Insights into the evolution of the ErbB receptor family and their ligands from sequence analysis
© Stein and Staros; licensee BioMed Central Ltd. 2006
Received: 09 June 2006
Accepted: 06 October 2006
Published: 06 October 2006
In the time since we presented the first molecular evolutionary study of the ErbB family of receptors and the EGF family of ligands, there has been a dramatic increase in genomic sequences available. We have utilized this greatly expanded data set in this study of the ErbB family of receptors and their ligands.
In our previous analysis we postulated that EGF family ligands could be characterized by the presence of a splice site in the coding region between the fourth and fifth cysteines of the EGF module and the placement of that module near the transmembrane domain. The recent identification of several new ligands for the ErbB receptors supports this characterization of an ErbB ligand; further, applying this characterization to available sequences suggests additional potential ligands for these receptors, the EGF modules from previously identified proteins: interphotoreceptor matrix proteoglycan-2, the alpha and beta subunit of meprin A, and mucins 3, 4, 12, and 17. The newly available sequences have caused some reorganizations of relationships among the ErbB ligand family, but they add support to the previous conclusion that three gene duplication events gave rise to the present family of four ErbB receptors among the tetrapods.
This study provides strong support for the hypothesis that the presence of an easily identifiable sequence motif can distinguish EGF family ligands from other EGF-like modules and reveals several potential new EGF family ligands. It also raises interesting questions about the evolution of ErbB2 and ErbB3: Does ErbB2 in teleosts function differently from ErbB2 in tetrapods in terms of ligand binding and intramolecular tethering? When did ErbB3 lose kinase activity, and what is the functional significance of the divergence of its kinase domain among teleosts?
The ErbB family of receptors is a diverse set of Type I receptor tyrosine kinases ubiquitously distributed throughout the animal kingdom. In vertebrates there are four family members, ErbB 1/EGF receptor, ErbB2/neu/HER2, ErbB3/HER3, and ErbB4/HER4, while in invertebrates only one receptor has been identified. The vertebrate ligands are more numerous and varied than the receptors and include, epidermal growth factor, transforming growth factor α, heparin-binding epidermal growth factor, amphiregulin, betacellulin, epiregulin, epigen, neuregulin 1–4, tomoregulin/TMEFF 1–2, and neuroglycan-C. In invertebrates, one ligand has been identified in Caenorhabditis, lin-3, while four ligands have been identified in Drosophila, vein, gurken, spitz, and keren.
We previously carried out an evolutionary analysis of the ErbB receptor and ligands , which was based on a more limited sequence data set than is currently available. In our analysis the order of gene duplications leading to the four mammalian receptors was supported by the known functions and interactions of the receptors, while the segregation of the mammalian ligands into EGF receptor ligands and ErbB3/ErbB4 ligands mirrored the receptor segregation. In addition, sequence comparison between different species and receptors suggested regions of the receptors that might lead to specific differences in function between the four different receptors.
Recent genomic sequencing from a variety of species should allow for a substantial expansion of the previous analysis, which focused mainly on the mammalian and specifically the human receptors. The completed or partial genomic sequences from zebrafish, fugu, tetraodon, xenopus, and chicken among other species, allow for the examination of sequence variation of additional branches of the vertebrates beyond the mammalian lineage and how these different branches compare to each other. Comparison of these additional sequences confirms our previous description of the gene duplication events for the receptors, while the additional ligands generate a more populated ligand tree that yields new perspectives about receptor specificity.
