MOLECULAR REPRODUCTION AND DEVELOPMENT 27:54-59 (1990)
An Evolutionarily Conserved TGF-a/Insulin-DegradingEnzyme MARSHA RICH ROSNER The Ben May Institute and the Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois
INTRODUCTION Receptor-mediated signal transduction can be regulated by at least three mechanisms. The first mechanism is synthesis of the receptor or the ligand that activates the receptor. The second mechanism is biochemical modification of the receptor or the ligand in order to render i t active or inactive. A third mechanism for regulating signal transduction is degradation. The focus of this paper is the regulation of growth factor levels by their degradation. This is a field about which we know little, largely because it has been assumed that ligands such as growth factors bind to receptors, are endocytosed, and then are degraded in the lysosomes. There are reports of enzymes that initiate degradation of growth factors such as epidermal growth factor (EGF). EGF, for example, appears to be degraded in discrete steps (Planck et al., 1984) by a t least two pathways. What this paper discusses is what one might call the first member of such a class of growth factor-degrading enzymes. We have called it here a TGF-dinsulin-degrading enzyme. This may, however, be a misnomer; I a m sure that it degrades other substrates as well. This enzyme is also referred to by the acronym IDE, for insulin-degrading enzyme.
INSULIN/TGF-a-DEGRADING ENZYME We discovered the insulin/TGF-&-degrading enzyme in Drosophila while searching for the EGF receptor in nonmammalian organisms. We had decided to use a n evolutionary approach to study EGF receptor interactions so that we could study structural and functional conservation, obtain developmental profiles, and do genetic analyses. We started our search by using a n antihuman EGF receptor antibody that cross reacted with receptors from other species. The antibody, prepared by Stuart Decker a t Rockefeller University, recognizes the cytoplasmic domain of the human and EGF receptor. It has been subsequently demonstrated to react with the neu protein and recognizes a highly conserved kinase domain within the receptor. Initially, we used Western blotting to detect cross-reacting proteins in several species. We probed Drosophila, nematodes, and yeast. Dro-
0 1990 WILEY-LISS, INC.
sophila was chosen for further study because it gave amazingly distinct cross-reactive bands, and we began our studies with the Drosophila embryonal cell line, Kc. We prepared membranes from Kc cells, resolved the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroeluted the proteins, and blotted them with our antihuman EGF receptor antibody. With this procedure, a band of 170 kDa was detected in membranes from A431 cells. A band of 190 kDa was detected in membranes of the Kc line and in membranes from Schneider L2 cells, another Drosophila cell line. Shilo and coworkers have since shown that the 190 kDa band is the Drosophila homolog of the mammalian EGF receptor or neu protein (Schejter et al., 1986). We also found that a second band of 100-110 kDa was recognized by the antihuman EGF receptor antibody (Thompson et al., 1985). To determine whether this 110 kDa band was also a n EGF receptor, we tested to see whether it could be cross linked to EGF. We immunoprecipitated the membrane proteins from Kc cells with anti human EGF receptor antibody, cross linked the proteins with 1251-labeledEGF using disuccinimidyl suberate, and resolved them by SDS-gel electrophoresis. When membranes from 3T3 cells were treated with this protocol, two protein bands of 170 and 150 kDa were cross linked to EGF. However, in the Kc cell membrane preparations, instead of being cross linked to a 190 kDa protein, the EGF was cross linked to the protein band of approximately 100 kDa that we had previously found was recognized by the anti-EGF receptor antibody. In Schneider L2 cells, a protein band of 110 kDa was also cross linked to EGF. The binding of labeled EGF to the 110 kDa protein could be competed with unlabeled EGF. What was surprising to us was that unlabeled insulin was also a n effective competitor for EGF binding to the 110 kDa protein. We did the opposite experiment and found that unlabeled EGF or insulin could also compete with Iz5Ilabeled insulin for cross linking to the 110 kDa protein. In contrast, the EGF receptor does not interact with insulin. If lz51-labeledEGF was cross linked to the EGF receptor, it could be competed out with comparable concentrations of unlabeled insulin. Similarly, the anti-
TGF-dINSULIN-DEGRADING ENZYME EGF receptor antibody did not immunoprecipitate insulin receptor t h a t had been cross linked to insulin. We measured the affinity of the 100 kDa protein for several growth factors. Whereas insulin and insulinlike growth factor-I1 (IGF-11) bound with reasonable affinity (Kd = lop7 M for insulin and lo-' M for IGF11), IGF-I had a lower affinity, and glucagon barely bound at all. We also examined the binding affinities of EGF and related proteins. TGF-a that was synthesized by Dr. James Tam at Rockefeller University bound with high affinity to the protein (Kd = 10p8-10-9 M), whereas EGF bound weakly (Kd = lop6 M) and nerve growth factor (NGF) bound with lower affinity than EGF. Platelet-derived growth factor (PDGF) did not bind, nor did TGF-P. The difference in binding affinity of EGF and TGF-a to the 110 kDa protein is a property t h a t is not characteristic of EGF receptors, except for the chicken receptor, which binds TGF-a with high affinity (Lax et al., 1988). To ensure that our results did not reflect differences in the ligands themselves, we tested our ligands for binding to the human EGF receptor in competitive binding assays and found no appreciable difference in the affinity of EGF relative to synthetic TGF-a. We also tested the hypothesis t h a t we were selecting a fraction of the population of protein in Drosophila that bound only TGF-a and that there might be another population with properties similar to that of the EGF receptor. However, when we looked a t the TGF-a cross linked proteins either by using antibody initially or by cross linking the entire population without first using antibody, we found t h a t the Drosophila cell membranes contained only the one TGF-a binding protein. In summary, we have found a Drosophila protein of 110 kDa that is immunoprecipitated by antihuman EGF receptor antiserum. It binds mammalian EGF, TGF-a, insulin, IGF-I, and IGF-I1 with affinities ranging from to lo-' M, but does not bind PDGF or TGF-P. This protein is also the only detectable TGF-a binding protein in Drosophila Kc cells.
RELATIONSHIP BETWEEN THE 110 kDa PROTEIN AND THE INSULIN RECEPTOR Initially, we thought that this TGF-ahnsulin binding protein could be a receptor. One possibility was that this protein might be the insulin receptor homolog in Drosophila. At about the same time, Rosen and colleagues had been characterizing the insulin receptor in Drosophila and found t h a t expression of the insulin receptor protein was developmentally regulated in embryos and t h a t i t was also expressed in embryonic cell lines (Petruzelli et al., 1985). Like our insulin/TGF-ol binding protein, the a-subunit of the embryonic insulin receptor homolog in Drosophila has a molecular weight of 110,000 and binds mammalian insulin. We tested the hypothesis that the TGF-aiinsulin binding protein was the insulin receptor (Garcia et al., 1987). Cell extracts were prepared and cross linked to
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1251-insulin, and the cross linked extracts were fractionated on a wheat germ agglutinin column, which should bind the glycosylated insulin receptor. We determined whether our anti-EGF receptor antibody would immunoprecipitate any of the proteins in the various fractions from the column. To identify our protein, we cross linked the proteins in each fraction to labeled TGF-a as well a s insulin. Three fractions were examined: 1)what was loaded onto the column, 2) what bound to the column, and 3) what did not bind. All of the material that was immunoprecipitable with the anti-EGF receptor antibody did not bind to the column. Some insulin binding activity came through the column without binding. Some insulin binding activity was detected in the eluate. However, the only fraction that bound TGF-a was the flow-through material, and binding of TGF-a could be competed by insulin in this fraction. In contrast, the insulin binding fraction that was retained by the wheat germ agglutinin column did not bind TGF-a. Thus, there were two proteins with similar molecular weights and insulin binding activities in the Kc cell extract: the Drosophila homolog of the a-subunit of the embryonal insulin receptor and the T G F d i n s u l i n binding protein that we had identified. We examined the distribution of the TGF-a binding protein in Kc cells by immunocytochemistry and found the protein to be concentrated in the cytoplasmic fraction. Comparison of the protein isolated from cytoplasmic and membrane fractions by two-dimensional gel analysis suggested that the proteins were identical. The fact that the TGF-dinsulin binding protein was primarily cytoplasmic suggested that this protein was not a receptor for growth factors.
