino acid TGF-a, a hydrophobic transmembrane domain, and a 35 residue cytoplasmic domain. A similar transmembrane character has also been proposed for the related vaccinia virus growth factor (VVGF) precursor (Stroobant et al., 1985) and the EGF precursor (Gray et al., 1983; Scott et al., 1983). In the latter case, the extracellular domain is much longer and could function as a receptor for an un- known ligand (Pfeffer and Ullrich, 1985). Experimental evidence indicates that the TGF-a precursor is indeed syn- thesized as a transmembrane molecule and that the fre- quently encountered size heterogeneity of the TGF-a spe- cies is due to differential proteolytic cleavages in the external precursor domain and to N- and 0-glycosylation of the larger forms (Bringman et al., 1987; Gentry et al., 1987; Teixido et al., 1987). The C-terminal cytoplasmic segment is rich in cysteines and undergoes covalent at- tachment of palmitate at one or more cysteines. Pulse- chase experiments also indicated that at least part of the proteolytic processing of TGF-a precursor takes place in the Golgi apparatus or in the secretory vesicles (Bringman et al., 1987).

The previous findings raised several new questions, which led to the work reported here. First, we evaluated the effectiveness of the proteolytic processing of TGF-a and whether it is possible to detect the TGF-a precursor on the cell surface. We were able to visualize by immuno- fluorescence that TGF-a was present at the cell surface not only of recombinant cell lines overproducing TGF-a, but also of tumor cell lines expressing the endogenous TGF-a gene. This led us to examine whether the trans- membrane form of TGF-a is able to interact with the EGF/TGF-a receptor. We therefore mutated the proteolytic cleavage sites downstream of the 50 amino acid TGF-a in the precursor, and overexpressed this modified precursor sequence in EGF/TGF-a receptor-deficient Chinese ham- ster ovary (CHO) cells. An enriched preparation of solubi- lized TGF-a precursors induced tyrosine autophosphory- lation of the EGFITGF-a receptors in intact cells and enabled NRK fibroblasts to form colonies in soft agar in the presence of TGF-8. Cell contact between the TGF-a precursor-overexpressing cells and cells expressing high numbers of EGF/TGF-a receptors indicated that cell-cell contact was sufficient to induce the tyrosine autophos- phorylation of the receptor. These data indicate that membrane-bound TGF-a can activate the EGFITGF-a

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Figure 1. Indirect lmmunofluorescence with Anti-TGF-a Antibody TGF-al

(a) C5-4-1 cells overexpressing and secreting TGF-a were incubated with the monoclonal anti-TGF-a antibody TGF-al and rhodamine-conjugated anti-mouse IgG goat antibody at 0%. The specific staining on the cell surface indicated the presence of TGF-a transmembrane forms. (b) Staining of C5-4-I cells at 37“C showed internalization of the membrane TGF-a-antibody complexes. The perinuclear inclusions probably cor- respond to lysosomes. (c, d) Indirect immunofluorescence of the fibrosarcoma cell line HT1080 (c) and the renal carcinoma cell line 7860 (d) at 37X, after the TGF-a precur- sor-antibody complexes had been internalized

receptor and thus that the transmembrane form of TGF-a is biologically active.

Results

lmmunofluorescent Detection of Membrane-Bound TGF-a We have previously described the introduction of a mam- malian TGF-a expression vector into CHO cells that are deficient in dihydrofolate reductase expression and lack functional EGF receptors (Bringman et al., 1987). One of the transformants, C5-4-1 Amp804 had undergone se- quential amplification of the integrated plasmid sequences, which resulted in the secretion of relatively high levels of TGF-a. These cells were stained by indirect immunofluo- rescence using the murine monoclonal antibody TGF-al, raised against the correctly folded 50 amino acid form of human TGF-a (Bringman et al., 1987), and a second rhodamine-coupled antibody. Live cells kept on ice showed an even staining over the surface, which was more intense at the edges (Figure la), indicating the existence of TGF-a forms at the cell surface. This characteristic staining was not seen when the C5-4-1 Amp800 cells were incubated

with an unrelated control monoclonal antibody, nor with the antibody TGF-al preincubated with an excess of TGF- a (data not shown). Parental CHO cells incubated with TGF-a antibody were also immunofluorescence negative (data not shown). This staining is also not due to binding of TGF-a to the receptors, since CHO cells lack EGF/TGF- a receptors (Livneh et al., 1986; Clark et al., 1988) and since binding of TGF-a to the receptors on Rat-l cells did not result in immunofluorescent staining (data not shown). The specificity of the antibody for a membrane form of TGF-a was also confirmed by comparing the 1251-labeled TGF-al antibody and a control monoclonal antibody against the herpes gD protein (data not shown).

