A unified model of c-erbB receptor homo- and heterodimerisation.

The c-erbB receptor tyrosine kinase family plays an important role in cell regulation. Receptor activation proceeds by the formation of receptor homo- and/or hetero-dimers and is promoted by the binding of a cognate ligand at the cell surface. While some experimental work has demonstrated that the formation of heterodimers can influence a cellular response, the extent of heterodimerisation has not been accurately assessed: the assortment of receptors and ligands gives rise to a complex combinatorial system for which intuitive prediction of homo- and hetero-dimerisation is difficult. We present a mathematical model which combines observations for homo-dimerisation with the additional interactions arising from the presence of multiple c-erbB receptors. We provide a simple explanation for the apparently conflicting results for binding studies carried out with either solubilised receptors, vesicles or cells and our model predicts binding behaviour which is compatible with published experimental findings for cells expressing either one or two c-erbB receptors. This model establishes the basis for interpretation of ligand binding experiments, where variations in the apparent ligand affinity can be attributed to changes in receptor expression or ligand preferences according to the binding profile.

Modulation of the receptor binding affinity of amphiregulin by modification of its carboxyl terminal tail.

Amphiregulin (AR), a heparin-binding, epidermal growth factor (EGF) receptor ligand has homology with EGF but exhibits a lower affinity for the EGF receptor than EGF. As the mature form of AR is truncated at the C terminus and lacks a conserved leucine residue known to be essential for high affinity binding of EGF to the EGF receptor, wild-type AR (AR1-84), a C-terminally extended AR construct incorporating six residues from the predicted coding sequence of AR (AR1-90) and a similarly extended construct with a Met86 to Leu substitution (AR1-90(leu86)) were expressed as recombinant proteins in yeast, purified by heparin affinity and C18 reverse phase chromatography and their relative biological activities determined. The growth factors were tested in mitogenesis and EGF receptor autophosphorylation assays and their relative order of potencies was found to be leu86 > met86 > wt. The AR1-90(leu86) construct was found to be 50- to 100-fold more active than wild type AR1-84 consistent with previously reported studies of the role of the equivalent C-terminal leucine in EGF or TGF alpha. Significantly, the C-terminally extended form of AR, AR1-90, which utilized six residues from the predicted coding sequence, was 10-times more active than wild type AR1-84. This difference in activity of the C-terminally extended form of AR may be of biological significance since differential proteolytic processing of the AR precursor in vivo could result in production of multiple forms of the growth factor with differing affinities for the EGF receptor and hence differing biological potencies.

Targeting the epidermal growth factor receptor for therapy of carcinomas.

As a group, the carcinomas represent a substantial proportion of all human malignancies, but, with relatively few exceptions, current treatments are ineffective. Modification of existing chemotherapeutic agents has not led to significant improvements in the survival of carcinoma patients, and development of new therapeutic strategies is imperative. It is now becoming apparent that activation of the epidermal growth factor receptor (EGF-R) has much wider implications than a straightforward stimulation of cell division. The pleiotropic effects of EGF-R signalling may influence tumour behaviour and the response of carcinomas to treatment; these are important considerations for the development of new therapies that aim to exploit the expression or modulate the function of the EGF-R in these tumours.

Functional inferences from reconstructed evolutionary biology involving rectified databases--an evolutionarily grounded approach to functional genomics.

If bioinformatics tools are constructed to reproduce the natural, evolutionary history of the biosphere, they offer powerful approaches to some of the most difficult tasks in genomics, including the organization and retrieval of sequence data, the updating of massive genomic databases, the detection of database error, the assignment of introns, the prediction of protein conformation from protein sequences, the detection of distant homologs, the assignment of function to open reading frames, the identification of biochemical pathways from genomic data, and the construction of a comprehensive model correlating the history of biomolecules with the history of planet Earth.

Induction of anchorage-independent growth by amphiregulin.

We have previously shown that the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AR) exhibits low potency as a result of its C-terminal truncation. This led us to investigate whether its inability to promote anchorage-independent growth (AIG) of normal cells arose because of its compromised interaction with EGFR. Wild type AR(1-84) was tested in AIG and mitogenesis assays using NRK-49F or NR6/HER fibroblasts. In contrast to NR6/HER cells, the response of NRK-49F fibroblasts to AR was much lower than expected. As the effect of AR was heparin-insensitive, contributions from heparan sulphate proteoglycan interactions could not explain the differing sensitivities of the cells. Comparison of the effects of AR on two additional cell lines indicated that low EGFR number correlated with AR insensitivity: this suggested that the low potency of AR precluded activation of sufficient receptors to elicit a response. Consistent with this proposal, a modified form of AR (AR[1-90(leu86)]) with enhanced potency was able to induce AIG of NRK-49F fibroblasts. Thus, the ability of AR to promote AIG is determined both by ligand potency and the EGFR complement of cells.