RELATIONSHIP BETWEEN THE 110 kDa PROTEIN AND A PROTEASE An enzyme from mammalian cells of 110 kDa termed the insulin-degrading enzyme (IDE) is also able to bind and cross link to insulin. However, there had never been any suggestion that this enzyme might interact with EGF- or TGF-a-related factors. To determine whether our 110 kDa protein might correspond to the IDE, we looked for IDE in Drosophila and found such a n activity. We purified the insulin-degrading activity from Kc cells using several column chromatography steps, which included DEAE cellulose, Sephadex G200, hydroxylapatite, butyl-agarose, and chromatofocusing (Garcia et al., 1988). We obtained a purified protein of 110 kDa that bound and degraded insulin. Insulin could be cross linked to the protein, and insulin competed for binding. The 110 kDa IDE from Drosophila had properties very similar to those of its mammalian homologue (summarized in Table 1).In fact, the Drosophila and mammalian enzymes are so closely related that they can be purified using the same purification scheme. A comparison of the properties of the two enzymes shows
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M. RICH ROSNER
TABLE 1. Similarities Between the Drosophila and Mammalian IDEs Subunit M, Insulin degradation Affinity labeling with insulin Inhibition by sulfhydryl reagents Inhibition by glutathione Inhibition by bacitracin Inhibition by EDTA Optimal pH Isoelectric point Assay temperature ("C) Km for insulin (kM) S value
Drosophila 110
+ + ++ +
7-8 5.3 37 0.1 7.2
Mammalian 110
+ + ++ +
6.5-8.5 5.3 37 0.029-0.13 Not determined
that they have almost identical molecular weights, they both degrade insulin, and they can be affinitylabeled with insulin. Thus the IDE is a n evolutionarily conserved enzyme. Although the IDE is a metalloproteinase, i t is also inhibited by certain inhibitors of cysteine proteases such as N-ethyl maleimide, a sulfhydryl reagent, and bactitracin (summarized in Garcia et al., 1988). This inhibitor profile is shared by both the Drosophila and mammalian IDEs and is not characteristic of other characterized metalloproteinases (Kuo et al., 1990). The Drosophila and mammalian enzymes are inhibited by extensive treatment with EDTA (Shii e t al., 1986; Gehm and Rosner, unpublished results). However, the metal ion chelator phenanthroline is a n even more effective inhibitor of the enzyme. Zinc will restore the activity after EDTA or phenanthroline treatment, indicating that metal ion binding is required for enzyme activity. The optimal pH range of the IDE is between 7 and 8. I t is a nonlysosomal, cytosolic enzyme. The isoelectric point for both Drosophila and mammalian enzymes was 5.3. Although Drosophila grows a t room temperature, the maximum activity of the Drosophila IDE was 37"C, which is the same a s that for the mammalian enzyme. The K, of the Drosophila IDE for mammalian insulin is 0.1 pM comparable to the K, reported for the mammalian IDE (Gehm and Rosner, unpublished results). In collaboration with Dr. William Duckworth of the University of Nebraska Medical Center, we determined the sites on insulin that are cleaved by the Drosphila IDE (Duckworth et al., 1989). The IDE, unlike proteases such as trypsin, seems to have a conformational specificity. Once the IDE binds to insulin, it initiates a number of cleavages around that region. Thus, all the bonds cleaved by the enzyme are in close proximity (A chain residues 13-15; B chain residues 10, 11, 14-16), with the exception of residues B24B26. Surprisingly, all the sites on porcine insulin cleaved by the Drosophila IDE correspond to a subset of the cleavage sites of the mammalian enzyme. In sum, we have characterized a n IDE from Drosophila that is highly conserved in its molecular
TABLE 2. Similarities Between the InsulidTGF-cu Binding Protein and the Drosophila IDE Relative molecular mass (kD) Affinity labeling Insulin EGF TGF-a Immunocross reactivity Anti EGF-receptor antiserum Anti-Drosophila IDE antiserum Degradation Insulin EGF
Binding protein 110
IDE 110
+ + + + +
+ + + + +
weight, in its catalytic properties, and in its ability to degrade mammalian substrates. We then showed that this enzyme was the insulin/TGF-a binding protein that we initially found in Drosophila Kc cells (Garcia e t al., 1989b). Although I will not describe all the studies used to establish the identity of the IDE with the TGF-a binding protein, I will describe one piece of evidence, a n insulin degradation assay. We incubated the enzyme with 1251-labeledinsulin and used trichloroacetic acid (TCA) to precipitate intact insulin. Insulin that has been degraded will stay in the supernatant. We then used anti-EGF receptor antibody to determine whether it was able to precipitate the insulin-degrading activity. We found that the antibody that specifically recognizes the insulin/ TGF-a binding protein immunoprecipitated the insulin-degrading activity. The evidence for the identity of the TGF-a binding protein and the IDE is summarized in Table 2. Both proteins have the same molecular weights; can be affinity-labeled with insulin, EGF, or TGF-a, and degrade insulin. Finally, they are immunologically cross reactive.