Incubation of live cells with TGF-al antibody on ice, fol- lowed by the rhodamine-conjugated antibody incubation at 37% for 1 hr, resulted in fluorescence that was not ho- mogeneous on the surface but was concentrated in circu- lar inclusions, many of them surrounding the nucleus (Fig- ure lb). This suggested that the fluorescent antibody complexes were internalized in endosomes and in lyso- somes. Lysosomal staining of the cells with acridine or- ange revealed a similar staining pattern of perinuclear inclusions (data not shown), but double staining with acri-

Transmembrane TGF-a Is Biologically Active 693

90 GTG - CTG

Figure 2. Site-Directed Mutagenesis of Two Cleavage Sites of the TGF-a Precursor

The arrows indicate the cleavage sites. The two proteolytic cleavage sites between the C-terminus of the 50 amino acid TGF-a and the cell membrane were changed by site-directed mutagenesis to prevent the release of fully processed TGF-a. The cleavage site at the N-terminus of the 50 amino acid TGF-a remained functional. N-glycosylation takes place onto the asparagine residue of the NST triplet.

dine orange and with the rhodamine-based double anti- body immunofluorescence to verify this localization was technically not possible. Analysis of the time course using 1251-labeled TGF-al antibody substantiated the gradual internalization of the antigen-antibody complexes and showed a subsequent and slower release of the 12? label into the medium (data not shown). This internalization and the possible lysosomal localization of the antigen-anti- body complexes have been seen with various membrane proteins and may be characteristic for this class of mole- cules.

As shown before (Bringman et al., 1987), the transmem- brane form of TGF-a could not be immunoprecipitated with our antibodies, probably due to a lack of recognition under the conditions used. The immunofluorescence pat- tern using the 37% incubation was considerably more sensitive than the surface staining on ice without internali- zation. We therefore chose this method to evaluate the staining of tumor cells that have endogenous TGF-a ex- pression as measured by Northern blot analysis (Derynck et al., 1987). Figures lc and Id show the staining of the fibrosarcoma cell line HT1080 and the renal carcinoma cell line 7860. Both cells show the punctate immunofluo- rescence seen with the overproducing cell line (X-4-1 AmpBOO, albeit at a much lower intensity. Control experi- ments indicate that this staining is not due to TGF-a inter- acting with its receptor, presumably since ligand-receptor

interaction makes the TGF-a epitope inaccessible to the antibody. These data thus indicate the presence of TGF-a on the surface of these cells.

Cell Lines Overexpressing Uncleaved TGF-a Precursors Two potential cleavage sites are located downstream of the 50 amino acid TGF-a unit (Derynck et al., 1984; Bring- man et al., 1987). One cleavage takes place between Ala and Val-Val immediately following the C-terminus of the fully processed form of TGF-a, while the other cleavage site is located 8 amino acids downstream following the di- basic Lys-Lys residues. It is likely that these two different cleavages needed for the release of the different TGF-a species are caused by two different proteases. To inves- tigate whether the uncleaved TGF-a precursor can inter- act with the EGF/TGF-a receptor, we changed these cleav- age sites so that the normal release of active TGF-a could not take place. Using site-directed mutagenesis, the co- dons for Ala-Val-Val were mutated to Ile-Leu-Leu, while the Lys-Lys codons were converted to Arg-lle. The proteolytic cleavage sequence at the N-terminus of the 50 amino acid TGF-a was not modified (Figure 2).

The resulting coding sequence for the modified TGF-a precursor was then introduced under the control of the SV40 early promoter, yielding a plasmid identical to pMTE4 (Rosenthal et al., 1986) except for the mutated codons. This plasmid was transfected into CHO dhfr- cells. The immunofluorescent staining patterns with the TGF-a antibody at the cell surface at 0°C and 37°C were indistinguishable from the patterns described above for C5-4-1 Amp800 (data not shown). One of the transfor- mants, 813-4-1, underwent sequential amplification of the integrated plasmid sequences with increasing levels of methotrexate up to 1000 nM and was then enriched for the highest expression levels using fluorescence-activated cell sorting (FACS). The relative expression levels were monitored by FACS analysis using fluorescein-conjugated TGF-al. The expression levels of the transmembrane TGF-a precursors could not be quantitated in our ELISA; therefore they were determined by FACS analysis in con- junction with the F/P ratio for the antibody used (see Ex- perimental Procedures). The number of TGF-a precursors of the highest-producing cells was calculated in this way to be 235,000 per cell. ELISA determination (Bringman et al., 1987) showed that these cells did not release any de- tectable TGF-a, i.e., less than 0.2 nglml, into the medium.

Preparation and Characterization of Solubilized TGF-a Precursors The ability of the transmembrane form of TGF-a to activate the EGF/TGF-a receptor and its biological activity were evaluated with partially purified, solubilized TGF-a precur- sors. The cells producing the modified transmembrane forms of TGF-a were lysed in detergent, and the solubi- lized proteins were purified over an immunoaffinity matrix containing the immobilized monoclonal antibody TGF-al. Gel electrophoresis revealed that the eluted fractions were considerably enriched for two prominent bands at approximately 23 and 15 kd (Figure 3). Glycoprotein analy-

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A 42.7-

21.5-

14.4-

- LEXSXXPL

-) VVSHXNDX

a b Figure 3. Glycoprotein Staining and N-Terminal Sequence Analysis of the Solubilized TGF-a Precursor Preparation

