FfAME:org :: Stephen Chamberlin's Publications

Stephen Chamberlin's Publications

Expanded Genetic Alphabets in the Polymerase Chain Reaction
Yang, ZY; Chen, F; Chamberlin, SG; Benner, SA
Angew. Chem. Int. Ed. 49 (1) 177-180 (2010)

Analysis of transitions at two-fold redundant sites in mammalian genomes. Transition redundant approach-to-equilibrium (TREx) distance metrics
Li, T; Chamberlin, SG; Caraco, MD; Liberles, DA; Gaucher, EA; Benner, SA
BMC Evol. Biol. 6 25 (2006)
<Abstract>

Background: The exchange of nucleotides at synonymous sites in a gene encoding a protein is believed to have little impact on the fitness of a host organism. This should be especially true for synonymous transitions, where a pyrimidine nucleotide is replaced by another pyrimidine, or a purine is replaced by another purine. This suggests that transition redundant exchange ( TREx) processes at the third position of conserved two-fold codon systems might offer the best approximation for a neutral molecular clock, serving to examine, within coding regions, theories that require neutrality, determine whether transition rate constants differ within genes in a single lineage, and correlate dates of events recorded in genomes with dates in the geological and paleontological records. To date, TREx analysis of the yeast genome has recognized correlated duplications that established a new metabolic strategies in fungi, and supported analyses of functional change in aromatases in pigs. TREx dating has limitations, however. Multiple transitions at synonymous sites may cause equilibration and loss of information. Further, to be useful to correlate events in the genomic record, different genes within a genome must suffer transitions at similar rates. Results: A formalism to analyze divergence at two fold redundant codon systems is presented. This formalism exploits two-state approach-to-equilibrium kinetics from chemistry. This formalism captures, in a single equation, the possibility of multiple substitutions at individual sites, avoiding any need to "correct" for these. The formalism also connects specific rate constants for transitions to specific approximations in an underlying evolutionary model, including assumptions that transition rate constants are invariant at different sites, in different genes, in different lineages, and at different times. Therefore, the formalism supports analyses that evaluate these approximations. Transitions at synonymous sites within two-fold redundant coding systems were examined in the mouse, rat, and human genomes. The key metric (f(2)), the fraction of those sites that holds the same nucleotide, was measured for putative ortholog pairs. A transition redundant exchange ( TREx) distance was calculated from f(2) for these pairs. Pyrimidine-pyrimidine transitions at these sites occur approximately 14% faster than purine-purine transitions in various lineages. Transition rate constants were similar in different genes within the same lineages; within a set of orthologs, the f(2) distribution is only modest overdispersed. No correlation between disparity and overdispersion is observed. In rodents, evidence was found for greater conservation of TREx sites in genes on the X chromosome, accounting for a small part of the overdispersion, however. Conclusion: The TREx metric is useful to analyze the history of transition rate constants within these mammals over the past 100 million years. The TREx metric estimates the extent to which silent nucleotide substitutions accumulate in different genes, on different chromosomes, with different compositions, in different lineages, and at different times.

Solution structure of the mEGF/TGF alpha(44-50) chimeric growth factor
Chamberlin, SG; Brennan, L; Puddicombe, SM; Davies, DE; Turner, DL
Euro. J. Biochem. 268 (23) 6247-6255 (2001)
<Abstract>

The solution structure of the growth factor chimera mEGF/TGF alpha (44-50) 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.

Beyond BLAST: Paleogenomics tools to infer function to genetic sequences.
Benner, S; Chamberlin, S
Am. J. Hum. Genet. 67 (4) 260-260 (2000)

Functional inferences from reconstructed evolutionary biology involving rectified databases. An evolutionarily-grounded approach to functional genomics.
Benner, SA; Chamberlin, SG; Liberles, DA; Govindarajan, S; Knecht, L
Res. MicroBiol. 151 (2) 97-106 (2000)
<Abstract>

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.

A unified model of c-erbB receptor homo- and heterodimerisation
Chamberlin, SG; Davies, DE
Biochim. Biophys. Acta 1384 (2) 223-232 (1998)
<Abstract>

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. (C) 1998 Elsevier Science B.V. All rights reserved.