FUNCTION OF THE INSULIN/TGF-a-DEGRADING ENZYME Several lines of evidence suggest that the IDE is the primary enzyme involved in the initiation of insulin degradation in mammalian cells (summarized in Duckworth et al., 1989). As is illustrated below, in certain cell systems this enzyme is largely responsible for removing intact insulin from the medium of the cell. However, we discovered the Drosophila IDE by looking for EGF and TGF-a binding proteins. So, could this enzyme have a broader range of specificity? Does i t regulate growth factor levels? If it does play a role in growth control, could it also play a role in differentiation? We first determined whether the Drosophila IDE, which is able to bind EGF, can also degrade EGF (Table 3; Garcia et al., 1989a).Initially, we used a TCA precipitation assay to monitor 1251-insulindegradation. In this assay, unlabeled insulin blocked the activity,
TGF-&/INSULIN-DEGRADINGENZYME TABLE 3. Comparative Substrate Specificities and Antigenic Properties of the Mammalian and DrosoDhila IDES Mammalian IDE Affinity labeling + 1251-TGF-a + 1251-insuIin '"I-EGF Inhibition of TGF-a or insulin binding + TGF-a Insulin + EGF + Growth factor degradation + TGF-a Insulin +EGF Inhibition of insulin or TGF-a degradation TGF-a + Insulin + EGF + Immunologic crossreactivity Antihuman IDE +Anti-Drosophila IDE Anti-human EGF receptor -
Drosophila IDE
+ + I
+ + I
+ ++ + + -
+ +
whereas other proteins did not block it. When the same experiment was done with labeled EGF, the EGF was only slightly degraded and unlabeled insulin did not block this EGF-degrading activity. Thus, the EGF degradation that we observed was nonspecfic and was not really meaningful. The sensitivity of the EGF degradation assay can be increased by using a n EGF receptor binding assay. Again, no degradation was detectable. Thus the enzyme does not degrade EGF. This result was not surprising in that the enzyme's affinity for EGF is not very high. By contrast, the Drosophila enzyme does degrade human TGF-a, for which i t has a higher affinity (Table 3). The degradation of TGF-a was specifically blocked by unlabeled insulin. We then investigated the mammalian IDE to determine whether i t also has the property of binding and degrading TGF-a (Table 3; Garcia e t al., 1989a). We attempted to affinity-label the IDE in mouse 3T3 cells with 1251-EGFand 1251-TGF-a.We found that the mammalian enzyme did not bind EGF. By contrast, the enzyme bound TGF-a, and this binding was inhibited by competition with insulin. It was also inhibited slightly by competition with unlabeled EGF. Using the TCA precipitation assay, we showed that the mammalian enzyme also degraded TGF-a, and degradation was blocked with unlabeled insulin. We compared the ability of the mammalian enzyme to degrade insulin and recombinant TGF-a, which was generously donated by Dr. Rik Derynck of Genentech. The recombinant TGFa did not have quite as high a n affinity as the synthetic TGF-a; thus its affinity for the enzyme was about the same as that of insulin. Both substrates were degraded to a comparable extent by the IDE.
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These results raise the possibility that there is a nonlysosomal mechanism for specifically degrading TGF-a as well as insulin in mammalian cells. The observation that TGF-a and insulin are both degraded by the IDE suggests that insulin could block the degradation of TGF-a in vivo and vice versa. The inhibitory curves for degradation of insulin by the Drosophila enzyme reveal that insulin and TGF-a are comparable inhibitors of IDE activity. Whereas EGF also inhibits insulin degradation by the Drosophila enzyme, it is not as effective as TGF-a. In addition, EGF inhibits insulin degradation by the mammalian enzyme to a n even lesser degree than it does the Drosophila enzyme. Thus the IDE may provide a mechanism for interaction between the insulin and TGF-a families in the sense that high levels of one of these ligands may affect degradation of the other one. To address the question of whether the IDE functions similarly in vivo as i t does in vitro, we are in the process of characterizing the degradation products of TGFa in vitro and then looking for these same products in vivo. The second approach that we are taking is to clone the enzyme and determine whether expression of TGF-a can be modulated with cloned enzyme. The third approach is to inhibit degradation in vivo by using specific inhibitors. The insuliniTGF-a-degrading enzyme has a characteristic spectrum of inhibitors that affect both cysteine proteases and the metalloproteases. By using phenanthroline and bacitracin as selective inhibitors, one can get a n idea of whether the IDE might be responsible for specific effects in vivo. We exposed human hepatoma (HepG2) cells to 1251-labeled insulin or 1251-labeled TGF-a and looked for degradation of ligand in the medium by measuring loss of binding activity. HepG2 cells degraded insulin, and the degradation could be completely blocked with cold insulin or the inhibitor bacitracin. In this cell system, therefore, the insulin/ TGF-a-degrading enzyme appears to be a major mechanism for degrading insulin. This result is supported by previous studies (Shii and Roth, 1986) demonstrating that antibodies to the IDE can inhibit at least 60% of intracellular insulin degradation by HepG2 cells. An interesting consequence of inhibiting the IDE is that the insulin in the medium remains undegraded. Thus the net effect of blocking the IDE is that the level of undegraded ligand in the medium increases. If the same experiment is done with 1251-TGF-a,then partial inhibition (-50%) of TGF-a degradation is observed. Again, the net effect of adding the IDE inhibitors is to raise the level of undegraded TGF-a in the medium. Similar results were obtained with phenanthroline and other metalloprotease inhibitors, and the ID,,+ for inhibition of insulin and TGF-a degradation were comparable (Gehm and Rosner, submitted). Thus the substrate and inhibitor profiles of the TGF-a-degrading enzyme in vivo parallel those of the IDE in vitro. These results suggest that the human IDE or a sim-
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M. RICH ROSNER
hIDE
134 ALDRFGQFFIAPLFTPSATEREINAVNSEHEiCNLPSDLWRIKQVNRHLAK 183 IIIIl:lli:.IlI..I..:II:1II:IIIlI1: .I - 1 1 : I::: :. 1 6 1 ALDRFAQFFLCPLE'DESCKDREVNAVDSEHEK"DAk7RLFQLEKATGN 210
dIDE
184 PDHAYSKFGSGXKTTQTEMPKS~KNIDVRDELLKFTSS 220
hIDE
211 PKHPFSKFGTGNKYTLETRPNQEGIDVRQELLKFHSA 2 4 7
dIDE
I.I::IIII.III
I
... I . ' . . I I I I : I I I I I
I.
Fig. 1. Partial comparison of deduced amino acid sequence of the Drosophila and human IDEs.
ilar enzyme can mediate degradation of TGF-a and insulin in vivo. These observations also raise a number of other possibilities. First, there may be multiple pathways for degradation of growth factors, such as EGF or TGF-a, that bind to the same receptor. Second, growth factor-degrading enzymes might be a mechanism for regulating the extracellular levels of one growth factor by another. For example, high extracellular insulin might lead to decreased TGF-a degradation. Finally, these results suggest that the insuliniTGF-a-degrading enzyme may play a role in controlling cell growth.
A ROLE FOR THE INSULIN/TGF-a-DEGRADING ENZYME IN DIFFERENTIATION A similar approach based on protease inhibitors was used by Kayalar and colleagues to investigate a possible role for the IDE in regulating muscle cell differentiation. The results of their studies suggest that IDE or a n enzyme with a n inhibitor profile like that of the IDE is required for differentiation of muscle cells (Kayalar and Wong, 1989). If IDE activity can influence the onset of differentiation, then i t is also possible that expression of the enzyme is regulated during development. To explore this possibility, we used the Drosophila system (Stoppelli et al., 1988). The IDE is expressed in the embryo, in the larvae, and in the adult head and body of the fruit fly as well a s in cultured Kc and Schneider L2 cells. When we looked at the relative expression of the enzyme in Drosophila, we found that it was expressed a t very low levels in the embryo compared to the adult organism. As a control, we put the IDE in extracts from the different adult and developmental stages to show that it is not differentially degraded in the various extracts during the experiment. We have compared the level of expression of the enzyme in embryo and adult in a number of ways. As well a s using the TGF-a cross linking assay, we have done immunoblotting with Drosophila antibody to the IDE and measured insulin-degrading activity. The difference in IDE expression between embryo and adult that we measured with these assays is about tenfold. The level of expression increases gradually from embryo to larva through pupa to adult. These results are consistent with a role for the IDE in differentiation. This enzyme is widely distributed in
Drosophila, it appears to be developmentally regulated, and the pattern of its expression suggests a specific role for this protein in the later stages of development.