Two aliquots of the TGF-a precursor preparation were run on SDS- polyacrylamide gels. One lane was stained with Ccomassie blue (a), and the other was glycoprotein fluorescent labeled with dansylhydra- zine (b). N-terminal sequences revealed that the TGF-a precursor prep- aration contained two forms of transmembrane TGF-a: one larger, glycosylated form with the signal peptide cleaved off, and one smaller form, which started with the N-terminus of the 50 amino acid TGF-a.

sis using fluorescent labeling with dansylhydrazine indi- cated that the larger one was glycosylated and the smaller one was not (Figure 3). N-terminal sequence determina- tion of the TGF-a precursor preparation bound to a re- verse-phase Cl8 microsequencing column and of the individual protein bands transferred to polyvinylidene difluoride (PVDF) membranes indicated that these bands corresponded to TGF-a precursors (Figure 3). The larger species represented the glycosylated precursor without the signal peptide, thus establishing the cleavage site for the signal peptide. The smaller protein corresponded to the nonglycosylated precursor cleaved at the N-terminus of the 50 amino acid TGF-a unit. Since we could not rely on the receptor binding assay nor on the TGF-a ELISA for quantitation of the TGF-a precursor species, we measured the concentration of these proteins on the basis of their amino acid compositions. To rule out the possibility that the 50 amino acid form of TGF-a was responsible for any demonstrated activities of TGF-a precursors, we checked the precursor preparation in a Western blot. Analysis of an overloaded gel did not detect any smaller forms of TGF-a, indicating that a preparation of 1425 pmol of TGF-a precur- sors contained less than 1.8 pmol of fully processed TGF-a (data not shown).

The Transmembrane TGF-a Precursors Activate EGF/TGF-a Receptors The biological activity of the transmembrane TGF-a forms was assessed by examining the induction of the tyrosine autophosphorylation of the receptors. Cells overexpress-

C

1 2 3 4 5 Figure 4. Induction of Tyrosine Autophosphorylation of EGFfTGF-a Receptors by Solubilized Transmembrane TGF-a Precursors

A431 and HERc cells were incubated for different times with different concentrations of solubilized TGF-a precursors. After cell lysis EGF receptors were immunoprecipitated with the monoclonal antibody 108.1. Proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose. Tyrosine phosphorylation was detected with the anti-phosphotyrosine antibody 5E2 and 1251-labeled protein A. (A) A431 cells (2 x 10s) 5 min. Lane 1, negative control; lane 2, 36 pmollml (200 nglml) TGF-a; lane 3, 0.95 nmollml TGF-a precursors; lane 4, 1.52 nmollml TGF-a precursors; lane 5, 2.66 nmollml TGF-a precursors. (B) HERc cells (1 x 106), 5 min. Lane 1, negative control; lane 2, 36 pmollml (200 nglml) TGF-a; lane 3, 0.38 nmollml TGF-a precursors; lane 4, 1.52 nmollml TGF-a precursors; lane 5. 2.66 nmol/ml TGF-a precursors. (C) A431 cells (2 x 10s). 15 min. Lane 1. negative control; lane 2, 36 pmollml (200 nglml) TGF-a; lane 3, 0.95 nmollml TGF-a precursors; lane 4. 1.52 nmollml TGF-a precursors; lane 5, 3.8 nmollml TGF-a precursors.

ing the EGF/TGF-a receptors were incubated with various concentrations of the solubilized TGF-a precursor prepa- ration. Either A431 carcinoma cells or NIH 3T3 fibroblasts transfected with an EGF receptor cDNA expression plas- mid (HERc cells; Lee et al., 1989) were used as target cells. The treated cells in monolayer were lysed and the EGF/TGF-a receptors were immunoprecipitated with the monoclonal antibody 108.1, directed against the extracel- lular domain of the EGF receptor (Honegger et al., 1987). Tyrosine phosphorylation was then visualized after West- ern blot analysis using monoclonal anti-phosphotyrosine antibody 5E2 (B. Fendly, unpublished data).

After establishing a concentration curve and the time course for the 50 amino acid TGF-a with either cell line, the assays were performed with the quantitated TGF-a precursor preparation. In our assay conditions at least 5-10 nglml of the 50 amino acid TGF-a was required to in- duce a detectable level of tyrosine phosphorylation (data not shown). Exposure of the two cell types with increasing concentrations of the transmembrane forms of TGF-a for 5 min resulted in tyrosine phosphorylation of the receptors (Figure 4). The effect seen with 2.66 nmollml of the precur- sors was approximately equivalent to the effect of 36 pmol/ml (200 nglml) of fully processed TGFa. Exposure

Transmembrane TGF-a Is Biologically Active 695

Table 1. Formation of Colonies in Soft Agar by TGF-a Precursors in the Presence of TGF-6

Number of Colonies

Conditions Exp. 1 Exp. 2

No growth factors 1.8 pmollml (10 nglml) TGF-a 0.1 pmollml (2.5 ng/ml)TGF-8 1.8 pmollml TGF-a + 0.1 pmollml TGF-8 12 pmollml TGF-a precursor + 0.1 pmollml TGF-p 24 pmollml TGF-a precursor + 0.1 pmollml TGF-8 48 pmollml TGF-a precursor + 0.1 pmollml TGF-8 95 pmollml TGF-a precursor + 0.1 pmollml TGF-6 190 pmollml TGF-a precursor + 0.1 pmollml TGF-5 285 pmollml TGF-a precursor + 0.1 pmollml TGF-6 380 pmollml TGF-a precursor + 0.1 pmollml TGF-6

