In Vivo Analysis of Argos Structure-Function
SEQUENCE REQUIREMENTS FOR INHIBITION OF THE DROSOPHILA EPIDERMAL GROWTH FACTOR RECEPTOR*

Robert HowesDagger , Jonathan D. Wasserman, and Matthew Freeman§

From the Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The Drosophila Argos protein is the only known extracellular inhibitor of the epidermal growth factor receptor (EGFR). It is structurally related to the activating ligands, in that it is a secreted protein with a single epidermal growth factor (EGF) domain. To understand the mechanism of Argos inhibition, we have investigated which regions of the protein are essential. A series of deletions were made and tested in vivo; furthermore, by analyzing chimeric proteins between Argos and the activating ligand, Spitz (a transforming growth factor-alpha -like factor), we have examined what makes one inhibitory and the other activating. Our results reveal that Argos has structural requirements that differ from all known EGFR activating ligands; domains flanking the EGF domain are essential for its function. We have also defined the important regions of the atypical Argos EGF domain. The extended B-loop is necessary, whereas the C-loop can be replaced with the equivalent Spitz region without substantially affecting Argos function. Comparison of the argos genes from Drosophila melanogaster and the housefly, Musca domestica, supports our structure-function analysis. These studies are a prerequisite for understanding how Argos inhibits the Drosophila EGFR and provide a basis for designing mammalian EGFR inhibitors.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The epidermal growth factor receptor (EGFR)1 controls many aspects of animal growth, development, and cell proliferation (1). How these different roles are coordinated and their relative importance in normal development is not yet well understood, but it is clear that overactivation of the receptor in mammals is implicated in many forms of cancer (2-5). This overactivation can be caused by amplification of the receptor gene, by activating mutations in the receptor, or frequently by overexpression of activating ligands, which causes inappropriate autocrine signaling. The EGFR is a tyrosine kinase; it has an extracellular ligand binding domain and an intracellular kinase domain. As with other receptor tyrosine kinases, it forms dimers upon activation, and these then transphosphorylate each other, causing the activation of downstream signal transduction pathways (6). There are four members of the EGFR family in mammals (ErbB1-ErbB4), and they act in a complex set of overlapping functions, both as homo- and heterodimers (7). They are activated by several classes of ligand, including EGF itself, transforming growth factor-alpha (TGFalpha ), neuregulins, and others. The motif responsible for receptor activation in all known ligands is an EGF domain, comprising six characteristically spaced cysteines and a few other essential amino acids (8). Antagonists of the EGFR might have therapeutic potential, and much effort has been made to understand ligand binding and activation, to help design such inhibitors (9-14). Although several important regions have been identified, effective antagonists have remained elusive because of the apparent inability to uncouple receptor binding and activation; all mutants that still bind efficiently are activating.

Like its mammalian counterparts, the Drosophila EGFR regulates many aspects of development (15-21). In flies, there is only one such receptor and it is equally similar to all four of the erbB genes of mammals, suggesting that it is the prototype receptor of the mammalian family (22, 23). Its activating ligands fall into the recognized classes of mammalian ligands; Spitz and Gurken are TGFalpha -like molecules, whereas Vein resembles the neuregulins (24-26). There is also a unique extracellular inhibitor of the fly EGFR called Argos. Argos has the hallmarks of an EGFR ligand in that it is a secreted protein with a single EGF domain (albeit atypical) (27-29), and it has been shown to inhibit EGFR activation both in tissue culture and in the developing fly (30). EGFR activity is required for many aspects of fly development (20). Of relevance to this work, EGFR triggers the differentiation of cells in the eye and the wing vein (21, 31). Loss of activity in the pathway (e.g. by loss-of-function mutations in EGFR or its activating ligands, or hyperactivity of Argos) causes loss of these structures; gain of activity (e.g. by loss-of-function mutations in argos) causes extra cells in the eye and wing veins.