CLONING THE TGF-dINSULINDEGRADING ENZYME To address the question of its role in growth and development in more detail, we decided to clone the Drosophila IDE. We used a number of different approaches. The most successful approach was to sequence some tryptic peptides from the enzyme and use these sequences to make oligonucleotide probes. We made two sets of oligonucleotide probes and screened about lo6 clones. From these screens, we found one weakly positive clone, which was about 2.8 kb long. We used this clone to rescreen the Drosophila library and then found three full-length clones of about 3.5 kb each. Using these clones, we can detect a major transcript of 3.6 kb by Northern analysis of mRNA from Kc cells. The human IDE gene has recently been cloned (Affholter e t al., 19881, and one of the two human transcripts is about the same size as the transcript we observed in Drosophila. To determine whether our clone encodes the Drosophila TGF-alinsulin-degrading enzyme, we have done in vitro translation using both reticulocyte and wheat germ systems. When we used the sense transcript in these systems, we observed a protein of 110 kDa, which was absent when we used the antisense transcript. The 110 kDa protein produced by in vitro translation from the sense transcript can be immunoprecipitated with anti-IDE antibody. Thus, the evidence indicates that we have a full-length cDNA clone for the Drosophila IDE, and this clone is able to express this protein in vitro (Kuo et al., submitted). Comparison of the deduced amino acid sequences from the human and Drosophila IDEs indicates that there is 50% identity (Fig. 1; for complete sequence, see Kuo et al., submitted). Even more striking is the fact that there is 70% similarity between these two sequences. Thus, there is very high conservation between the Drosophila and human enzymes. Gene bank searches revealed one other enzyme that shares significant sequence homology (27% identity, 48% similarity) with the human and Drosophila IDEs:
TGF-dINSULIN-DEGRADINGENZYME bacterial protease 111. Bacterial protease I11 is also a 110 kDa protein and a metalloproteinase, which is found in the periplasmic space. Although it is capable of degrading the insulin B chain, its function in bacteria is not yet known. In sum, these results suggest that the IDE is a member of a new evolutionarily conserved family of enzymes whose role in growth and development is just beginning to be elucidated.
ACKNOWLEDGMENTS I would like to acknowledge the colleagues who have done this work. Karol Thompson, a graduate student, first identified the Drosophila IDE, and J. Victor Garcia and M. Patrizia Stoppelli, postdoctoral associates, did the initial characterization and purification of the enzyme. Dr. Wen-Liang Kuo has cloned the Drosophila enzyme. Dr. Barry Gehm has been studying the in vivo degradation of TGF-a. Dr. William Duckworth of the University of Nebraska Medical Center collaborated on the characterization of the insulin cleavage sites. This work was supported in part by the U S . Public Health Service, National Institutes of Health, the American Diabetes Association, and the University of Chicago Diabetes Research and Training Center. QUESTIONS AND ANSWERS Q: This does not seem to be a glycosylated or secreted protein. Where is it located in the cell? A: You are right that i t is not glycosylated. In fact, the wheat germ experiment was done to distinguish it from the insulin receptor that is glycosylated and is able to bind to the lectin columns. The enzyme is primarily in the cytosol as far as we can tell. It is also membrane-associated and might be in vesicles. We initially found i t associated with the membrane. We have never detected it on the outer membrane of the cells. In Drosophila, we have evidence that it might be secreted. The IDE also has a topological problem t h a t I think you have alluded to, which is how does this enzyme get in contact with insulin and TGF-a? It has been found in preacidic endosomes of human cells following insulin binding to its recpetor and internalization. The IDE may be secreted and then reinternalized and thus come into contact with insulin. Q: What is your evidence that this enzyme actually degraded TGF-a? A: We observe a n increase in TCA solubility of the label attached to TGF-a. Also, the TGF-a does not bind its receptor after incubation with the enzyme, so i t is no longer a functional growth factor.
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Q: Does the enzyme degrade the insulin or TGF-a receptors? A: We have not done that experiment directly. As far as we can tell, it does not degrade the receptor. The question that we want to ask is whether the enzyme affects the function of the receptor by decreasing the concentration of its ligand. Q: How specific is this enzyme for TGF-a and insulin? A: I t degrades IGF-I1 as well a s IGF-I, but it is not very active on the latter substrate. I t does not degrade bovine serum albumin (BSA) and many other randomly chosen proteins that we have tried. The enzyme was called a n insulin-degrading enzyme because it seemed to be very specific for the insulin family. Although I believe there may be other substrates that we have not found, the enzyme also shows a great deal of specificity by degrading TGF-a and not EGF. EGF and TGF-a are very similar in structure, yet we have not been able to detect significant degradation of EGF by the IDE. Q: Have you used immunocytochemistry to localize the enzyme in the cells? A: We attempted to do some immunocytochemistry with the antibody, but i t did not work out. There has been one study with human cells, and the results suggest that the enzyme is localized in the cytoplasm. However, I think t h a t the question of where this protein is localized is still open.
REFERENCES Affholter JA, Fried VA, Roth RA (1988); Science 242:1415-1418. Duckworth WC, Garcia JV, Liepnieks J J , Hamel FG, Hermodson MA, Frank BH, Rosner MR (1989): Biochemistry 28:2471-2477. Garcia JV, Fenton BW, Rosner MR (1988): Biochemistry 27:42374244. Garcia JV, Gehm BD, Rosner MR (1989a): J Cell Biol109:1301-1307. Garcia JV, Stoppelli MP, Decker SJ, Rosner MR (1989b): J Cell Biol 108:177-182. Garcia JV, Stoppelli MP, Thompson KL, Decker SJ, Rosner MR (1987): J Cell Biol 105:449-456. Kayalar C, Wong WT 11989): J Biol Chem 26423928-8934. Kuo WL, Gehm BD, Holmgren R, Rosner MR (1990): (submitted). Lax I, Johnson A, Hawk R, Sap J , Bellot F, Winkler M, Ullrich A, Vennstrom B, Schlessinger J, Givol D (1988): Mol Cell Biol, 8:19701978. Petruzelli L, Herrera R, Garcia R, Rosen OM (1985): Cancer Cells [Cold Spring Harbor Laboratory] Cold Spring Harbor, pp. 3:115121. Planck SR, Finch JS, Magun BE (1984): J Biol Chem 259:3053-3057. Schejter ED, Segal D, Glaser L, Shilo BZ (1986): Cell 46:1091-1101. Shii K, Roth RA (1986): Proc Natl Acad Sci USA 83:4147-4151. Shii K, Yokono K, Baba S, Roth RA (1986): Diabetes 35575-683. Stoppelli MP, Garcia JV, Decker SJ, Rosner MR (1988): Proc Natl Acad Sci USA 85:3469-3473. Thompson KL, Decker SJ,Rosner MR (1985): Proc Natl Acad Sci USA 82:8443-8447.