011 010 o/o

1731163 o/o 113 414

38141 132030 297/241

010 o/o o/o

2301232 o/o 011 5110

24137 2041153 339/426 3901447

Duplicate values are shown for each experiment. Only the colonies that consisted of at least 30 cells were counted

of A431 cells for 15 min (Figure 4C) showed an approxi- mately equal level of tyrosine phosphorylation with 3.8 nmollml TGF-a precursors. These data indicate that the solubilized TGF-a precursors can activate the EGFITGF-a receptors in intact live cells, as assessed by tyrosine auto- phosphorylation. Yet, quantitation data suggest that the transmembrane forms are approximately 50- to lOO-fold less active than the 50 amino acid form of TGF-a.

The activity of the transmembrane TGF-a forms was fur- ther evaluated by the ability to induce anchorage indepen- dence of NRK cells in the presence of TGF-f3. Soft-agar colony formation of these cells can be equally induced by the fully processed forms of both EGF and TGF-a (Mas- sag& 1983; Derynck et al., 1984) and was one of the original assays leading to the discovery of TGF-a (De Larco and Todaro, 1978). As shown in Table 1, the prepara- tion of transmembrane TGF-a forms was able to induce anchorage independence in this assay in a dose-depend- ent way. Comparison with the dose response for the 50

2min IOmin 15min r-----i - + .- + -

A -‘-I.-

2 min 5 min 10min I- -r-I- + - + - + 20min - +

Figure 5. Induction of Tyrosine Autophosphorylation of EGF/TGF-a Receptors by Intact Recombinant Cells Overexpressing Transmem- brane TGF-a Precursors

HERc cells (1 x 10s) were incubated for different times with 5 x 10s (A) or 10 x lo6 (B) cells of a control CHO cell line transfected with the dihydrofolate reductase expression plasmid alone (lanes -) or of a CHO cell line overexpressing transmembrane TGF-a precursors (lanes

+)-

amino acid TGF-a reveals that, in this assay as well, the preparation of solubilized precursors is about lOO-fold less active.

Cells Overexpressing the Transmembrane TGF-a Precursors Activate EGF Receptors We next wanted to demonstrate the activation of the EGF/TGF-a precursors in an assay using intact live cells. The assay system depended on the direct cell contact between two cell types, one expressing EGF receptors and the other expressing TGF-a precursors. The receptor- overexpressing HERc cells, which unlike the A431 cells do not synthesize TGF-a, were grown as a monolayer culture. A defined number of the TGF-a precursor-overproducing 613-4-l cells, which lack the EGF/TGF-a receptors, or cells transfected with a control plasmid were then, after re- moval of the conditioned medium, layered onto the HERc cells and incubated for various times. The tyrosine auto- phosphorylation of the receptors was evaluated as in the previous experiments. Similar to results in the above- mentioned experiments, the greatest stimulation of the tyrosine phosphorylation of receptors by TGF-a precur- sors was seen at early time points, ranging from 2-20 min, using 5 or 10 x lo6 transfected CHO cells (Figure 5). Control experiments using the TGF-a ELISA did not indi- cate the release of short forms of proteolytically processed TGF-a into the medium (data not shown). These data indi- cate that transmembrane TGF-a precursors on intact cells can activate receptors on neighboring cells via direct cell contact.

Discussion

A variety of transformed and normal cells express the TGF-a gene (Derynck, 1988). The precursor for TGF-a is a glycosylated transmembrane protein that is cleaved in its extracellular domain and gives rise to several secreted species of biologically active TGF-a (Bringman et al., 1987). We have previously determined that certain cell lines show relatively high levels of TGF-a mRNA yet do not secrete detectable amounts of TGF-a into the medium (Derynck et al., 1987). We therefore evaluated whether

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this discrepancy can be explained by membrane-bound TGF-a forms at the cell surface. Our immunofluorescence data using a specific monoclonal antibody indicate that membrane-bound forms of TGF-a are present on the sur- face of the TGF-a-producing cell lines examined and that this specific immunofluorescence is not due to interaction of secreted TGF-a with the cell surface. Internalization of the antibody-antigen complexes, which is characteristic for many membrane proteins, occurs at 37%. The more intense immunofluorescence pattern following internali- zation allowed the detection of membrane forms of TGF-a in tumor cells that naturally produce relatively low levels of TGF-a.

These findings led us to the question of whether the transmembrane TGF-a precursor can activate the EGF/ TGF-a receptor. We modified the two natural cleavage sites that result in the release of TGF-a, and expressed this precursor, which cannot undergo its natural proteo- lytic cleavage, in receptor-negative cells. The interaction of TGF-a precursors with the EGF/TGF-a receptor and the subsequent activation were assayed by tyrosine auto- phosphorylation of the receptor. Activation of the receptor tyrosine kinase, which follows ligand-receptor interaction and results in tyrosine autophosphorylation of the recep- tor, is generally considered the crucial step in inducing the multiple intracellular events leading to mitosis (Yarden and Ullrich, 1988; Schlessinger, 1988). This phosphoryla- tion is induced by the fully processed forms of EGF and TGF-a (Ushiro and Cohen, 1980; Downward et al., 1984; Reynolds et al., 1981).