We would like to understand the mechanism of EGFR inhibition by Argos for theoretical and practical reasons. Our goal is to identify which parts of the molecule are necessary for its function to help design mammalian EGFR antagonists. To these ends, we have explored which parts of the Argos protein are essential for its inhibitory function. We have also compared it with the activating ligand, Spitz, to understand the structural elements that make one an activator and the other an inhibitor of the EGF receptor. With a similar goal, two groups have recently reported attempts to make chimeras between human EGF and the EGF domain of Argos, but neither succeeded in producing an inhibitor of the human EGFR (32, 33). Our results suggest several reasons for this; although the atypical EGF domain is indeed a key element of Argos's inhibitory functions, other regions of the protein are also essential. These findings distinguish Argos from all other EGF-like factors, which appear to interact with the EGFR solely through the EGF domain; they also suggest that rational design of EGFR inhibitors should eventually be possible.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmid Construction-- The plasmid pSKargos was created by cloning a 972-base pair EcoRI-BamHI and a 735-base pair BamHI-XmnI argos cDNA (corresponding to nucleotides 1428-3128 in Freeman et al. (27)) into pBluescript (Stratagene) digested with EcoRI and EcoRV. This was cloned directionally into pKB267PL (gift of M. Mlodzik, EMBL, Heidelberg, Germany) for expression in the eye under the control of the sevenless enhancer. Ectopic expression of constructs was also achieved using the Gal4/UAS system (34). The argos cDNA was cloned as a 1786-base pair EagI-Asp718 fragment from pSKargos into NotI-Asp718-digested pUAST (34) to produce the plasmid pUAS-Argos.

All mutant plasmids were created using PCR-based mutagenesis with Pfu polymerase (Stratagene) and cloned into pBluescript SKII+ (Stratagene). Each mutant was sequenced to confirm that the expected mutations were present and that no errors had been incorporated during the PCR reaction. Standard protocols were used in all the cloning steps (35). See Figs. 1A, 2A, and 3B for coordinates of residues altered.

The constructs NTD1, NTD2, NTD3, ASA, and SAS were cloned into pKB267PL. The following constructs were cloned into pUAST: NTD1, NTD2, NTD3, NTD4, CTD3, CTD4, CTD5, CTD6, AS, SA, A3S4A, A4S6A, S3A4S, and S4A6S.

For expression in S2 cells, constructs were cloned into the pRmHa3 metallothionein vector (36). Proteins encoded by the pMtNTD2, pMtNTD3, and pMtNTD4 plasmids cannot be detected by anti-Argos antibodies so a Myc tag (EQKLISEEDLN) was introduced, by PCR mutagenesis, between Pro-29 and Leu-30 (37). This produced the plasmids pMtNTD2myc, pMtNTD3myc, and pMtNTD4myc, respectively. Details of all cloning strategies are available upon request.

All constructs were introduced into Drosophila by P-element-mediated transformation using standard techniques into host embryos of the genotype yw. Several independent transformants were selected for each transposon.

Cloning a Musca Homologue of Argos-- PCR reactions (200 µM dNTPs, 1 × Taq buffer, 10 pmol of each primer, 10 ng of genomic DNA, and 5 × PCR Compatible Loading Dye; Ref. 38) were performed on a Stratagene RoboCycler Gradient 96 using the following conditions: 2.5 min at 94 °C followed by 35 cycles of 90 s at 94 °C, 90 s at 37-48 °C or 44-66 °C, and 90 s at 52 °C. A final extension of 5 min at 72 °C was also used. The forward primer GARACICCITGGATHGARMG (standard degenerate code; I = inosine) corresponds to peptide ETPWIER, and the reverse primer (GTIARIGTCCAIGTRTARTC) corresponds to peptide DYTWT(L/F)T. PCR products were gel-purified and cloned into pGEM-T (Promega) according to manufacturer's directions. At least three independent clones per PCR product were sequenced. The fragment isolated by PCR was used to probe a M. domestica adult female cDNA library (generously provided by Daniel Bopp, University of Zurich, Zurich, Switzerland) by standard techniques; a full-length cDNA was isolated and sequenced. Alignment of sequences was performed using ClustalW 1.6 (39).

Histology-- Scanning electron micrographs were performed as described previously (40). Cobalt sulfide staining of pupal retinae was performed using the method of Wolff and Ready (41).