Results and discussion
Our earlier analysis suggested that EGF family ligands could be distinguished from non-ligand EGF motifs based on the presence of a splice site between the fourth and fifth cysteines within the six cysteine EGF-module and the placement of this module in close proximity to the transmembrane region of the potential ligand . Since our last analysis, several new ligands have been identified. One of these ligands, identified from a mouse keratinocyte expressed sequence tag library, has been termed epigen . The EGF-module occurs prior to a putative transmembrane region and examination of its chromosomal location indicates a splice site between the fourth and fifth cysteines. Two other ligands are very similar and have been called either tomoregulin 1 and 2 or TMEFF (transmembrane with an egf and two follistatin domains) 2 and 1 [3, 4]. Both of these ligands also have the proposed splice site and location relative to a putative transmembrane region. A report suggested that the EGF-module from neuroglycan-C is a ligand for ErbB3  and it has the proposed splice site and location relative to a putative transmembrane region. The chicken homologue to neuroglycan-C, CALEB, is noted in the databank to be chicken EGF (accession # CAA70459), but was first identified as a neural member of the EGF family and was shown to be associated with glial and neuronal tissues . In the invertebrates, keren was identified in Drosophila as a close homologue to the previously identified spitz . Of the newly discovered ligands, only keren, like its extensively characterized homologue spitz, does not have the proposed splice site, which likely reflects the general reduction of introns in the Drosophila genome.
In addition to the previously described ligands and the newly described ligands, this study has also identified additional EGF modules in previously described proteins that have the splice site between the fourth and fifth cysteines and are near putative transmembrane domains. These modules occur in mucin 3, 4, 12, and 17, meprin 1α and 1β, and interphotoreceptor matrix proteoglycan 2. Only one of these proteins, mucin 4, has been directly implicated in the activity of the ErbB receptor family. It has been shown that mucin 4 down regulates the signaling ability of ErbB2, though not as a secreted ligand, but as a membrane bound protein . Whether the other candidate ligands that we have identified act as direct ErbB receptor ligands or are capable of modulating their activity remains to be determined.
List of Ligands
c, ch, co, d, es, f, h, m, ma, o, p, r, ra, rh, rt(2), t, xt, z
ae, c, ca, ch, co, d, es, f, h, m, p, r, ra rh, s, t, xt, z
epidermal growth factor (EGF)
ae, c, ch, ct, d, dn, et, f, h, m, me, o, p, r, t, xt, z
c, ch, co, d, h, m, o, r, ra, xl(2), xt, z
c, ch, co, d, f, h, m, me(2), o, r, ra, rh, rt(2), t, xt, zf
da, de, di, dm, dp, dr, ds, dw, dy
heparin-binding epidermal growth factor (HB-EGF)
c, eg, ch, co, d, f, gm, h, m, ma, me, o, p, r, ra, rt, st, t, xt, z
interphotoreceptor matrix proteoglycan-2 (IMP2)
c, ch, co, d, f, h, m, o, r, rh, t, xt, z
da, de, dg, di, dj, dm, dp, dr, ds, dv, dw, dy, g
meprin 1α (MEP1α)
ae, c, ch, d, dn, f, h, m, o, r, rh, t, xl, xt, z
meprin 1β (MEP1β)
c, ch, co, d, f, h, m, o, p, r, rh, t, xl(2), xt, z
mucin 3 (MUC3)
co, h, m, r, rh, rt, xt(3), z
mucin 4 (MUC4)
c, co, d, h, m, o, r, xt
mucin 12 (MUC12)
c, co, d, h, m, ra, rh
mucin 17 (MUC17)
d, et, h, m, o, r, rh
ch, co, d, dn, gu, h, m, ma, o, p, r, ra, rt, xl, z
c, ch, co, d, h, m, o, r, xl, z
c, ch, co, d, et, f, gu, h, m, o, r, rh, t, z
c, ch, co, d, et, f, gu, h, m, o, r, rh, z
c, ch, co, d, es, f, gu, h, m, o, p, r, t, xt, z(2)
c, ch, co, f, gu, h, m, me, o, p, r, rh, rt, xt
ab, c, ch, co, d, f, h, m, me(2), o, r, rh, sh, xt, z
an, da, de, dg, di, dj, dm, dp, dr, ds, dw, dv, dy, hb, 1, tc, yf
c, ch, co, d, dn, es, et, f, h, m, o, p, r, xl, xt, z(2)
c, ch, co, d, f, h, m, o, p, r, rh, rt, t(2), xl, xt, z
transforming growth factor α (TGFα)
aa, ae, c, ch, co, d, dn, f, h, m, ma, o, or, p, r, ra, rh, sh, t, xl(2), xt(2), z
an, da, de, dg, di, dj, dm, dp, dr, ds, dv, dy, hb, yf
viral growth factor
ar, be; bp(2); cl(2), cp, ep(5), fp(2), gp(2), ls(2), mp, my, rf, rp, sa, sp(3), va(4), vc(5), yl