Structure prediction in a post-genomic environment: A secondary and tertiary structural model for the initiation factor 5A family
Gerloff, DL; Joachimiak, M; Cohen, FE; Cannarozzi, GM; Chamberlin, SG; Benner, SA
Biochem. Biophys. Res. Comm. 251 (1) 173-181 (1998)
<Abstract>

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 land 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. (C) 1998 Academic Press.

Structure-function studies of ligand-induced epidermal growth factor receptor dimerization
Neelam, B; Richter, A; Chamberlin, SG; Puddicombe, SM; Wood, L; Murray, MB; Nandagopal, K; Niyogi, SK; Davies, DE
Biochemistry 37 (14) 4884-4891 (1998)
<Abstract>

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 (AR(1-84)), which has a truncated C-tail and lacks a conserved leucine residue, was ineffective unless used at >1 mu M. 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/TGF alpha(44-50) and the EGF point mutant L47A) and a C-terminally extended form of AR (AR(1-90)) with those of two other unrelated EGF mutants (I23T and L15A). The potency of these ligands was in the order EGF > I23T > mEGF/TGF alpha(44-50) > L47A = L15A much greater than AR(1-90) > AR(1-84). 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.

Determination of solution structures of paramagnetic proteins by NMR
Turner, DL; Brennan, L; Chamberlin, SG; Louro, RO; Xavier, AV
Euro. Biophys. J. Biophys. Lett. 27 (4) 367-375 (1998)
<Abstract>

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 from 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.

Targeting the epidermal growth factor receptor for therapy of carcinomas
Davies, DE; Chamberlin, SG
Biochem. Pharmacol. 51 (9) 1101-1110 (1996)
<Abstract>

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.

The interaction of a chimeric EGF containing the C-tail of TGF alpha with the EGF receptor reveals hidden ligand complexities.
Puddicombe, S; Wood, L; Chamberlin, S; Davies, D
FASEB J. 10 (6) 2171-2171 (1996)

The mitogenic effect of low affinity ligands is enhanced by overexpression of EGF receptors
Davies, D; Adam, R; Murray, M; Niyogi, S; Chamberlin, S
FASEB J. 10 (6) 2172-2172 (1996)

Amphiregulin is a poor effector of ligand-induced EGF receptor dimerisation.
Richter, A; Neelam, B; Chamberlin, S; Davies, D
FASEB J. 10 (6) 2173-2173 (1996)

A high intensity signal is required for EGF receptor mediated morphogenesis and migration.
Solic, N; Murray, M; Chamberlin, S; Niyogi, S; Davies, D
FASEB J. 10 (6) (1996)

A mathematical model of c-erbB receptor dimerisation.
Chamberlin, S; Davies, D
FASEB J. 10 (6) (1996)

Induction of anchorage-independent growth by amphiregulin
Adam, RM; Chamberlin, SG; Davies, DE
Growth Factors 13 193-203 (1996)
<Abstract>

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 NRG/HER fibroblasts. In contrast to NRG/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 significance of valine 33 as a ligand-specific epitope of transforming growth factor alpha
Puddicombe, SM; Chamberlin, SG; MacGarvie, J; Richter, A; Drummond, DR; Collins, J; Wood, L; Davies, DE
J. Biol. Chem. 271 (26) 15367-15372 (1996)
<Abstract>

Although binding of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) 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, me have synthesized analogues of TGF alpha 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 TGF alpha 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 TGF alpha by the chicken EGFR, we assessed the mitogenicity of the TGF alpha hinge mutants, as well as the other EGFR ligands, using chicken embryo fibroblasts. The preference of the chicken EGFR for TGF alpha 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 TGF alpha for chicken embryo fibroblasts. EGF and amphiregulin, both with hydrogen bond donor functionalities at their hinge, displayed markedly decreased in potency by comparison with TGF alpha. We propose that EGFR ligands can be subclassified into TGF alpha-like and EGF-like and that this is of functional significance, identifying a potential mechanism whereby EGFR can discriminate between its ligands.

The interaction of an epidermal growth factor transforming growth factor alpha tail chimera with the human epidermal growth factor receptor reveals unexpected complexities
Puddicombe, SM; Wood, L; Chamberlin, SG; Davies, DE
J. Biol. Chem. 271 (48) 30392-30397 (1996)
<Abstract>

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-alpha(44-50)) results in complex features that belie this initial simplistic assumption, Comparison of EGF and mEGF/TGF-alpha(44-50) 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-alpha(44-50) 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.