The interaction of an epidermal growth factor/transforming growth factor alpha tail chimera with the human epidermal growth factor receptor reveals unexpected complexities.

It has been assumed that substitution of homologous regions of transforming growth factor alpha (TGF-alpha) into epidermal growth factor (EGF) can be used to probe ligand-receptor recognition without detrimental effects on ligand characteristics for the human EGF receptor (EGFR). We show that a chimera of murine (m) EGF in which the carboxyl-terminal tail is substituted for that of TGF-alpha (mEGF/TGF-alpha44-50) results in complex features that belie this initial simplistic assumption. Comparison of EGF and mEGF/TGF-alpha44-50 in equilibrium binding assays showed that although the relative binding affinity of the chimera was reduced 80-200-fold, it was more potent than EGF in mitogenesis assays using NR6/HER cells. This superagonist activity could not be attributed to differences in ligand processing or to binding to other members of the c-erbB family. It appeared to be due, in part, to choice of an EGFR-overexpressing target cell where high receptor number compensated for the low affinity of the ligand; it also appeared to be related to the ability of the chimera to activate the EGFR tyrosine kinase. Thus, when EGFR autophosphorylation was measured, mEGF/TGF-alpha44-50 was more potent than EGF, despite its low affinity. When tested using chicken embryo fibroblasts, substitution of the TGF-alpha carboxyl-terminal tail into mEGF failed to enhance its binding affinity for chicken EGFRs; however, the chimera was intermediate in potency between TGF-alpha and mEGF in mitogenesis assays. Our results suggest a contextual requirement for EGFR recognition which is ligand-specific. Further, the unpredictable responses to chimeric ligands underline the complex nature of the processes of ligand recognition, receptor activation, and the ensuing cellular response.

Determination of solution structures of paramagnetic proteins by NMR.

Standard procedures for using nuclear Overhauser enhancements (NOE) between protons to generate structures for diamagnetic proteins in solution from NMR data may be supplemented by using dipolar shifts if the protein is paramagnetic. This is advantageous since the electron -nuclear dipolar coupling provides relatively long-range geometric information with respect to the paramagnetic centre which complements the short-range distance constraints NOEs. Several different strategies have been developed to date, but none of these attempts to combine data from NOEs and dipolar shifts in the initial stages of structure calculation or to determine three dimensional protein structures together with their magnetic properties. This work shows that the magnetic and atomic structures are highly correlated and that it is important to have additional constraints both to provide starting parameters for the magnetic properties and to improve the definition of the best fit. Useful parameters can be obtained for haem proteins from Fermi contact shifts; this approach is compared with a new method based on the analysis of dipolar shifts in haem methyl groups with respect to data from horse and tuna ferricytochromes c. The methods developed for using data from NOEs and dipolar shifts have been incorporated in a new computer program, PARADYANA, which is demonstrated in application to a model data set for the sequence of the haem octapeptide known as microperoxidase-8.

The adaptive evolution database (TAED).

BACKGROUND: The Master Catalog is a collection of evolutionary families, including multiple sequence alignments, phylogenetic trees and reconstructed ancestral sequences, for all protein-sequence modules encoded by genes in GenBank. It can therefore support large-scale genomic surveys, of which we present here The Adaptive Evolution Database (TAED). In TAED, potential examples of positive adaptation are identified by high values for the normalized ratio of nonsynonymous to synonymous nucleotide substitution rates (KA/KS values) on branches of an evolutionary tree between nodes representing reconstructed ancestral sequences. RESULTS: Evolutionary trees and reconstructed ancestral sequences were extracted from the Master Catalog for every subtree containing proteins from the Chordata only or the Embryophyta only. Branches with high KA/KS values were identified. These represent candidate episodes in the history of the protein family when the protein may have undergone positive selection, where the mutant form conferred more fitness than the ancestral form. Such episodes are frequently associated with change in function. An unexpectedly large number of families (between 10% and 20% of those families examined) were found to have at least one branch with high KA/KS values above arbitrarily chosen cut-offs (1 and 0.6). Most of these survived a robustness test and were collected into TAED. CONCLUSIONS: TAED is a raw resource for bioinformaticists interested in data mining and for experimental evolutionists seeking candidate examples of adaptive evolution for further experimental study. It can be expanded to include other evolutionary information (for example changes in gene regulation or splicing) placed in a phylogenetic perspective.

Solution structure of the mEGF/TGFalpha44-50 chimeric growth factor.