Fly Strains-- All crosses were performed at 25 °C. For rescue of argos eye mutants, y w;P[w+;UAS-constructX]/+;argosgil5(or gil33)/TM3 Sb males were crossed to w/w;+;sev-Gal4 argosgil5/argosgil5 females. Red-eyed, Sb+ progeny (genotype w;P[w+;UAS-constructX]/+;sev-Gal4 argosgil5(or gil33)/argosgil5) were compared with their white-eyed Sb+ siblings (genotype w;+;sev-Gal4 argosgil5(or gil33)/argosgil5). Rescue of spitz eye mutants was performed in a similar manner.

Tissue Culture-- Schneider line 2 (S2) cells were grown in Schneider's medium (Sigma), supplemented with 15% fetal clone serum (HyClone Laboratories, Logan, UT), with air as the gas phase (42). Stable cell lines were established using calcium phosphate-DNA co-precipitation (43). The pMt constructs described above were cotransfected with the pMK33 plasmid (44), conferring hygromycin resistance, followed by selection in 200 µg/ml hygromycin B (Calbiochem). All cell lines were used as pools of transfected cells for expression purposes.

Cells were incubated with 750 µM CuSO4 in serum-free Schneider's medium to induce expression from the copper-inducible metallothionein promoter. Culture supernatants were collected after 72 h and assayed for protein production by Western blotting, following standard procedures, using either monoclonal anti-Argos or monoclonal anti-Myc primary antibodies (45-47).

Expression of Proteins in Flies-- Embryos of the genotype HSP-70-Gal4/UAS-(Argos mutant) were heat-shocked for 1 h at 38 °C and allowed to recover for 3 h. Extracts were Western-blotted with monoclonal anti-Argos by standard techniques.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

N- and C-terminal Truncations of Argos-- Argos is 444 amino acids long, and the EGF domain of about 50 residues is close to the C terminus (Fig. 1A) (27-29). We have shown previously that this domain is necessary for inhibition (30). To examine the function of the rest of the protein and to discover whether the EGF domain is sufficient for Argos function, we have made a series of N-terminal and C-terminal deletions (Fig. 1A). All the constructs retained the N-terminal 31 residues of Argos, which include the signal peptide required for secretion. All were assayed in vivo by expression in flies using P-element-mediated transformation; their ability to rescue argos reduction-of-function mutations was assessed. We chose this test of function over the tissue culture assay as we have found it more reliable; it is an unambiguous test of activating or inhibiting activity (see below). Furthermore, the in vivo assays are, by definition, performed under native physiological conditions; our understanding of the Argos mechanism is not sufficient to be sure that all normal components are present in the tissue culture assay. To estimate the relative potency of different deletion constructs, we examined the rescue of strong (argosgil5/argosgil5), medium (argosgil5/argosgil33), and weak (argosgil33/argosgil33) mutations that affect eye development. Full-length Argos expressed in this assay completely rescues even the strong mutant phenotype (46).


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Fig. 1.   In vivo activity of N- and C-terminal truncations of Argos. A, the N- and C-terminal deletions that we tested are shown against a schematic of the full-length Argos protein. The coordinates correspond to the amino acid numbering given in Freeman et al. (27). The signal peptide is shown in black; A1 and A2 are two domains that resemble a portion of the Argos EGF domain; they correspond to amino acids 141-164 and 307-343, as numbered in Fig. 5. The six cysteines of the EGF domain span residues 370-413. B-G, scanning electron micrographs of Drosophila eyes, of the following genotypes: B, wild type; C, argosgil5/argosgil5; D, sev-NTD2;argosgil5/argosgil5; E, sev-NTD3; argosgil33/argosgil5; F, argosgil33/argosgil5; G, sev-CTD6; argosgil33/argosgil5. The regular hexagonal array of facets in the wild-type compound eye (B) is severely disrupted when argos is reduced (C), but this phenotype is rescued by expression of NTD2. The bigger deletion, NTD3, is able to rescue partially (E) the less rough eye caused by moderate argos mutation (F), whereas the small C-terminal truncation, CTD6, has no rescuing activity (G).