MODULATION OF THE RECEPTOR-BINDING AFFINITY OF AMPHIREGULIN BY MODIFICATION OF ITS CARBOXYL-TERMINAL TAIL
Adam, R; Drummond, DR; Solic, N; Holt, SJ; Sharma, RP; Chamberlin, SG; Davies, DE
Biochim. Biophys. Acta 1266 (1) 83-90 (1995)
<Abstract>

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 (AR(1-84)), a C-terminally extended AR construct incorporating six residues from the predicted coding sequence of AR (AR(1-90)) and a similarly extended construct with a Met(86) to Leu substitution (AR1-90((leu86))) were expressed as recombinant proteins in yeast, purified by heparin affinity and C-18 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 leu(86) > met(86) > wt. The AR1-90((leu86))) construct was found to be 50- to 100-fold more active than wild type AR(1-84), 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, AR(1-90), which utilized six residues from the predicted coding sequence, was 10-times more active than wild type AR(1-84). 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.

CONTRIBUTION OF THE TRANSFORMING GROWTH-FACTOR-ALPHA B-LOOP BETA-SHEET TO BINDING AND ACTIVATION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR
Richter, A; Drummond, DR; Macgarvie, J; Puddicombe, SM; Chamberlin, SG; Davies, DE
J. Biol. Chem. 270 (4) 1612-1616 (1995)
<Abstract>

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(21-40) 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 us ing 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.

CONSTRAINED PEPTIDE ANALOGS OF TRANSFORMING GROWTH-FACTOR-ALPHA RESIDUES CYSTEINE-21-32 ARE MITOGENICALLY ACTIVE - USE OF PROLINE MIMETICS TO ENHANCE BIOLOGICAL POTENCY
Chamberlin, SG; Sargood, KJ; Richter, A; Mellor, JM; Anderson, DW; Richards, NGJ; Turner, DL; Sharma, RP; Alexander, P; Davies, DE
J. Biol. Chem. 270 (36) 21062-21067 (1995)
<Abstract>

Two proline mimetics, the enantiomers of 2-aza-bicyclo[2,2,1]heptane-3-carboxylic acid, have been incorporated in place of Pro(30) into synthetic peptides based on the B-loop beta-sheet sequence of human transforming growth factor-alpha (TGF-alpha) (residues Cys(21)-Cys(32)). 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 EC(50) values in the range 130-330 mu M; 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.

SYNTHESIS OF FRAGMENTS OF TRANSFORMING GROWTH-FACTOR-ALPHA INCORPORATING EXO-2-AZABICYCLO[2,2,1]HEPTANE-3-CARBOXYLIC ACIDS AS PROLINE SUBSTITUTES
Mellor, JM; Richards, NGJ; Sargood, KJ; Anderson, DW; Chamberlin, SG; Davies, DE
Tet. Lett. 36 (37) 6765-6768 (1995)
<Abstract>

Two novel amino acids, the enantiomers of exo 2-azabicyclo[2,2,1]heptane-3-carboxylic acid have ben independently synthesized by the aza Diels Alder reaction using a chiral auxiliary. The two acids have been advanced via solid phase synthesis to afford linear sequences, which have been cyclized to afford cyclic disulfide peptides, analogues of fragments of TGF alpha. The structures of these and other fragments have been firmly established.

MULTIDOMAIN BINDING OF TRANSFORMING GROWTH FACTOR-ALPHA TO THE EPIDERMAL GROWTH-FACTOR RECEPTOR
Richter, A; Conlan, JW; Ward, ME; Chamberlin, SG; Alexander, P; Richards, NGJ; Davies, DE
Biochemistry 31 (40) 9546-9554 (1992)
<Abstract>

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 (hTGFalpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGFalpha 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 hTGFalpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGFalpha, derived from H-1 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 hTGFalpha 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 hTGFalpha 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. Such a model is consistent with recent investigations of the interaction of EGF-R and its ligands using physical methods, which have indicated changes in the solution conformation of the receptor upon ligand binding [Greenfield, C., Hiles, I., Waterfield, M. D., Federwisch, M., Wollmer, A., Blundell, T. L., & McDonald, N. (1989) EMBO J. 8, 4115-41231. We anticipate that the receptor-binding assay reported in this study might also be more generally applicable in probing the interaction of other biologically important peptides, and proteins, with cellular receptors of similar structure to that of EGF-R.