The solution structure of the growth factor chimera mEGF/TGFalpha44-50 has been determined using an extended version of the dyana procedure for calculating structures from NMR data. The backbone fold and preferred orientation of the domains of the chimera are similar to those found in previous studies of EGF structures, and several H-bonds used as input constraints in those studies were found independently in the chimera. This shows that the modified activity of the chimera does not result from a major structural change. However, the improved precision of the structure presented here allows the origin of some unusual chemical shifts found in all of these compounds to be explained, as well as the results obtained from some site-specific mutants. Further studies of the properties of this chimeric growth factor should help to elucidate the mechanism(s) of hetero- and homodimerization of the c-erbB receptors.

The Adaptive Evolution Database (TAED).

BACKGROUND: Developing an understanding of the molecular basis for the divergence of species lies at the heart of biology. The Adaptive Evolution Database (TAED) serves as a starting point to link events that occur at the same time in the evolutionary history (tree of life) of species, based upon coding sequence evolution analyzed with the Master Catalog. The Master Catalog is a collection of evolutionary models, including multiple sequence alignments, phylogenetic trees, and reconstructed ancestral sequences, for all independently evolving protein sequence modules encoded by genes in GenBank [1]. RESULTS: We have estimated from these models the ratio of nonsynonymous to synonymous nucleotide substitution (Ka/Ks), for each branch in their respective evolutionary trees of every subtree containing only chordata or only embryophyta proteins. Branches with high Ka/Ks values represent candidate episodes in the history of the family where the protein may have undergone positive selection, a phenomenon in molecular evolution where the mutant form of a gene must have conferred more fitness than the ancestral form. Such episodes are frequently associated with change in function. We have found that an unexpectedly large number of families (between 10 and 20% of those families examined) have at least one branch with a notably high Ka/Ks value (putative adaptive evolution). As a resource for biologists wishing to understand the interaction between protein sequences and the Darwinian processes that shape these sequences, we have collected these into The Adaptive Evolution Database (TAED). CONCLUSIONS: Placed in a phylogenetic perspective, candidate genes that are undergoing evolution at the same time in the same lineage can be viewed together. This framework based upon coding sequence evolution can be readily expanded to include other types of evolution. In its present form, TAED provides a resource for bioinformaticists interested in data mining and for experimental evolutionists seeking candidate examples of adaptive evolution for further experimental study.

Contribution of the transforming growth factor alpha B-loop beta-sheet to binding and activation of the epidermal growth factor receptor.

We have exploited the differences in binding affinities of the chicken epidermal growth factor (EGF) receptor for EGF and transforming growth factor alpha (TGF alpha) to study the role of the B-loop beta-sheet of these ligands in receptor recognition and activation. Although EGF and TGF alpha share similar secondary and tertiary structures imposed by three highly conserved intramolecular disulfide bonds, they have only 30-40% overall sequence identity. The B-loop beta-sheet is the major structural element in EGF and TGF alpha, but sequence similarity in this region is low. To investigate its role in receptor binding, we constructed two chimeric growth factors (mEGF/hTGF alpha 21-30 and mEGF/hTGF alpha 21-32) composed of the murine EGF (mEGF) amino acid sequence with residues 21-30 of the B-loop beta-sheet replaced by the equivalent residues of human TGF alpha (hTGF alpha); in chimera mEGF/hTGF alpha 21-32, asparagine 32, which lies at the boundary of the amino and carboxyl domains of mEGF, was also replaced by its hTGF alpha counterpart (valine). In initial studies using unpurified medium, it was found that the recombinant growth factors exhibited differing mitogenic potencies (mEGF/hTGF alpha 21-32 > mEGF/hTGF alpha 21-30 > mEGF) when assayed on chicken fibroblasts, even though they were equivalent in mitogenesis assays using cells expressing the human EGF receptor. After purification, mEGF/hTGF alpha 21-32 was found to be 50 times more potent than mEGF in the chick fibroblast mitogenesis assay and exhibited a 10-fold increase in relative affinity for the chicken EGF receptor; both growth factors still exhibited equivalent mitogenic and receptor binding activity when tested on cells expressing human EGF receptors. We conclude that the B-loop beta-sheet of hTGF alpha is an important determinant of EGF receptor binding affinity and biological activity.

Structure prediction in a post-genomic environment: a secondary and tertiary structural model for the initiation factor 5A family.

Two predictions have been prepared for the fold of initiation factor 5A (IF5A) starting from a set of homologous sequences. In the first, a secondary structural model was predicted for the protein in 1994, when only eleven homologs (and no eubacterial homologs) had been sequenced. The second was made recently, after genome projects had generated a total of 33 sequences for the protein family from species of all three kingdoms of life. With the second set of sequences, but not with the first, it was possible to predict that the N-terminal domain of the protein folds in a possibly open beta-barrel/sandwich core structure, with a short helix capping one side of the barrel. We place the pair of predictions in the public domain before an experimental structure is known. This example illustrates the impact of genome sequencing projects on structure prediction from sequence alignments.

Multidomain binding of transforming growth factor alpha to the epidermal growth factor receptor.

Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGF alpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGF alpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGF alpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGF alpha, derived from 1H NMR measurements [Kline, T.-P., Brown, F.K., Brown, S.C., Jeffs, P.W., Kopple, K.D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGF alpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGF alpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

The significance of valine 33 as a ligand-specific epitope of transforming growth factor alpha.

Although binding of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) to the EGF receptor (EGFR) is mutually competitive, their binding is not identical, and their biological activities are not always equivalent. To probe for ligand-specific interactions, we have synthesized analogues of TGFalpha with modifications to the residue lying between the fourth and fifth cysteines (the "hinge"). Although this residue lies in a structurally conserved region of the protein, it is not conserved within the EGFR ligand family. Our results show that in TGFalpha there is a preference for a bulky hydrophobic hinge residue; this contrasts with EGF, for which a hydrogen bond donor functionality is preferred. Sequence analysis of the human EGFR ligands revealed that the nature of the hinge residue correlated with the sequence in the B-loop beta-sheet. As this region is an important determinant in recognition of TGFalpha by the chicken EGFR, we assessed the mitogenicity of the TGFalpha hinge mutants, as well as the other EGFR ligands, using chicken embryo fibroblasts. The preference of the chicken EGFR for TGFalpha hinge mutants with hydrophobic side chains paralleled that of the human EGFR. Betacellulin and heparin-binding EGF-like growth factor also possess an hydrophobic hinge; both were at least as potent as TGFalpha for chicken embryo fibroblasts. EGF and amphiregulin, both with hydrogen bond donor functionalities at their hinge, displayed markedly decreased in potency by comparison with TGFalpha. We propose that EGFR ligands can be subclassified into TGFalpha-like and EGF-like and that this is of functional significance, identifying a potential mechanism whereby EGFR can discriminate between its ligands.

Structure-function studies of ligand-induced epidermal growth factor receptor dimerization.

We present a novel 96-well assay which we have applied to a structure-function study of epidermal growth factor receptor dimerization. The basis of the assay lies in the increased probability of EGFRs being captured as dimers by a bivalent antibody when they are immobilized in the presence of a cognate ligand. Once immobilized, the antibody acts as a tether, retaining the receptor in its dimeric state with a resultant 5-7-fold increase in binding of a radiolabeled ligand probe. When the assay was applied to members of the EGF ligand family, murine EGF, transforming growth factor alpha, and heparin-binding EGF-like growth factor were comparable with human EGF (EC50 = 2nM); betacellulin, which has a broader receptor specificity, was slightly less effective. In contrast, amphiregulin (AR1-84), which has a truncated C-tail and lacks a conserved leucine residue, was ineffective unless used at >1 microM. We further probed the involvement of the C-tail and the conserved leucine residue in receptor dimerization by comparing the activities of two genetically modified EGFs (the chimera mEGF/TGFalpha44-50 and the EGF point mutant L47A) and a C-terminally extended form of AR (AR1-90) with those of two other unrelated EGF mutants (I23T and L15A). The potency of these ligands was in the order EGF > I23T > mEGF/TGFalpha44-50 > L47A = L15A >> AR1-90 > AR1-84. Although AR was much worse than predicted from its affinity, this defect could be partially rectified by co-localization of the immobilizing antibody with heparin. Thus, it seems likely that AR cannot dimerize the EGFR unless other accessory molecules are present to stabilize its functional association with the EGFR.

Constrained peptide analogues of transforming growth factor-alpha residues cysteine 21-32 are mitogenically active. Use of proline mimetics to enhance biological potency.

Two proline mimetics, the enantiomers of 2-aza-bicyclo[2,2,1]heptane-3-carboxylic acid, have been incorporated in place of Pro30 into synthetic peptides based on the B-loop beta-sheet sequence of human transforming growth factor-alpha (TGF-alpha) (residues Cys21-Cys32). The peptides were further modified by inclusion of an N-terminal phenylalanine and constrained by formation of an intramolecular disulfide bond. While no mitogenic response was observed in the parental NR6 cell line, the peptides stimulated DNA synthesis in NR6/HER cells (NR6 fibroblasts transfected with the human epidermal growth factor receptor). Induction of DNA synthesis was dose dependent, with EC50 values in the range 130-330 microM; in the presence of low doses of TGF-alpha, the mitogenic effect of the peptides was additive, up to the plateau response achieved by maximal doses of TGF-alpha alone. These effects are consistent with the peptides acting via the same mechanism as TGF-alpha. Analysis of the structure of the peptides by NMR indicated that the presence of the mimetics significantly increased the propensity of the peptidyl-proline bond to adopt the cis conformation. These data confirm the role of the beta-sheet in receptor activation, and emphasize the importance of presentation of peptides in an appropriate conformation for recognition.