In one approach we semi-purified the transmembrane forms of TGF-a. Sequence analysis confirmed the pres- ence of two forms of TGF-a precursors and established the signal peptide cleavage site, in contrast to a previous report suggesting that the signal peptide was not cleaved from the precursor (Teixid6 et al., 1987). Detergent-solubil- ized proteins form micelles, in which the hydrophobic seg- ments are embedded (Jones et al., 1987). Thus it can be assumed that a major fraction of transmembrane TGF-a forms are oriented with the hydrophobic region inside the micelles and the 50 amino acid sequence at the outside of the micelles, similar to their normal configuration at the cell surface. This is supported by their interaction with the TGF-al antibody used in the immunoaffinity chromatogra- phy. We show here that the detergent-solubilized trans- membrane TGF-a is able to interact with the receptors at the surface of intact cells and to induce the tyrosine auto- phosphorylation. In addition, the transmembrane TGF-a induces soft-agar colony formation of NRK cells in the presence of TGF-p, a biological activity common to the fully processed forms of EGF and TGF-a (Massagu6, 1983; Derynck et al., 1984).

We can therefore conclude that the transmembrane forms of TGF-a can be biologically active, although quanti- tation suggests that the solubilized transmembrane forms are 50-to lOO-fold less active than the fully processed 50 amino acid form. This lower activity of the solubilized TGF- a preparation could be artifactual and related to the deter- gent solubilization or the method of preparation. This could then result in a partial inactivation of these forms or

in a decrease of their affinity for the receptor. For example, a significant loss of affinity for the ligand has been ob- served for the EGFITGF-a receptor when the receptor is subjected to detergent solubilization (Greenfield et al., 1988). The described effects cannot be due to contamina- tion with smaller TGF-a forms, since they were not de- tected in the cell medium nor in the transmembrane form preparation.

In the other approach we incubated the TGF-a precur- sor-overproducing cells with EGFlTGF-a receptor-over- producing cells. These experiments indicate that, again, the transmembrane forms of TGF-a can productively inter- act with the receptor and induce subsequent tyrosine au- tophosphorylation. We do not know if in intact cells the transmembrane forms of TGF-a are also 50- to lOO-fold less active than the fully processed form, because of in- herent difficulties in assessing their specific activities in the cell contact experiments. At least 5-10 rig/ml of the 50 amino acid TGF-a is needed to obtain a detectable tyro- sine phosphorylation of the receptor in these assays. The modification of the proteolytic cleavage sites in the TGF-a precursor and the inability to detect proteolytically cleaved TGF-a species released into the medium make it unlike- ly that the tyrosine phosphorylation is due to secreted TGF-a.

It has been shown that the EGF receptor is internalized following binding of the ligand (Schlessinger, 1988). The functional activation of the receptor by the transmem- brane TGF-a forms suggests that the internalization may not be needed; however, on the basis of our data such in- teraction may be less efficient than with the secreted, 50 amino acid TGF-a. Alternatively, we want to stress that we cannot rule out that during cell-cell contact a proteolytic cleavage may take place, thus making internalization pos- sible, even though we have modified the natural cleavage sites and do not detect released TGF-a in the medium. It is indeed possible that a membrane-bound protease cleaves the TGF-a precursor during or following interac- tion with the receptor.

The data presented in this report thus indicate that the transmembrane form of TGF-a can functionally interact with the EGF/lGF-a receptor and consequently that re- lease of the growth factor into the medium is not needed in order to activate the receptor. Such interactions be- tween cells could conceivably play a role in various sys- tems in vivo, such as proliferation of skin keratinocytes and solid tumors in which many fibroblasts are inter- spersed with the transformed cells. It is thus possible that TGF-a-producing tumor cells transfer a mitogenic signal to the normal fibroblasts not only by release of the growth factor but also by cell-cell contact between the transmem- brane form of TGF-a and the corresponding receptor. These interactions could possibly also take place in an au- tocrine fashion, not only at the cell surface, but perhaps even in the secretory vesicles or the Golgi apparatus.

TGF-a is not the only known growth factor derived from a transmembrane precursor. All members of this structur- ally related family of growth factors, i.e., TGF-a (Derynck et al., 1984; Lee et al., 1985b), EGF (Gray et al., 1983; Scott et al., 1983). and VVGF (Stroobant et al., 1985), are

Transmembrane TGF-a Is Biologically Active 697

derived from larger precursors with structural features characteristic of transmembrane forms. The colony stim- ulating factor CSF-1 or M-CSF is also part of a precursor with transmembrane configuration (Fiettenmier et al., 1987; Rettenmier and Roussel, 1988). Finally, tumor ne- crosis factor, which in various systems can function as a growth factor, is made as a membrane protein that subse- quently undergoes cleavage (Kriegler et al., 1988). It is thus likely that all these transmembrane precursors are not mere inactive molecules from which the active growth factors are to be excised, but that they themselves can be considered as active growth factor units.