Deletion of a considerable portion of the N terminus of Argos (the first 194 amino acids of the mature protein; corresponding to NTD2) does not affect its ability to rescue strong eye mutations (Fig. 1, C and D). The next deletion in the series, NTD3, has the N-terminal 271 amino acids removed (not including the 31 residues in the signal peptide domain) and also has some rescuing activity (Fig. 1E), implying that the remaining 142 amino acids retain substantial inhibitory function. This assay is an unambiguous test of its inhibitory function, as conversion of the mutant protein into an activator would have led to enhancement of the rough-eye phenotype instead of its suppression. The construct with the longest deletion, NTD4, which truncates the protein very close to the EGF domain (Fig. 1A), has no activity. We have subsequently shown, however, that it is not secreted (see below) so we cannot ascertain whether the protein has intrinsic function.

The Argos C terminus is longer than that of known EGFR activating ligands; it extends 30 amino acids beyond the last cysteine of the EGF domain. Truncations within this region disrupt Argos function completely. Removing only the last 10 amino acids (CTD6) renders the protein unable to rescue any of the mutants (Fig. 1, F and G); furthermore, CTD5 and CTD6 have no activity in any other assay of Argos function (data not shown).

We draw the following conclusions from these deletion mutants. First, the N-terminal 271 amino acids are not essential for the inhibitory function of Argos, although they are necessary for its full potency. Because of the apparent failure of NTD4 to be secreted, we were unable to ascertain in this experiment whether sequences immediately N-terminal to the EGF domain are required (however, other evidence suggests that they are; see below). Second, the extended C-terminal domain of Argos, beyond the EGF motif, is essential for its function. The precise composition of the C terminus of other EGF-related growth factors is important for their activity. Argos is, however, distinct by virtue of the length of the C-terminal domain needed: 30 amino acids compared with, for example, 6 in amphiregulin (48) .

Chimeras between Argos and Spitz-- To examine more directly the elements that make Spitz activating and Argos inhibitory, we made a series of chimeras between the two proteins and assayed their function. The ability to rescue (or enhance) an argos or spitz mutant eye phenotype was assessed. In a separate in vivo assay, the chimeras were ectopically expressed in the developing eye and wing, and their effects compared with those of wild-type Spitz and Argos.

In one set of chimeras, we swapped the entire EGF domains of Spitz and Argos (Fig. 2). The SAS and ASA pair (in which only the EGF domains themselves were swapped; Fig. 2A, i and ii) were designed to test the possibility that the EGF domain is solely an EGFR binding domain, with no role in activation or inhibition. If this were the case, the EGF domains would be interchangeable and SAS might behave as Spitz, and ASA as Argos. In the SA and AS pair (Fig. 2A, iii and iv), we swapped the whole C termini of Spitz and Argos, including the EGF domains. If the Argos EGF domain and the adjacent C terminus were sufficient to confer its inhibitory function, the SA chimera (Spitz N terminus with the Argos EGF domain and C terminus; Fig. 2A, iv) might be expected to behave as Argos. Similarly, since the N terminus of Argos appears not to contain sufficient information to confer inhibitory function (see above), the AS chimera (Argos N terminus with the Spitz EGF domain and C terminus of the secreted form; Fig. 2A, iii) might retain Spitz activity . 


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Fig. 2.   Activity of chimeras between intact EGF domains of Argos and Spitz. A, the structures of the chimeras ASA, SAS, AS, and SA (i-iv) are shown with that of full-length Argos and the mature form of Spitz (like TGFalpha , Spitz is synthesized as a transmembrane protein but is only an active ligand when proteolytically cleaved from the cell surface; Refs. 68 and 69). B-E show Drosophila wings. B, a wild-type wing has a stereotypical pattern of five longitudinal veins (L1-L5). C, overexpression of wild-type Argos (using the GAl4/UAS system (34), driven by the Gal4 line MS1096 (70), which is expressed in the developing wing blade) causes the loss of most of the wing veins, due to inhibition of the EGFR pathway. D, conversely, increased signaling, caused by overexpression of the EGFR, also under MS1096 control, causes the formation of extra veins. E, a similar extra-vein phenotype is caused by the overexpression, under MS1096 control, of the AS chimera; note the extra vein material at the wing margin (examples indicated with arrowhead). F and G are scanning electron micrographs of eyes of the genotypes spiscp1/spiscp1 (F) and spiscp1/spiscp1; sev-Gal4/UAS-AS (G). The expression of the AS chimera partially rescues the rough eye caused by spitz reduction (compare with the regular array of facets in a wild-type eye; Fig. 1B).