An Evolutionarily Conserved TGF-a/Insulin-DegradingEnzyme MARSHA RICH ROSNER The Ben May Institute and the Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois
INTRODUCTION Receptor-mediated signal transduction can be regulated by at least three mechanisms. The first mechanism is synthesis of the receptor or the ligand that activates the receptor. The second mechanism is biochemical modification of the receptor or the ligand in order to render i t active or inactive. A third mechanism for regulating signal transduction is degradation. The focus of this paper is the regulation of growth factor levels by their degradation. This is a field about which we know little, largely because it has been assumed that ligands such as growth factors bind to receptors, are endocytosed, and then are degraded in the lysosomes. There are reports of enzymes that initiate degradation of growth factors such as epidermal growth factor (EGF). EGF, for example, appears to be degraded in discrete steps (Planck et al., 1984) by a t least two pathways. What this paper discusses is what one might call the first member of such a class of growth factor-degrading enzymes. We have called it here a TGF-dinsulin-degrading enzyme. This may, however, be a misnomer; I a m sure that it degrades other substrates as well. This enzyme is also referred to by the acronym IDE, for insulin-degrading enzyme.
INSULIN/TGF-a-DEGRADING ENZYME We discovered the insulin/TGF-&-degrading enzyme in Drosophila while searching for the EGF receptor in nonmammalian organisms. We had decided to use a n evolutionary approach to study EGF receptor interactions so that we could study structural and functional conservation, obtain developmental profiles, and do genetic analyses. We started our search by using a n antihuman EGF receptor antibody that cross reacted with receptors from other species. The antibody, prepared by Stuart Decker a t Rockefeller University, recognizes the cytoplasmic domain of the human and EGF receptor. It has been subsequently demonstrated to react with the neu protein and recognizes a highly conserved kinase domain within the receptor. Initially, we used Western blotting to detect cross-reacting proteins in several species. We probed Drosophila, nematodes, and yeast. Dro-
0 1990 WILEY-LISS, INC.
sophila was chosen for further study because it gave amazingly distinct cross-reactive bands, and we began our studies with the Drosophila embryonal cell line, Kc. We prepared membranes from Kc cells, resolved the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroeluted the proteins, and blotted them with our antihuman EGF receptor antibody. With this procedure, a band of 170 kDa was detected in membranes from A431 cells. A band of 190 kDa was detected in membranes of the Kc line and in membranes from Schneider L2 cells, another Drosophila cell line. Shilo and coworkers have since shown that the 190 kDa band is the Drosophila homolog of the mammalian EGF receptor or neu protein (Schejter et al., 1986). We also found that a second band of 100-110 kDa was recognized by the antihuman EGF receptor antibody (Thompson et al., 1985). To determine whether this 110 kDa band was also a n EGF receptor, we tested to see whether it could be cross linked to EGF. We immunoprecipitated the membrane proteins from Kc cells with anti human EGF receptor antibody, cross linked the proteins with 1251-labeledEGF using disuccinimidyl suberate, and resolved them by SDS-gel electrophoresis. When membranes from 3T3 cells were treated with this protocol, two protein bands of 170 and 150 kDa were cross linked to EGF. However, in the Kc cell membrane preparations, instead of being cross linked to a 190 kDa protein, the EGF was cross linked to the protein band of approximately 100 kDa that we had previously found was recognized by the anti-EGF receptor antibody. In Schneider L2 cells, a protein band of 110 kDa was also cross linked to EGF. The binding of labeled EGF to the 110 kDa protein could be competed with unlabeled EGF. What was surprising to us was that unlabeled insulin was also a n effective competitor for EGF binding to the 110 kDa protein. We did the opposite experiment and found that unlabeled EGF or insulin could also compete with Iz5Ilabeled insulin for cross linking to the 110 kDa protein. In contrast, the EGF receptor does not interact with insulin. If lz51-labeledEGF was cross linked to the EGF receptor, it could be competed out with comparable concentrations of unlabeled insulin. Similarly, the anti-
TGF-dINSULIN-DEGRADING ENZYME EGF receptor antibody did not immunoprecipitate insulin receptor t h a t had been cross linked to insulin. We measured the affinity of the 100 kDa protein for several growth factors. Whereas insulin and insulinlike growth factor-I1 (IGF-11) bound with reasonable affinity (Kd = lop7 M for insulin and lo-' M for IGF11), IGF-I had a lower affinity, and glucagon barely bound at all. We also examined the binding affinities of EGF and related proteins. TGF-a that was synthesized by Dr. James Tam at Rockefeller University bound with high affinity to the protein (Kd = 10p8-10-9 M), whereas EGF bound weakly (Kd = lop6 M) and nerve growth factor (NGF) bound with lower affinity than EGF. Platelet-derived growth factor (PDGF) did not bind, nor did TGF-P. The difference in binding affinity of EGF and TGF-a to the 110 kDa protein is a property t h a t is not characteristic of EGF receptors, except for the chicken receptor, which binds TGF-a with high affinity (Lax et al., 1988). To ensure that our results did not reflect differences in the ligands themselves, we tested our ligands for binding to the human EGF receptor in competitive binding assays and found no appreciable difference in the affinity of EGF relative to synthetic TGF-a. We also tested the hypothesis t h a t we were selecting a fraction of the population of protein in Drosophila that bound only TGF-a and that there might be another population with properties similar to that of the EGF receptor. However, when we looked a t the TGF-a cross linked proteins either by using antibody initially or by cross linking the entire population without first using antibody, we found t h a t the Drosophila cell membranes contained only the one TGF-a binding protein. In summary, we have found a Drosophila protein of 110 kDa that is immunoprecipitated by antihuman EGF receptor antiserum. It binds mammalian EGF, TGF-a, insulin, IGF-I, and IGF-I1 with affinities ranging from to lo-' M, but does not bind PDGF or TGF-P. This protein is also the only detectable TGF-a binding protein in Drosophila Kc cells.
RELATIONSHIP BETWEEN THE 110 kDa PROTEIN AND THE INSULIN RECEPTOR Initially, we thought that this TGF-ahnsulin binding protein could be a receptor. One possibility was that this protein might be the insulin receptor homolog in Drosophila. At about the same time, Rosen and colleagues had been characterizing the insulin receptor in Drosophila and found t h a t expression of the insulin receptor protein was developmentally regulated in embryos and t h a t i t was also expressed in embryonic cell lines (Petruzelli et al., 1985). Like our insulin/TGF-ol binding protein, the a-subunit of the embryonic insulin receptor homolog in Drosophila has a molecular weight of 110,000 and binds mammalian insulin. We tested the hypothesis that the TGF-aiinsulin binding protein was the insulin receptor (Garcia et al., 1987). Cell extracts were prepared and cross linked to
55
1251-insulin, and the cross linked extracts were fractionated on a wheat germ agglutinin column, which should bind the glycosylated insulin receptor. We determined whether our anti-EGF receptor antibody would immunoprecipitate any of the proteins in the various fractions from the column. To identify our protein, we cross linked the proteins in each fraction to labeled TGF-a as well a s insulin. Three fractions were examined: 1)what was loaded onto the column, 2) what bound to the column, and 3) what did not bind. All of the material that was immunoprecipitable with the anti-EGF receptor antibody did not bind to the column. Some insulin binding activity came through the column without binding. Some insulin binding activity was detected in the eluate. However, the only fraction that bound TGF-a was the flow-through material, and binding of TGF-a could be competed by insulin in this fraction. In contrast, the insulin binding fraction that was retained by the wheat germ agglutinin column did not bind TGF-a. Thus, there were two proteins with similar molecular weights and insulin binding activities in the Kc cell extract: the Drosophila homolog of the a-subunit of the embryonal insulin receptor and the T G F d i n s u l i n binding protein that we had identified. We examined the distribution of the TGF-a binding protein in Kc cells by immunocytochemistry and found the protein to be concentrated in the cytoplasmic fraction. Comparison of the protein isolated from cytoplasmic and membrane fractions by two-dimensional gel analysis suggested that the proteins were identical. The fact that the TGF-dinsulin binding protein was primarily cytoplasmic suggested that this protein was not a receptor for growth factors.