The interaction between these transmembrane growth factor precursors and their receptors is reminiscent of the interaction between cell adhesion molecules. In the latter class of proteins, there is interaction between two trans- membrane molecules. While only in few cases, some of the resulting changes in cellular physiology have been identified. It is likely that these cellular contacts constitute forms of communication that involve signal transmission in one or two directions. The interaction between a trans- membrane growth factor precursor and the receptor could also be considered as a form of intercellular communica- tion similar to the cell adhesion molecules and resulting in a modulation of cell proliferation. It is probable that only at a later stage in evolution did the introduction of specif- ic proteolytic cleavages occur to generate and release growth factors, which were less restricted and perhaps more versatile and potent in their growth modulation ac- tivities.

Note: The induction of receptor autophosphorylation in the cell contact experiments described above is followed after 20-30 min by a strong increase in c-fos mRNA. These results indicate that the activation of the receptors results in other intracellular responses, related to the mito- genie activity.

Experimental Procedures

Mammalian Cell Growth, Transfection, and Gene Amplification CHO cells deficient in the synthesis of dihydrofolate reductase (Urlaub and Chasin. 1980) were grown and transfected as previously described (Rosenthal et al., 1986). Amplification of the integrated plasmid se- quences was achieved by culturing the cells in increasing levels of methothrexate (from 100-1000 nM) in selective medium. A431 cells were grown in a composite medium containing 50% Ham’s F12 and 50% high-glucose DMEM (GIBCO) with 2 mM L-glutamine, 10 mM HEPES (pH 7.2), 100 U/ml penicillin G, 100 pglml streptomycin, and 5% fetal bovine serum (GIBCO). EGF receptor-overexpressing NIH 3T3 fibroblasts (HERc; Lee et al., 1989) were a gift from Dr. A. Ullrich and were grown in low-glucose DMEM (GIBCO) with 2 mM L-

glutamine, 100 U/ml penicillin G, 100 pg/ml streptomycin, and 10% fe- tal bovine serum. HT1080 and 7860 cells were grown in high-glucose DMEM with 2 mM L-glutamine, 100 U/ml penicillin G, 100 pglml strep- tomycin, and 5% fetal bovine serum. NRK 49F fibroblasts were grown in 50% low-glucose DMEM and 50% Ham’s F12.

Monoclonal Anti-TGF-a Antibody and TGF-a ELISA The monoclonal antibody TGF-al was raised against Escherichia coli-derived, 50 amino acid long human TGF-a with the correct disul- fide bridge formation. The characterization of this antibody is de- scribed in Bringman et al. (1987). The double-sandwich ELISA for TGF- CI uses the monoclonal antibody TGF-al and the polyclonal antiserum 34D and has a minimal detection level of 0.1-0.4 nglml (Bringman et al., 1987).

Indirect lmmunofluorescence CHO, HT1080. and 7860 cells were grown to confluence in double-well chamber slides (Lab Tek, Miles). The cultures were rinsed twice with PBS, 10% fetal bovine serum, then incubated at O°C for 1 hr in 0.2 ml of PBS, 10% fetal bovine serum, containing 20 bglml of the TGF-al monoclonal antibody or a control monoclonal antibody against human growth hormone. After two washes the cultures were incubated for 1 hr at 0°C or 3PC with a 1:30 dilution of rhodamine-conjugated anti- mouse IgG goat antibody (GAMrhodlgG; Cappel). The cells were washed twice again and then fixed for 3 min in 50% and subsequently 100% ice-cold methanol. Slides were air dried, the chambers were re- moved, and coverslips were mounted with Gelvatol (Monsanto). A Zeiss Photoscope Ill was used for fluorescence microscopy and pho- tomicrography.

Use of Q%Labeled Antibodies The purified monoclonal antibodies TGF-al and the anti-herpes gD were labeled with 12sl using chloramine T (Bringman et al., 1987). Transfected or control CHO cells were grown to confluence in 35 mm wells (6-well plates, Falcon). The cells were washed twice in PBS, 10% fetal bovine serum, then incubated on ice in the presence of 1251-TGF- al antibody or lz51-anti-herpes gD. The medium was then removed, and cells were washed four times with PBS, 10% fetal bovine serum. Subsequently they were incubated on ice or at 37°C. At several time points the amount of lz51 label at the cell surface, in the cell lysate, or released into the medium was determined. The surface-bound 1251-antibody was determined by washing the cells four times in PBS, 10% fetal bovine serum, followed by exposure to 0.5 ml of 0.2 M acetic acid, 0.5 M NaCl for 6 min at room temperature. A second rinse was performed with another 0.5 ml of acid solution. Acid-released 1251 was then counted and was indicative of the amount of surface-bound anti- body. The level of lz51-antibody in the cell lysate was determined after lysing the cells for 1 hr in 1 N NaOH (Haigler et al., 1980). The amount of degraded antibody released by the cells was determined by harvest- mg the medium and determining the amount of trichloroacetic acid-soluble material.