Neither SAS nor ASA has any activity; they neither suppress nor enhance the argos mutant phenotype. This suggests that the EGF domain is more than simply an EGFR binding domain; instead, it participates in specifying whether the receptor is activated or inhibited. SA also has no Argos activity (nor, as expected, is it Spitz-like). This implies that sequences N-terminal to the Argos EGF domain are necessary for its function.

The AS chimera activates the EGF receptor, suggesting its function is similar to Spitz, not Argos. When overexpressed in the wing, it produced the characteristic extra vein phenotype of EGFR activation (Fig. 2, B, D, and E). In this same assay, overexpression of Argos causes the opposite phenotype, namely loss of wing veins (Fig. 2C). These results indicate that the large N-terminal region of Argos is not sufficient to convert Spitz into an inhibitor. AS, however, rescues spitz eye mutants only partially (Fig. 2, F and G), suggesting that Spitz's potency is reduced by the addition of 355 amino acids of the Argos N terminus.

A second set of chimeras focused on the structural differences between the Argos and Spitz EGF domains themselves. The spacing between the six essential cysteines in EGF domains varies only slightly. In particular, between C3 and C4 (the B-loop), there are usually 10 amino acids in EGF-like ligands, and the range is 8-13 in all known EGF repeats (8). In Argos, there are 20 residues between C3 and C4, and this extension might account for Argos's unusual inhibitory function (Fig. 3A). Indeed, although the function of this B-loop remains uncertain, many studies have implicated it in EGF binding and activation of the receptor (11, 49-51). The C-loop of EGF domains, between cysteines 5 and 6, is also critical for the function of EGF-like ligands (10). In Argos, the C-loop is also atypical. It is only five residues long, compared with eight in Spitz and all the mammalian activating ligands, and it has two extra positive charges (Fig. 3A). To examine the functional importance of the Argos B- and C-loops, we constructed four chimeras between Argos and Spitz in which the B- and C-loops were exchanged (Fig. 3B, i-iv), and tested whether they activated or inhibited the EGFR.


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Fig. 3.   Activity of chimeras between regions of the EGF domains of Argos and Spitz. A, alignment of the EGF domains of Argos, Spitz, human EGF, and TGFalpha . The putative pattern of disulfide linking between cysteines 1 and 6 is shown; this causes the domain to have three loops, A, B, and C. Note that Argos has an extended B-loop and a reduced C-loop, compared with the activating ligands. All four factors have the six cysteines and the residues Gly-39 and Arg-41 (shaded, numbered according to human EGF), which are known to be essential for receptor binding. B, the structures of the four intra-EGF chimeras A3S4A, S3A4S, A4S6A, and S4A6S (i-iv); Argos sequences are shown in black, Spitz in white. Note that these diagrams show only the EGF domain of each chimera; the color of the arrowheads indicates the source of the rest of the protein. C, scanning electron micrograph of an eye expressing UAS-A4S6A under the control of the GMR-Gal4 driver (21), which is expressed in all the cells of the developing eye; the eye is severely disrupted in size and pattern. D, cobalt sulfide-stained retina from a pupa overexpressing A4S6A; compared with a wild-type retina (E), loss of cone cells (arrowhead) and the surrounding lattice of pigment cells is apparent. This phenotype is caused by reducing EGFR signaling (21). F, a wing overexpressing A4S6A under the control of the MS1096 Gal4 driver. Wing veins are lost as when wild-type Argos is similarly misexpressed (compare with Fig. 2, B and C). G, overexpression of the A3S4A chimera, under the control of the GMR-Gal4 driver, has no effect on eye development, indicating that replacement of the Argos B-loop causes the protein to be non-functional.