There are several additional features of the tree that are worth noting. One is the placement of the viral ligands within the tree. The orthopox ligands segregate with the EGF/EPR pair, avipox segregates with AR/HB-EGF, while the leporipox, yatapox, and capripox ligands segregate with NRG4. This segregation mirrors the ligand binding properties of the shope fibroma and myxoma growth factors (leporipox) that were found to bind to ErbB3 in the presence of ErbB2, though the shope fibroma growth factor was also able to bind to ErbB1, while vaccinia growth factor (orthopox) bound to ErbB1 . The variola growth factor (orthopox) was also found to only interact with ErbB1 . The different positions and binding specificities of the viral ligands raise questions of viral evolution, specifically with regard to viral hosts and reservoirs and when the different viruses acquired the different ligands. Additionally, the sequence analysis and tree generation suggests that the proteins termed muc3 for rat and mouse in NCBI (AAB83956 and AAH46639, respectively, but there are multiple accession numbers for mouse) are actually muc17 as has been detected in the automated protein screens for mouse (XP_355711). In addition the teleost amphibian mucins 3, 12, and 17 segregate separately from the rest of the mucins 3, 12, and 17. The branching pattern of these three mucins is comparable to a recent analysis of mucin phylogeny using different domains from the mucins .
Another feature of the tree is the apparent pairing of the ligands, suggestive of gene duplication events. Within the EGF receptor ligand branches these pairs include TR1/TR2, TGFα/BTC, AR/HB-EGF, and EPR/EGF. One interesting point about these apparent gene duplications is the differential receptor specificity for binding within each pair (Table 1). With the exception of the tomoregulins, which do not appear to follow this pattern, within each pair one is more specific for the EGF receptor (TGFα, AR, and EGF), while the other has a broader receptor specificity (BTC, HB-EGF, and EPR). Although, the functional significance of this apparent cross-specificity between ligand pairs is still unclear, it is suggestive of co-evolution of the ligands and receptors and the retained interdependent function after gene duplication in this family of receptors and ligands.
The TR1/TR2 pair has a different branching pattern (Fig. 4B), with both ligands in the teleost lineage segregating together and both tetrapod ligands segregating together. This pattern of branching could suggest independent gene duplications after the divergence of the two lineages or one gene duplication event that created the two ligands that then diverged with the divergence of the teleosts and tetrapods. It is noteworthy that the sequences labeled TR2 in the teleost lineage are two residues shorter than teleost and tetrapod TR1 and tetrapod TR2, which are the same length (Fig. 4C), supporting a difference in the requirement for sequence constancy between the two lineages, but it is unclear how this relates to the potential gene duplication events. In this comparison there are only sequences from teleosts and tetrapods, inclusion of sequences from additional orders might help differentiate these different possibilities. These different patterns of ligand evolution for the AR/HB-EGF and TR1/TR2 pairs argue against the indiscriminate extrapolation of function that the ligand might have in teleosts to its function in higher vertebrates, though this does not preclude a ligand from divergent lineages from having similar functions.
List of Receptors
an, c, cb, ce, ch, ci, co, cv, d, dm, dp, ds, ef, em, f, h, hb, m, op, p, r, rh, sm, t, xt, xx, yf, z
ch, co, d, f, h, m, ma, op, r, rh, t, xt, z
c, ch, co, d, f(2), h, m, o, op, r, rh, t(2), xt, z(2)
c, ch, d, f(2), h, m, r, rh, t(2), xt, z
In our previous analysis we noted the high conservation (~90% identity) between individual ErbB2 receptor sequences with two regions having less overall identity . Both of these less conserved regions align with sequences in the EGF receptor that are in close proximity to bound ligand [35, 36, 39, 40]. The addition of sequences from more diverse species does not yield new insights into the unconserved region located at the subdomain III-subdomain IV junction (Fig. 5, labeled A2), but does yield more insight into the region located in subdomain I (Fig. 5, labeled A1). This unconserved region, compared to the other three receptors, was noted as an insert in ErbB2. Interestingly, this insert does not occur in the teleosts or amphibians, suggesting that this insert occurred after the divergence of the amphibians and amniotes. It is not clear what role this insert might have in the loss of ligand binding, but it raises the question of whether the teleost or amphibian ErbB2 receptor is capable of binding ligand or whether it functions similarly to the mammalian receptor, as a dimerization partner without ligand.