Plasmid Construct Encoding the Modified TGF-a Precursor A BamHI-Hindlll insert containing the sequence coding for the TGF-a precursor was isolated from the plasmid pMTE4ARV(Derynck, unpub- lished data) and subcloned into M13mp18. The mutated oligonucleo- tides incorporated the modified codons flanked at both sides by 12 nucleotide long sequences complementary to the single-stranded template. Oligonucleotide-based site-directed mutagenesis was done as described (Zoller and Smith, 1985) and verified by dideoxynucleo- tide sequencing (Smith, 1980). The mutated cDNA fragment was then inserted into the expression vector such that the expression vector was identical to the pMTE4E plasmid (Rosenthal et al., 1986) except for the mutated codons for the two proteolyiic cleavage sites.

FACS Analysis The murine monOclonal antibody TGF-al was conjugated with fluores- cein as follows. Five milligrams of the purified TGF-al antibody in 0.5 ml was dialyzed in SpectroPor 2 membrane against PBS overnight at 4OC and subsequently for 8 hr against 50 mM bicarbonate-buffered sa- line, 150 mM NaCl (pH 9.2). and then incubated overnight with 100 pglml FITC (Research Organics) in the same buffer. The conjugation reaction was stopped by dialysis against PBS for 12-24 hr with four buffer changes (Wofsy et al., 1980).

The expression levels of the CHO cells transfected with the expres- sion vector for the TGF-a precursor were analyzed on a Bedon Dickin- son FACS IV (Mountain View, CA). A minimum of 10,000 cells was mea- sured for each sample analyzed. FITC was excited with 200 mW of 488 nm light from a 5 W argon ion laser. The cells were incubated for 1 hr on ice with 20 Kg/ml FITC-labeled TGF-al in 200 ~1 PBS, 2% heat inac- tivated fetal bovine serum, then washed twice in PBS, 2% heat inacti- vated fetal bovine serum, and resuspended before analysis. Using a standard curve for the fluorescence intensity, established with the Quantitative Fluorescein Microbead Standards Kit (Flow Cytometry Standards Corporation), the mean fluorescence channel values of the samples were converted into equivalent soluble fluorescent mole- cules. These numbers were divided by the F/P ratio for the antibody used to give the final numbers of surface antigens per cell. The F/P

Cell 698

ratio had been determined with Simply Cellular Microbeads (Flow Cytometry Standards Corporation) according to the manufacturer’s directions and was 1.09 for the antibody used. This analysis led to the estimation that the amplified and sorted population of transfected B13- 4-1 contained 235,000 TGF-a precursors per cell. The preparative sort- ing of the upper 1% of the transfected and amplified population was done on the same FACS IV.

Preparation of Solubillzed TGF-a Precursor Thirteen milligrams of the purified antibody TGF-al was coupled to 5 ml of CNBr-activated Sepharose 48 (Pharmacia) according to the manufacturers specifications. This immunoaffinity matrix and a precolumn matrix of activated Sepharose 48 were washed with differ- ent buffers to prepare them for chromatography as described (Springer, 1987). 813-4-l cells in 40-80 roller bottles (850 cmz, Corn- ing) were lysed for 10 min in 12 ml per roller bottle of 10 mM ethanola- mine, 10 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 20 uglml leupeptin, 20 uglml aprotinin (pH 10.0). The clarified lysate was applied to the prewashed columns for 48 hr at 4OC and at a flow rate of 25 mllhr. The immunoaffinity matrix was then washed with ten column vols of wash buffer (pH 8.0) and five column vols of Tris buffer (pH 8.0). Elution of the bound material was in 50 mM glycine, 0.1% Tri- ton X-100, 0.15 M NaCl (pH 2.5) (Springer, 1987). Fractions (935 @I) of the eluate were collected in 65 ul of 1 M Tris-HCI (pH 9.0). After separa- tion with denaturing polyacrylamide gels according to Laemmli (1970). the fractions were analyzed by silver staining with a protocol slightly changed from Morrissey (1981).

Western Blot Analysis Western blot analysis was performed using a polyclonal goat anti-TGF- a antibody (Biotope) according to the manufacturers directions, except that rz51-labeled protein A (Amersham) was used.

Glycopmtein Fluorescent Labeling Two aliquots of the solubilized TGF-a precursor preparation were sepa- rated with a denaturing polyacrylamide gel. The gel was cut in half, and one side was fixed in 50% methanol, 10% acetic acid, stained with 0.05% Coomassie brilliant blue R, 50% methanol, 10% acetic acid, de- stained with 5% methanol, 7% acetic acid, and air dried between cel- lophane sheets. The other side was glycoprotein fluorescent labeled with dansylhydrazine according to Estep and Miller (1986).

N-Terminal Sequence Analysis and Amino Acid Analysis Proteins were sequenced by Edman degradation on a prototype liquid- phase sequencer as described in E.P.O. 257735. Reagents were purchased from Beckman (Quadrol and phenyl isothiocyanate), ABI (trifluoroacetic acid and 12.5% trimethylamine), and Bardick and Jack- son (heptane, ethyl acetate, butyl chloride). Protein samples in solution were sequenced by direct application to a microsequencing column containing both control pore glass and 15 mm Cl8 reverse-phase pack- ing media (Baker). After loading, the microsequencing columns were washed with 10% acetonitrile (Baker) and 1% trifluoroacetic acid. The columns were then placed in the prototype sequencer and sequenced as described in E.P.O. 257735.