When the B- or C-loop of Spitz is replaced by the corresponding Argos sequence (S3A4S and S4A6S), the chimeras have no activity. This might be expected if these domains are critical for EGFR activation by Spitz, as they are in other activating ligands. Despite being unusual, the Argos C-loop does have the glycine and arginine residues (corresponding to Gly-39 and Arg-41 of human EGF) that are essential for the activating ligands (Fig. 3A). The failure of the Argos C-loop to replace functionally that from Spitz must therefore be due to more subtle reasons, e.g. its reduced length or increased positive charge. Spitz is not converted into an inhibitor by insertion of either the B- or C-loop from Argos, consistent with our finding that sequences outside the EGF domain are essential for Argos function. Surprisingly, Argos with the Spitz C-loop (which is very similar to that from human EGF) still functions as an inhibitor; its overexpression in the eye causes loss of cells and, in the wing, loss of veins, which are characteristic argos phenotypes (Fig. 3, C-F). Furthermore, this A4S6A chimera can rescue moderate argos eye mutations (data not shown). In contrast, insertion of the Spitz B-loop removes all activity from Argos (Fig. 3G). We conclude from these two chimeras that the Argos B-loop is essential for its ability to inhibit the EGFR but the C-loop is not, despite its atypical composition.

Expression and Secretion of the Mutant Proteins-- To ensure that the loss of function in vivo of the various deleted and chimeric proteins was not caused by a failure of secretion from the cell, we expressed each of the proteins in Drosophila S2 tissue culture cells and tested the medium for the accumulation of a protein of the expected size. Most of the mutant proteins are secreted, although with varying efficiency (Fig. 4). The only protein that cannot be detected in the medium is NTD4, which corresponds to the most extensive N-terminal deletion. Consequently, we do not know whether this "minimal Argos" has inhibitory activity.


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Fig. 4.   Secretion and expression of the mutant forms of Argos. The constructs encoding key mutants were expressed in Drosophila S2 cells in culture and the medium was assayed by Western blot for the presence of secreted protein of the appropriate length. A, the mutants in this panel were detected by an anti-Argos monoclonal antibody; all are secreted, although at somewhat different levels. Note that the secretion level does not correlate with the observed in vivo results (for example, NTD1 is fully active in vivo but is secreted less well than CTD5, which is inactive). Therefore, secretion efficiencies cannot account for our results. B, NTD2, NTD3, and NTD4 all lack the epitope recognized by the anti-Argos monoclonal so these were expressed as Myc epitope-tagged proteins and detected with an anti-Myc epitope antibody. NTD2myc and NTD3myc are secreted efficiently; NTD4myc can only be detected in the cell pellet, not in the supernatant, indicating that it is not secreted. C, the mutants were expressed in flies (see "Experimental Procedures") and detected by Western blot with monoclonal anti-Argos. We were only able to detect proteins that had the epitope for the Argos monoclonal since we do not have an antibody against Spitz that recognizes the chimeric proteins on Western blots. All the proteins are detected, indicating that they are produced and relatively stable in vivo. Note that, as with secretion levels, the variation in expression does not correlate with in vivo activity.

To check that the mutant proteins are synthesized and relatively stable in flies, we expressed them under the control of the inducible HSP-70 promoter and assayed their levels in embryonic extracts 3 h after induction (Fig. 4C). Of the proteins tested, all were expressed at substantial levels. These results are supported by in vitro data showing that the activity of the proteins correlates well with the in vivo data presented here.2 Only the two chimeras SA and SAS (Fig. 2A) have been neither detected by Western blot (due to the lack of a suitable antibody) nor assayed for in vitro activity. It is therefore possible that they lack activity for the trivial reason that they are not stably expressed, although we consider this unlikely given the stability of all the other chimeras.

Sequence Conservation between Drosophila and Musca-- By comparing gene sequences between different species, it is possible to deduce which parts of a protein are functional; these regions are under selective pressure to be conserved. We therefore cloned Argos from the housefly, Musca domestica, which diverged from Drosophila melanogaster between 100 and 150 million years ago (52, 53). We believe this to be a true argos homologue for the following reasons. First, the overall similarity to argos is clear, both in amino acid sequence and overall structure. Second, it is the only Musca gene related to argos that we have found, despite extensive PCR, cDNA library screening, and genomic Southern blots. Third, we have also cloned argos genes from insects more closely related to Drosophila3 and exactly the same pattern of conservation is seen. Fourth, this gene has several characteristics that are specifically argos-like, and quite dissimilar from all other EGF domain-containing proteins. The Drosophila and Musca argos genes are 62% identical, but this overall similarity masks very different degrees of conservation in different parts of the protein (Fig. 5). The most similar region is the C-terminal third of the protein (88% identical over the last 162 amino acids), which includes all the regions that our mutational analysis suggests are necessary for Argos function. In contrast, apart from a single highly conserved block of 41 amino acids, the N-terminal 281 amino acids are only 39% identical: low conservation for genes that diverged relatively recently, implying little selective pressure.