RELATIONSHIP BETWEEN THE 110 kDa PROTEIN AND A PROTEASE An enzyme from mammalian cells of 110 kDa termed the insulin-degrading enzyme (IDE) is also able to bind and cross link to insulin. However, there had never been any suggestion that this enzyme might interact with EGF- or TGF-a-related factors. To determine whether our 110 kDa protein might correspond to the IDE, we looked for IDE in Drosophila and found such a n activity. We purified the insulin-degrading activity from Kc cells using several column chromatography steps, which included DEAE cellulose, Sephadex G200, hydroxylapatite, butyl-agarose, and chromatofocusing (Garcia et al., 1988). We obtained a purified protein of 110 kDa that bound and degraded insulin. Insulin could be cross linked to the protein, and insulin competed for binding. The 110 kDa IDE from Drosophila had properties very similar to those of its mammalian homologue (summarized in Table 1).In fact, the Drosophila and mammalian enzymes are so closely related that they can be purified using the same purification scheme. A comparison of the properties of the two enzymes shows
56
M. RICH ROSNER
TABLE 1. Similarities Between the Drosophila and Mammalian IDEs Subunit M, Insulin degradation Affinity labeling with insulin Inhibition by sulfhydryl reagents Inhibition by glutathione Inhibition by bacitracin Inhibition by EDTA Optimal pH Isoelectric point Assay temperature ("C) Km for insulin (kM) S value
Drosophila 110
+ + ++ +
7-8 5.3 37 0.1 7.2
Mammalian 110
+ + ++ +
6.5-8.5 5.3 37 0.029-0.13 Not determined
that they have almost identical molecular weights, they both degrade insulin, and they can be affinitylabeled with insulin. Thus the IDE is a n evolutionarily conserved enzyme. Although the IDE is a metalloproteinase, i t is also inhibited by certain inhibitors of cysteine proteases such as N-ethyl maleimide, a sulfhydryl reagent, and bactitracin (summarized in Garcia et al., 1988). This inhibitor profile is shared by both the Drosophila and mammalian IDEs and is not characteristic of other characterized metalloproteinases (Kuo et al., 1990). The Drosophila and mammalian enzymes are inhibited by extensive treatment with EDTA (Shii e t al., 1986; Gehm and Rosner, unpublished results). However, the metal ion chelator phenanthroline is a n even more effective inhibitor of the enzyme. Zinc will restore the activity after EDTA or phenanthroline treatment, indicating that metal ion binding is required for enzyme activity. The optimal pH range of the IDE is between 7 and 8. I t is a nonlysosomal, cytosolic enzyme. The isoelectric point for both Drosophila and mammalian enzymes was 5.3. Although Drosophila grows a t room temperature, the maximum activity of the Drosophila IDE was 37"C, which is the same a s that for the mammalian enzyme. The K, of the Drosophila IDE for mammalian insulin is 0.1 pM comparable to the K, reported for the mammalian IDE (Gehm and Rosner, unpublished results). In collaboration with Dr. William Duckworth of the University of Nebraska Medical Center, we determined the sites on insulin that are cleaved by the Drosphila IDE (Duckworth et al., 1989). The IDE, unlike proteases such as trypsin, seems to have a conformational specificity. Once the IDE binds to insulin, it initiates a number of cleavages around that region. Thus, all the bonds cleaved by the enzyme are in close proximity (A chain residues 13-15; B chain residues 10, 11, 14-16), with the exception of residues B24B26. Surprisingly, all the sites on porcine insulin cleaved by the Drosophila IDE correspond to a subset of the cleavage sites of the mammalian enzyme. In sum, we have characterized a n IDE from Drosophila that is highly conserved in its molecular
TABLE 2. Similarities Between the InsulidTGF-cu Binding Protein and the Drosophila IDE Relative molecular mass (kD) Affinity labeling Insulin EGF TGF-a Immunocross reactivity Anti EGF-receptor antiserum Anti-Drosophila IDE antiserum Degradation Insulin EGF
Binding protein 110
IDE 110
+ + + + +
+ + + + +
weight, in its catalytic properties, and in its ability to degrade mammalian substrates. We then showed that this enzyme was the insulin/TGF-a binding protein that we initially found in Drosophila Kc cells (Garcia e t al., 1989b). Although I will not describe all the studies used to establish the identity of the IDE with the TGF-a binding protein, I will describe one piece of evidence, a n insulin degradation assay. We incubated the enzyme with 1251-labeledinsulin and used trichloroacetic acid (TCA) to precipitate intact insulin. Insulin that has been degraded will stay in the supernatant. We then used anti-EGF receptor antibody to determine whether it was able to precipitate the insulin-degrading activity. We found that the antibody that specifically recognizes the insulin/ TGF-a binding protein immunoprecipitated the insulin-degrading activity. The evidence for the identity of the TGF-a binding protein and the IDE is summarized in Table 2. Both proteins have the same molecular weights; can be affinity-labeled with insulin, EGF, or TGF-a, and degrade insulin. Finally, they are immunologically cross reactive.
