From the Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom
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ABSTRACT |
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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--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.
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INTRODUCTION |
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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- (TGF
),
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
TGF-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.
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EXPERIMENTAL PROCEDURES |
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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.
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RESULTS |
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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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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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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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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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DISCUSSION |
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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 TGF 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 -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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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; TGF,
transforming growth factor-
; 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.
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REFERENCES |
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