The extracellular juxtamembrane region of ErbB4 also exhibits differences among species. In mammals this region exhibits alternative splicing  generating a long form and a short form (the long form is shown in Fig. 5, labeled B). There is a functional difference between the two isoforms, with the long form susceptible to inducible proteolytic cleavage, while the short form is insensitive to cleavage [41, 42]. Interestingly, only the short form is present in teleosts. The presence of the long form of ErbB4 in the tetrapods suggests an additional function or regulation of the ErbB4 receptor in tetrapods that is not present in teleosts.
It was noted previously that only one N-linked glycosylation site (N599, EGF receptor numbering) was conserved among all of the vertebrate receptors . Examination of the additional vertebrate receptor sequences currently available shows that all of these vertebrate sequences, except for the EGF receptor from X. tropicalis, contain this glycosylation site (Fig. 5, labeled C). It is unknown what role this glycosylation site might play in receptor maturation or function.
Since our previous analysis, the solution of crystal structures of the extracellular domains from the receptors [43–47] suggested a mechanism of ligand binding and receptor dimerization in which an intramolecular tether stabilizes the unliganded monomeric receptor and release of the tether allows a structural rearrangement permitting high affinity ligand binding and receptor dimerization . There are three main extracellular regions of the ErbB receptors that are involved in either tether formation or dimerization. Two regions are in the dimerization arm of subdomain II. One region in subdomain II is involved in both interactions; it makes contact with the second region in subdomain II from another monomer to form the dimer or with subdomain IV from the same monomer to form the tether. The residues in subdomain II of one monomer that are involved in interacting with the opposing subdomain II from a second monomer are Tyr246, Pro248, and Tyr251 (Fig. 5, residues labeled # (246) and & (248, 251); EGF receptor numbering). Tyr246 is conserved in all vertebrate receptors, while the amino acid at position 248 is Pro in EGF receptor, ErbB4, and teleost ErbB2, Lys in ErbB3, and predominantly Thr in ErbB2 from tetrapods. The amino acid at position 251 is Tyr in tetrapod EGF receptor, His in teleost EGF receptor, and Phe in ErbB2, ErbB3, and ErbB4. These three residues interact with several residues in the other monomer that include, Phe230, Phe263, Ala265, Tyr275, Cys283, and Arg285 (Fig. 5, residues labeled *; EGF receptor numbering). Positions 230 and 283 are invariant, while position 263 and 275 are either Phe or Tyr; 263 is Phe in EGF receptor and ErbB2, Tyr in ErbB4 and tetrapod ErbB3, and either Phe or Tyr in teleost ErbB3, while 275 is Tyr in EGF receptor and ErbB2 and Phe in ErbB3 and ErbB4. The amino acid at position 265 is Ala in EGF receptor, ErbB2, and ErbB4, while it is Gly in tetrapod ErbB3 and Ser in teleost ErbB3. Position 285 is Arg in EGF receptor, tetrapod ErbB3, and ErbB4, Leu in ErbB2 (except for zebrafish where it is Met), and Ser in teleost ErbB3 (except for one version in tetraodon where it is Arg). This pattern of amino acids at the positions that mediate the interaction between the two monomers most likely reflects the different preferences for homo- and heterodimerization. ErbB2 and ErbB3 exhibit little to no homodimerization; differences at these sites may contribute to the inability of these receptors to homodimerize.