FUNCTION OF THE INSULIN/TGF-a-DEGRADING ENZYME Several lines of evidence suggest that the IDE is the primary enzyme involved in the initiation of insulin degradation in mammalian cells (summarized in Duckworth et al., 1989). As is illustrated below, in certain cell systems this enzyme is largely responsible for removing intact insulin from the medium of the cell. However, we discovered the Drosophila IDE by looking for EGF and TGF-a binding proteins. So, could this enzyme have a broader range of specificity? Does i t regulate growth factor levels? If it does play a role in growth control, could it also play a role in differentiation? We first determined whether the Drosophila IDE, which is able to bind EGF, can also degrade EGF (Table 3; Garcia et al., 1989a).Initially, we used a TCA precipitation assay to monitor 1251-insulindegradation. In this assay, unlabeled insulin blocked the activity,
TGF-&/INSULIN-DEGRADINGENZYME TABLE 3. Comparative Substrate Specificities and Antigenic Properties of the Mammalian and DrosoDhila IDES Mammalian IDE Affinity labeling + 1251-TGF-a + 1251-insuIin '"I-EGF Inhibition of TGF-a or insulin binding + TGF-a Insulin + EGF + Growth factor degradation + TGF-a Insulin +EGF Inhibition of insulin or TGF-a degradation TGF-a + Insulin + EGF + Immunologic crossreactivity Antihuman IDE +Anti-Drosophila IDE Anti-human EGF receptor -
Drosophila IDE
+ + I
+ + I
+ ++ + + -
+ +
whereas other proteins did not block it. When the same experiment was done with labeled EGF, the EGF was only slightly degraded and unlabeled insulin did not block this EGF-degrading activity. Thus, the EGF degradation that we observed was nonspecfic and was not really meaningful. The sensitivity of the EGF degradation assay can be increased by using a n EGF receptor binding assay. Again, no degradation was detectable. Thus the enzyme does not degrade EGF. This result was not surprising in that the enzyme's affinity for EGF is not very high. By contrast, the Drosophila enzyme does degrade human TGF-a, for which i t has a higher affinity (Table 3). The degradation of TGF-a was specifically blocked by unlabeled insulin. We then investigated the mammalian IDE to determine whether i t also has the property of binding and degrading TGF-a (Table 3; Garcia e t al., 1989a). We attempted to affinity-label the IDE in mouse 3T3 cells with 1251-EGFand 1251-TGF-a.We found that the mammalian enzyme did not bind EGF. By contrast, the enzyme bound TGF-a, and this binding was inhibited by competition with insulin. It was also inhibited slightly by competition with unlabeled EGF. Using the TCA precipitation assay, we showed that the mammalian enzyme also degraded TGF-a, and degradation was blocked with unlabeled insulin. We compared the ability of the mammalian enzyme to degrade insulin and recombinant TGF-a, which was generously donated by Dr. Rik Derynck of Genentech. The recombinant TGFa did not have quite as high a n affinity as the synthetic TGF-a; thus its affinity for the enzyme was about the same as that of insulin. Both substrates were degraded to a comparable extent by the IDE.
57
These results raise the possibility that there is a nonlysosomal mechanism for specifically degrading TGF-a as well as insulin in mammalian cells. The observation that TGF-a and insulin are both degraded by the IDE suggests that insulin could block the degradation of TGF-a in vivo and vice versa. The inhibitory curves for degradation of insulin by the Drosophila enzyme reveal that insulin and TGF-a are comparable inhibitors of IDE activity. Whereas EGF also inhibits insulin degradation by the Drosophila enzyme, it is not as effective as TGF-a. In addition, EGF inhibits insulin degradation by the mammalian enzyme to a n even lesser degree than it does the Drosophila enzyme. Thus the IDE may provide a mechanism for interaction between the insulin and TGF-a families in the sense that high levels of one of these ligands may affect degradation of the other one. To address the question of whether the IDE functions similarly in vivo as i t does in vitro, we are in the process of characterizing the degradation products of TGFa in vitro and then looking for these same products in vivo. The second approach that we are taking is to clone the enzyme and determine whether expression of TGF-a can be modulated with cloned enzyme. The third approach is to inhibit degradation in vivo by using specific inhibitors. The insuliniTGF-a-degrading enzyme has a characteristic spectrum of inhibitors that affect both cysteine proteases and the metalloproteases. By using phenanthroline and bacitracin as selective inhibitors, one can get a n idea of whether the IDE might be responsible for specific effects in vivo. We exposed human hepatoma (HepG2) cells to 1251-labeled insulin or 1251-labeled TGF-a and looked for degradation of ligand in the medium by measuring loss of binding activity. HepG2 cells degraded insulin, and the degradation could be completely blocked with cold insulin or the inhibitor bacitracin. In this cell system, therefore, the insulin/ TGF-a-degrading enzyme appears to be a major mechanism for degrading insulin. This result is supported by previous studies (Shii and Roth, 1986) demonstrating that antibodies to the IDE can inhibit at least 60% of intracellular insulin degradation by HepG2 cells. An interesting consequence of inhibiting the IDE is that the insulin in the medium remains undegraded. Thus the net effect of blocking the IDE is that the level of undegraded ligand in the medium increases. If the same experiment is done with 1251-TGF-a,then partial inhibition (-50%) of TGF-a degradation is observed. Again, the net effect of adding the IDE inhibitors is to raise the level of undegraded TGF-a in the medium. Similar results were obtained with phenanthroline and other metalloprotease inhibitors, and the ID,,+ for inhibition of insulin and TGF-a degradation were comparable (Gehm and Rosner, submitted). Thus the substrate and inhibitor profiles of the TGF-a-degrading enzyme in vivo parallel those of the IDE in vitro. These results suggest that the human IDE or a sim-
58
M. RICH ROSNER
hIDE
134 ALDRFGQFFIAPLFTPSATEREINAVNSEHEiCNLPSDLWRIKQVNRHLAK 183 IIIIl:lli:.IlI..I..:II:1II:IIIlI1: .I - 1 1 : I::: :. 1 6 1 ALDRFAQFFLCPLE'DESCKDREVNAVDSEHEK"DAk7RLFQLEKATGN 210
dIDE
184 PDHAYSKFGSGXKTTQTEMPKS~KNIDVRDELLKFTSS 220
hIDE
211 PKHPFSKFGTGNKYTLETRPNQEGIDVRQELLKFHSA 2 4 7
dIDE
I.I::IIII.III
I
... I . ' . . I I I I : I I I I I
I.
Fig. 1. Partial comparison of deduced amino acid sequence of the Drosophila and human IDEs.
ilar enzyme can mediate degradation of TGF-a and insulin in vivo. These observations also raise a number of other possibilities. First, there may be multiple pathways for degradation of growth factors, such as EGF or TGF-a, that bind to the same receptor. Second, growth factor-degrading enzymes might be a mechanism for regulating the extracellular levels of one growth factor by another. For example, high extracellular insulin might lead to decreased TGF-a degradation. Finally, these results suggest that the insuliniTGF-a-degrading enzyme may play a role in controlling cell growth.
A ROLE FOR THE INSULIN/TGF-a-DEGRADING ENZYME IN DIFFERENTIATION A similar approach based on protease inhibitors was used by Kayalar and colleagues to investigate a possible role for the IDE in regulating muscle cell differentiation. The results of their studies suggest that IDE or a n enzyme with a n inhibitor profile like that of the IDE is required for differentiation of muscle cells (Kayalar and Wong, 1989). If IDE activity can influence the onset of differentiation, then i t is also possible that expression of the enzyme is regulated during development. To explore this possibility, we used the Drosophila system (Stoppelli et al., 1988). The IDE is expressed in the embryo, in the larvae, and in the adult head and body of the fruit fly as well a s in cultured Kc and Schneider L2 cells. When we looked at the relative expression of the enzyme in Drosophila, we found that it was expressed a t very low levels in the embryo compared to the adult organism. As a control, we put the IDE in extracts from the different adult and developmental stages to show that it is not differentially degraded in the various extracts during the experiment. We have compared the level of expression of the enzyme in embryo and adult in a number of ways. As well a s using the TGF-a cross linking assay, we have done immunoblotting with Drosophila antibody to the IDE and measured insulin-degrading activity. The difference in IDE expression between embryo and adult that we measured with these assays is about tenfold. The level of expression increases gradually from embryo to larva through pupa to adult. These results are consistent with a role for the IDE in differentiation. This enzyme is widely distributed in
Drosophila, it appears to be developmentally regulated, and the pattern of its expression suggests a specific role for this protein in the later stages of development.