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Fig. 5.   Alignment of the argos gene from Drosophila and Musca. The alignment of the two protein sequences is shown with gaps introduced to optimize the identity between the two; identical residues are highlighted in black and conservative substitutions in gray. The EGF domain is underlined, and the A1 and A2 repeats are indicated with a dashed underline. The residue corresponding to Arg-41 in human EGF is indicated with an asterisk. GenBankTM accession number of M. argos is AF038405.

The Argos EGF domain is 93% identical between Drosophila and Musca, and two of the four differences are conservative. It is surprising that one of the changes is of an arginine that is essential for all known EGFR binding ligands (Arg-41 in human EGF; asterisk in Fig. 5); it is replaced by a histidine in Musca. This same change has been tried in human EGF and reduces the activity of the protein by more than 100-fold (10). In support of our conclusion that regions flanking the EGF domain are essential for its function, they are also strikingly conserved; the C terminus is identical in the two species apart from two conservative changes, and the 87 amino acids N-terminal to the EGF domain are very similar. The one substantial block of identity within the highly diverged N-terminal half of the protein corresponds to one of two EGF-related motifs (A1 and A2) outside the true EGF domain. These both have some of the characteristically spaced cysteines of the Argos EGF domain but not the full complement to allow the presumed disulfide linking. A1 is 100% identical in the two species, and A2, which is adjacent to the true EGF domain, is also highly conserved. We do not know the significance of these structures, but their conservation suggests that they have a function. Nevertheless, the removal of A1 in our N-terminal truncation NTD2 has little effect on its potency as an inhibitor in vivo (Fig. 1D).

The comparison between Argos sequences from Drosophila and Musca provides a valuable complement to our mutational analysis. It supports the idea that the key functional parts of Argos include not only the unusual EGF domain, but also its flanking sequences; it also suggests that the idiosyncratic spacing of the cysteines within the EGF domain is significant (Fig. 6). In summary, our results are mutually consistent and point to there being substantial differences between the structural requirements of Argos and the known activating ligands of the EGFR.


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Fig. 6.   Summary of regions essential for Argos function. The full-length Drosophila protein is shown. Beneath, the regions of high conservation between Drosophila and Musca are indicated with the percentage of identical residues. The bottom line delineates which regions of the protein we have found to be essential in our structure-function analysis: the dashed line indicates the uncertainty of how much sequence N-terminal to the EGF domain is necessary, and the white box corresponds to the C-loop, which is replaceable with the corresponding sequence from Spitz.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The wide range of functions of the members of the EGF receptor family is reflected in their large number of different activating ligands (54-59). The mature forms of EGF and TGFalpha are polypeptides that comprise a single EGF domain and little else (8). The neuregulins are much larger; they include a C-terminal EGF domain and a long N-terminal region, which may modulate their action (59). A minimal EGF domain from the neuregulins is sufficient to bind and activate their receptor (60, 61). Amphiregulin and heparin-binding EGF have very basic domains immediately N-terminal to their EGF domains, which are necessary for heparin binding and full biological activity (62, 63). In this paper, we have shown that Argos, which is extended N- and C-terminally compared with EGF, and which is the only known EGF receptor inhibitor, has different structural requirements to any of the known activating ligands. For Argos to be an effective inhibitor, it needs its own EGF domain, the whole adjacent C terminus of the protein, and some of the region immediately N-terminal to the EGF domain (note that this region does not resemble the lysine-arginine rich heparin-binding regions of amphiregulin and heparin-binding EGF).