The tether is formed by the intramolecular interactions between subdomain II and subdomain IV. The residues involved in this interaction are Tyr246, Asp563, His566, and Lys585 (Fig. 5, residues labeled #, +; EGF receptor numbering). Tyr246 is the same residue involved in the dimer interface discussed above. The amino acids at positions 563 and 585 are invariantly Asp and Lys, respectively, while 566 is His in EGF receptor and tetrapod ErbB3, Phe in teleost ErbB2, variable in tetrapod ErbB2, His or Tyr in teleost ErbB3, and Asn in ErbB4. The high conservation of these residues suggests that tether formation occurs in all receptors, with the possible exception of tetrapod ErbB2. The potential lack of tether formation in tetrapod ErbB2 is consistent with the crystal structure obtained for ErbB2, which is in an untethered monomeric, but dimer-competent conformation. The observed conservation in teleost ErbB2 of residues involved in tether formation raises the question as to whether it has the ability to form the tether and therefore functions differently than tetrapod ErbB2. This issue was raised earlier in consideration of the insert present in the ligand binding region of tetrapod ErbB2 but not in teleost ErbB2.
Mutagenic analyses of the receptor have shown that tether formation is important in ligand affinity [43, 49, 50]. It has recently been shown that the extent of tethering of the monomeric receptor can be measured with an antibody (m806) that recognizes a sequence in the EGF receptor that is not accessible in either the tethered monomeric state or the dimeric state . In addition, alteration of the sugar moieties affects the tethered state, with a decrease in oligosaccharide processing present in mutant or overexpressed receptors leading to an increase in the amount of untethered receptor . This suggests a potential role of receptor processing in receptor signaling. Recently, it was shown that in A431 epidermoid carcinoma cells there is incomplete glycosylation at Asn579 (EGF receptor numbering) , a site that is conserved only in tetrapod EGF receptor (Fig. 5, residue labeled %). Mutagenesis of this consensus glycosylation site (Asn579Gln) showed that the receptor without glycosylation at this site was more untethered than wt EGF receptor and had altered ligand binding, suggesting that the tethered receptor is stabilized by the presence of the N-linked oligosaccharides at this site . This might suggest that compared to the other receptors in the family, the tetrapod EGF receptors may have acquired an additional method of regulating signaling by modulating the extent of intramolecular tethering by glycosylation at Asn579.
The other regions previously highlighted fall within the kinase region of the receptors. We noted a lack of conservation in two regions within the kinase domain of the human receptors that correspond to the C-helix and activation loop (Fig. 5, labeled D1 and D2, respectively). Comparison of these regions from the additional species in this study supports the lack of conservation between receptor subtypes and points to additional receptor subtype differences in these regions. For the EGF receptor, ErbB2, and ErbB4 there is complete conservation of sequences in the C-helix (Fig. 5, labeled D1) within each receptor; while the teleost ErbB3 sequences have very little conservation and the tetrapod ErbB3 sequences have nearly complete conservation. Within this region the consensus sequences from ErbB3 vary greatly from those of the other three receptors; the other three receptor subtypes are over 50% identical. Similar to the C-helix, the region in the activation loop exhibits high conservation within each receptor subtype, except for ErbB3 from teleosts, with ErbB3 sharing very little identity with the other receptors (Fig. 5, labeled D2).
The remaining region of the kinase domain that we previously examined corresponds to the c-terminal portion of the kinase domain. What was observed was not a lack of conservation within this domain, but what appeared to be receptor subtype specific differences in particular residues in this region (Fig. 5, labeled E). The present analysis supports the identification of these residues and extends this region further into the kinase domain. The intracellular portion of the receptors that has been reported to mediate high affinity binding [55–57] corresponds to this region in the kinase domain. It was thought that this region was involved in either direct protein interactions with the other kinase domain within the dimer or that this interaction was mediated by an accessory protein.