CLONING THE TGF-dINSULINDEGRADING ENZYME To address the question of its role in growth and development in more detail, we decided to clone the Drosophila IDE. We used a number of different approaches. The most successful approach was to sequence some tryptic peptides from the enzyme and use these sequences to make oligonucleotide probes. We made two sets of oligonucleotide probes and screened about lo6 clones. From these screens, we found one weakly positive clone, which was about 2.8 kb long. We used this clone to rescreen the Drosophila library and then found three full-length clones of about 3.5 kb each. Using these clones, we can detect a major transcript of 3.6 kb by Northern analysis of mRNA from Kc cells. The human IDE gene has recently been cloned (Affholter e t al., 19881, and one of the two human transcripts is about the same size as the transcript we observed in Drosophila. To determine whether our clone encodes the Drosophila TGF-alinsulin-degrading enzyme, we have done in vitro translation using both reticulocyte and wheat germ systems. When we used the sense transcript in these systems, we observed a protein of 110 kDa, which was absent when we used the antisense transcript. The 110 kDa protein produced by in vitro translation from the sense transcript can be immunoprecipitated with anti-IDE antibody. Thus, the evidence indicates that we have a full-length cDNA clone for the Drosophila IDE, and this clone is able to express this protein in vitro (Kuo et al., submitted). Comparison of the deduced amino acid sequences from the human and Drosophila IDEs indicates that there is 50% identity (Fig. 1; for complete sequence, see Kuo et al., submitted). Even more striking is the fact that there is 70% similarity between these two sequences. Thus, there is very high conservation between the Drosophila and human enzymes. Gene bank searches revealed one other enzyme that shares significant sequence homology (27% identity, 48% similarity) with the human and Drosophila IDEs:
TGF-dINSULIN-DEGRADINGENZYME bacterial protease 111. Bacterial protease I11 is also a 110 kDa protein and a metalloproteinase, which is found in the periplasmic space. Although it is capable of degrading the insulin B chain, its function in bacteria is not yet known. In sum, these results suggest that the IDE is a member of a new evolutionarily conserved family of enzymes whose role in growth and development is just beginning to be elucidated.
ACKNOWLEDGMENTS I would like to acknowledge the colleagues who have done this work. Karol Thompson, a graduate student, first identified the Drosophila IDE, and J. Victor Garcia and M. Patrizia Stoppelli, postdoctoral associates, did the initial characterization and purification of the enzyme. Dr. Wen-Liang Kuo has cloned the Drosophila enzyme. Dr. Barry Gehm has been studying the in vivo degradation of TGF-a. Dr. William Duckworth of the University of Nebraska Medical Center collaborated on the characterization of the insulin cleavage sites. This work was supported in part by the U S . Public Health Service, National Institutes of Health, the American Diabetes Association, and the University of Chicago Diabetes Research and Training Center. QUESTIONS AND ANSWERS Q: This does not seem to be a glycosylated or secreted protein. Where is it located in the cell? A: You are right that i t is not glycosylated. In fact, the wheat germ experiment was done to distinguish it from the insulin receptor that is glycosylated and is able to bind to the lectin columns. The enzyme is primarily in the cytosol as far as we can tell. It is also membrane-associated and might be in vesicles. We initially found i t associated with the membrane. We have never detected it on the outer membrane of the cells. In Drosophila, we have evidence that it might be secreted. The IDE also has a topological problem t h a t I think you have alluded to, which is how does this enzyme get in contact with insulin and TGF-a? It has been found in preacidic endosomes of human cells following insulin binding to its recpetor and internalization. The IDE may be secreted and then reinternalized and thus come into contact with insulin. Q: What is your evidence that this enzyme actually degraded TGF-a? A: We observe a n increase in TCA solubility of the label attached to TGF-a. Also, the TGF-a does not bind its receptor after incubation with the enzyme, so i t is no longer a functional growth factor.
59
Q: Does the enzyme degrade the insulin or TGF-a receptors? A: We have not done that experiment directly. As far as we can tell, it does not degrade the receptor. The question that we want to ask is whether the enzyme affects the function of the receptor by decreasing the concentration of its ligand. Q: How specific is this enzyme for TGF-a and insulin? A: I t degrades IGF-I1 as well a s IGF-I, but it is not very active on the latter substrate. I t does not degrade bovine serum albumin (BSA) and many other randomly chosen proteins that we have tried. The enzyme was called a n insulin-degrading enzyme because it seemed to be very specific for the insulin family. Although I believe there may be other substrates that we have not found, the enzyme also shows a great deal of specificity by degrading TGF-a and not EGF. EGF and TGF-a are very similar in structure, yet we have not been able to detect significant degradation of EGF by the IDE. Q: Have you used immunocytochemistry to localize the enzyme in the cells? A: We attempted to do some immunocytochemistry with the antibody, but i t did not work out. There has been one study with human cells, and the results suggest that the enzyme is localized in the cytoplasm. However, I think t h a t the question of where this protein is localized is still open.
REFERENCES Affholter JA, Fried VA, Roth RA (1988); Science 242:1415-1418. Duckworth WC, Garcia JV, Liepnieks J J , Hamel FG, Hermodson MA, Frank BH, Rosner MR (1989): Biochemistry 28:2471-2477. Garcia JV, Fenton BW, Rosner MR (1988): Biochemistry 27:42374244. Garcia JV, Gehm BD, Rosner MR (1989a): J Cell Biol109:1301-1307. Garcia JV, Stoppelli MP, Decker SJ, Rosner MR (1989b): J Cell Biol 108:177-182. Garcia JV, Stoppelli MP, Thompson KL, Decker SJ, Rosner MR (1987): J Cell Biol 105:449-456. Kayalar C, Wong WT 11989): J Biol Chem 26423928-8934. Kuo WL, Gehm BD, Holmgren R, Rosner MR (1990): (submitted). Lax I, Johnson A, Hawk R, Sap J , Bellot F, Winkler M, Ullrich A, Vennstrom B, Schlessinger J, Givol D (1988): Mol Cell Biol, 8:19701978. Petruzelli L, Herrera R, Garcia R, Rosen OM (1985): Cancer Cells [Cold Spring Harbor Laboratory] Cold Spring Harbor, pp. 3:115121. Planck SR, Finch JS, Magun BE (1984): J Biol Chem 259:3053-3057. Schejter ED, Segal D, Glaser L, Shilo BZ (1986): Cell 46:1091-1101. Shii K, Roth RA (1986): Proc Natl Acad Sci USA 83:4147-4151. Shii K, Yokono K, Baba S, Roth RA (1986): Diabetes 35575-683. Stoppelli MP, Garcia JV, Decker SJ, Rosner MR (1988): Proc Natl Acad Sci USA 85:3469-3473. Thompson KL, Decker SJ,Rosner MR (1985): Proc Natl Acad Sci USA 82:8443-8447.