Recently, the only other known inhibitor of an receptor tyrosine kinase has been identified. This factor, angiopoietin-2, is structurally very similar to the activating ligand for the Tie-2 receptor, which it blocks (64). Although Ang-2 binds the receptor, it fails to activate it, providing a simple explanation for its antagonistic function. The mechanism of Argos function must be more complex since it will inhibit ligand-independent activation of the receptor, which can be induced in tissue culture cells (30). This implies that Argos does not act by binding to and sequestering ligand, nor does it simply block the ligand binding site on the receptor. Receptor tyrosine kinases are activated by ligand-induced dimerization or oligomerization (65), suggesting a simple mechanism for blocking receptor activation; we hypothesize that Argos inhibits EGFR dimerization. Our discovery that sequences from outside the EGF domain are essential for its function suggests that these extra domains participate in the hypothesized block to receptor dimerization. Note that, although there is strong circumstantial evidence that Argos acts directly on the receptor, the biochemical demonstration has yet to be reported. It is, however, clear that the inhibition occurs at the receptor itself (30), so the general proposal that Argos acts by causing a block to EGFR dimerization could hold even if it works indirectly.

As well as identifying new functional domains of Argos, our results shed light on which parts of its EGF domain are essential for Argos to inhibit the receptor. The extended B-loop, which includes the C3-C4 stretch of amino acids, is an obvious candidate for being involved in Argos's unusual inhibitory action. We have shown that if this loop is replaced by the corresponding region of the Spitz EGF domain, which resembles human EGF, Argos function is abolished. This supports the idea that this region is necessary and participates in Argos inhibition. The B-loop in Musca Argos is of identical length and near-identical sequence to Drosophila Argos, consistent with its functional importance. Indeed, this spacing is also identical in Argos from the butterfly, Precis coenia, which diverged from Drosophila more than 200 million years ago,4 further supporting this conclusion. Interestingly, myxoma virus growth factor, which is related to EGF, has a slightly extended B-loop (13 amino acid residues) and activates the EGFR 200-fold less effectively than EGF (66). Despite intensive study, the role of this domain, which forms a beta -sheet in human EGF, is still not clear, but there is substantial evidence implicating it in receptor binding and/or activation (11, 49-51).

The C-loop of EGF domains also contains critical residues for EGFR activation (9, 13, 67). Both Spitz and Argos contain the two key amino acids (Gly-39 and Arg-41 from human EGF; Fig. 3A), but in other ways the Argos C-loop looks very different from all known activating ligands, including Spitz. It is shorter and has increased positive charge; it is also identical in Musca, so it is surprising that replacing it with the more typical Spitz C-loop has little effect on Argos function. Perhaps the C-loop is necessary for receptor binding, but not for conferring inhibitory properties, making it is replaceable by a similar loop from another Drosophila EGFR-binding protein, Spitz.

A naturally occurring EGFR inhibitor like Argos might have therapeutic value in humans. We have failed to isolate mammalian homologues of Argos by various techniques, and it is possible that an EGFR inhibitor has evolved only in insects. We think it more likely that the conservation of EGF-like domains is not high enough over long enough stretches (their main conserved features are the spacing of cysteines and a few other critical amino acids) to make them easy to clone over long evolutionary distances. (Drosophila diverged from vertebrates 600-1000 million years ago). We are more hopeful of engineering an antagonist based on known mammalian ligands, as has been tried by several groups (9, 14, 32, 33), and our goal is to exploit Argos and Spitz to gain the necessary insight into the mechanisms of EGFR activation and inhibition.

    ACKNOWLEDGEMENTS

We are grateful to Myriam Golembo and Ben-Zion Shilo for help with sequencing some of the constructs and Richard Smith for excellent technical assistance. Daniel Bopp and René Feyereisen generously sent us M. domestica cDNA libraries, and David Micklem and Michael Akam helped greatly with our attempts to clone Argos from various species. Tony Burgess at the Cambridge University Anatomy Department performed the scanning electron microscopy. We thank Sean Munro and Rob Kay for their comments on the manuscript.

    FOOTNOTES

* This work was supported in part by a grant from the UK/Israel Science and Technology Research Fund of the British Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Howard Hughes Medical Institute and Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305.

§ To whom correspondence should be addressed. Tel.: 44-1223-402351; Fax: 44-1223-412142; E-mail: mf1{at}mrc-lmb.cam.ac.uk.

1 The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; TGFalpha , transforming growth factor-alpha ; S2, Schneider's line 2; PCR, polymerase chain reaction.

2 R. Howes and M. Freeman, unpublished results.

3 J. D. Wasserman and M. Freeman, unpublished results.

4 M. Kengaku and C. Tabin, personal communication.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
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