Recently, a direct protein-protein interaction for this C-terminal region in kinase activation was found . Instead of forming a symmetric interaction that leads to kinase activation an asymmetric interaction was found in which only one of the kinase domains in the dimer is thought to be active at any one time. This asymmetric dimer occurs via the C-terminal region of one kinase that interacts with the C-helix and juxtamembrane region of the other kinase leading to the activation of this kinase within the dimer. These results elegantly explain certain characteristics of the ErbB receptor family, specifically the presence of the ligand-less dimerization partner ErbB2 and the kinase inactive, but functional ErbB3. While these results support the difference in the ErbB3 sequence in the C-helix compared to the other three receptors (Fig. 5, D1), the results do not explain the high conservation of these residues in tetrapod ErbB3. If this region is not needed for kinase activation, the high conservation of residues in this region would suggest that they may have another important functional role.
Examination of the ErbB receptor family and their ligands from both biochemical and evolutionary viewpoints yields insights into the functioning of the receptor and ligand families. The additional ligand sequences that have become available since our earlier analysis  support our characterization of an ErbB receptor ligand by the presence of a splice site in the coding region for the fourth and fifth cysteines and the placement of the EGF module near the transmembrane domain. These criteria were used to identify several potential new ErbB ligands in previously identified proteins. Except for the newly identified tomoregulins (which lack the conserved Arg before the sixth cysteine) the ligands segregate into canonical EGF receptor ligands and ErbB3/ErbB4 ligands. Except for the placement of the tomoregulins, this branching pattern is suggestive of an interesting co-evolution of the ligands and receptors.
Insight into the functioning of the ErbB receptors is gained by taking into account the evolution of the receptors. The additional receptor sequences used in this analysis support the previous conclusion that three gene duplication events led to the present set of four receptors in the tetrapods. The additional sequences also raise interesting questions about when ErbB2 lost its ligand binding capability and the role that it plays as a dimerization partner. Examination of residues involved in ligand recognition supports a general model of ligand binding, but x-ray crystal structures of ErbB3 and ErbB4 with bound ligands are needed to address whether the ErbB3/ErbB4 ligands bind similarly to their receptors and how subtle differences in ligand binding lead to differences in receptor signaling.
Protein sequences were obtained from GenBank at the National Center for Biotechnology Information, Ensembl, TIGR, or other public databases. Sequences were identified via Blast  searches utilizing full length receptors or EGF modules. For the ligands, only the EGF module was used because across the ligands this is the only conserved domain. These searches yielded a variety of sequences depending on the database being searched. Where these searches yielded predicted genes, comparisons of these genes to the human sequences were carried out to verify that the predicted genes were complete. This was especially important for receptor searches, since the automated gene predications can skip exons, especially short ones. The skipped exons were then identified in the parental DNA (contig, scaffold, or higher order sequence compilation) and these were then used to construct full length DNA sequences. Where only locations in the parental DNA were found, GENSCAN  was used to identify exons and splice sites. If in this procedure any exons were missed, the same procedure described above was carried out to obtain full length DNA sequences. The quality of the sequences used ranged from cDNA and est sequences up to at least 7X genomic coverage. This leads to the potential that proteins used in the analysis will have a certain error rate inversely proportional to the quality of the sequencing data. All DNA sequences (see Additional file 1 for accession numbers) were converted to amino acid sequences for subsequent analyses. Consensus sequences were derived by comparing the sequences at individual positions and calling that position conserved if the percentage of the most likely amino acid occurred above the desired threshold. In defining a consensus sequence, a residue only had to be in 75% of the sequences to take into account the potential errors in the sequences. The use of the 75% cutoff balances the potential for calling a residue conserved when it really is not against calling a residue not conserved due to poor sequence quality when it is conserved. Protein alignments were carried out using ClustalX  with no adjustment of the default parameters. Bootstrapping (500 replicates) was carried out using MEGA (version 3.1)  or the Phylip group of programs (version 3.5)  using neighbor-joining or minimum evolution methods and several models of amino acid substitution, including poisson correction and Jones, Taylor & Thornton (JTT). Several methods of analysis were carried out to minimize any potential problems of carrying out a phylogenic analysis on the short EGF module used in these analyses, though this does not guarantee the accuracy of the obtained trees.
We thank Drs. D. McCauley, D. Funk, and W. Eanes for critically reading an early version of this manuscript. This work was supported by a grant from the NIH (R01 GM55056).
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