For sequencing of PVDF-bound proteins (Immobilon, Millipore), the TGF-a precursor preparation was run on 15% SDS-polyacrylamide gels. Gels were soaked in blotting buffer (25 mM Tris base, 192 mM glycine, 15% methanol) for 15 min, while the PVDF sheets were soaked first in 100% methanol for 1 min, then in blotting buffer for 15 min. Pro- teins were transferred for 2 hr at 70 V. PVDF sheets were stained with 0.2% Coomassie blue in 50% methanol and 10% acetic acid for l-2 min, destained with 50% methanol and 10% acetic acid, and air dried. Proteins were sequenced by cutting out 1 mm strips of stained protein bands. The PVDF strips were packed into the center of a sequencing column and sandwiched between 1 cm of control pore glass and 1 cm of reverse-phase packing.

After sequencing, the columns were removed from the sequencer and placed in a Waters Pico.TAG workstation and hydrolyzed in 6 N HCI vapor at llO°C for 24 hr. After hydrolysis the columns were vacuum dried overnight. The amino acids were eluted off the column with sam- ple dilution buffer (Beckman) for amino acid analysis on a Beckman 6300.

EGF Receptor Phosphorylation Assay A431 cells were seeded at a density of 2 x 10s cells per 35 mm dish, grown for 1 day in 5% serum, and then changed to serum-free medium for 1 day. HERc cells were seeded at a density of 1 x 10s cells per 60 mm dish, grown overnight in 2% serum, and used the next day. Cells were incubated for 15 min at 3PC in 300 pl of serum-free medium with 0.3% BSA to which 200 ul of various concentrations of the TGF-a precursor preparation or the 50 amino acid TGFa were added. The in- cubation buffer was removed, and cells were lysed for 4 min on ice in 300 ul (400 ul for HERc cells) of 50 mM HEPES (pH 7.4) 1% Triton X-100,10 mM sodium pyrophosphate, 100 mM NaF, 4 mM EDTA, 2 mM sodium orthovanadate, 2 mM PMSF, 20 ug/uI aprotinin, 1 mM ZnClz. The supernatant was transferred to tubes, kept on ice for 5 min, and spun for 10 min in an Eppendorf centrifuge. The clarified lysate was then transferred into 750 pl(l ml for HERc cells) of wash buffer (50 mM HEPES [pH 7.5],150 mM NaCI, 100 mM NaF, 2 mM sodium orthovana- date, 0.1% Triton X-100, 10% glycerol, 1 mM ZnClz), 100 t~l of protein A solution (100 mglml), and 2-4 ul of monoclonal anti-EGF receptor an- tibody 108.1 (Honegger et al., 1987) and incubated at 4OC for 2-3 hr.

lmmunoprecipitates were washed three times with wash buffer, and SDS gel sample buffer was added and heated for 4 min at 95°C. The samples were fractionated by Z5O/o SDS-polyacrylamide gels, and the separated proteins were blotted onto nitrocellulose sheets (20 mM so- dium phosphate [pH 8.01; 1 A for 5 hr at 4OC). Western blot analysis was done with the mouse monoclonal anti-phosphotyrosine antibody 5E2, which is not cross-reactive with phosphoserine or phosphothreo- nine (5 Kg/ml; provided by B. Fendly, Genentech) and with 1251-labeled protein A (Amersham). Buffers were basically derived from lzumi et al. (1987) and slightly changed for our purposes.

Soft-Agar Colony Assay Two milliliters of 0.5% Noble agar (Difco) in high-glucose DMEM- Ham’s F12 with 10% fetal bovine serum was poured as lower agar in each well in 12-well plates. NRK 49F fibroblasts (1 x 104) were in- cubated with different amounts of reagents in 0.3 ml of medium for 5 min. Seven-tenths milliliter of 0.5% Noble agar was added, mixed, and layered on top of lower agar. After it solidified plates were incubated at JIOC for 8 days. One milliliter of iodonitrotetrazolium (50 mgllO0 ml; Sigma) was added overnight, and colonies of approximately more than 30 cells were microscopically evaluated (Rosenthal et al., 1986).

Cell Contact Experiments HERc cells (1 x 106) were grown overnight in 2% serum. TGF-a precursor-producing cells (813-4-l) and control CHO cells transfected with pSVdhfr, an expression plasmid containing the dihydrofolate reductase transcription unit, were detached with PBS containing 4 mM EDTA, counted, spun down, and resuspended in serum-free medium at concentrations of 5 and IO x 10s cells per 0.5 ml. One-half milliliter aliquots of the cell suspensions were layered on HERc monolayers for different times at 37pC. Supernatants were removed, and the experi- ment was carried out as for HERc cells in the above-described phos- phorylation assay.

Acknowledgments

We thank Dr. A. Ullrich and J. Lee for providing the HERc cells and anti- body 108.1. and B. Fendly for antibody 5E2. We also thank S. McCabe for her help with the FACS analysis and T. Bringman for help in the early stages of the work. We are grateful to Dr. J. Pouyssegur (Univer- site de Nice, France), Dr. M. Korc, and G. Bennett for advice.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 22, 1988; revised January 3, 1989.

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