Using members of the epidermal growth factor (EGF) family as well as site-directed recombinant human EGF mutants, we investigated how ligand binding properties influence endosomal sorting. Mouse EGF (mEGF), human EGF (hEGF), and transforming growth factor alpha (TGF alpha) bind to the human EGF receptor (EGFR) with similar affinities at pH 7.4. However, the binding properties of these ligands have substantially different pH sensitivities resulting in varying degrees of dissociation from the receptors at lower pH levels characteristic of endosomes. We employed a steady-state sorting assay to determine the fraction of ligand sorted to recycling versus degradation as a function of the number of intracellular ligand molecules in mouse B82 fibroblasts. mEGF, hEGF, and TGF alpha display significantly different steady-state endosomal sorting patterns which correspond to the extent of their dissociation at endosomal pH. Moreover, several recombinant hEGF mutants with differing affinities exhibit altered endosomal sorting compared to hEGF, demonstrating a similar direct relationship between ligand binding properties and endosomal sorting outcomes. Intracellular trafficking of the EGF ligands was also monitored by measuring the observed degradation rate constants. These likewise show marked differences that correlate with the differing pH sensitivities of the ligands' binding properties.

Successful use of growth factors in therapeutic and bioprocessing applications requires overcoming two attenuation mechanisms: growth factor depletion and receptor down-regulation. Current ameliorative strategies use physiologically inappropriate high growth-factor concentrations, along with periodic media refeeding in vitro and reinjection or controlled-release devices in vivo. We demonstrate a new approach derived from understanding how these attenuation mechanisms arise from ligand/receptor trafficking processes. Specifically, a recombinant epidermal growth factor (EGF) mutant with reduced receptor binding affinity is a more potent mitogenic stimulus for fibroblasts than natural EGF or transforming growth factor alpha because of its altered trafficking properties.

Multidrug-resistant strains of Shigella dysenteriae type 1 were implicated in three outbreaks and sporadic cases of dysentery in eastern India in 2002 and 2003. After a hiatus of 14 years, this pathogen reemerged with an altered antibiotic resistance pattern. In addition to ampicillin, co-trimoxazole, tetracycline, chloramphenicol, and nalidixic acid, all the recent strains were resistant to norfloxacin, lomefloxacin, pefloxacin, and ofloxacin and showed reduced susceptibility to ciprofloxacin. Pulsed-field gel electrophoresis identified a new clone of S. dysenteriae type 1 that was associated with the recent outbreaks and sporadic cases. Based on the spatial and temporal spread of multidrug-resistant S. dysenteriae type 1, we predict that this clonal type may spread further in this region.

Eight analogues of human epidermal growth factor (hEGF) having specific amino acid substitutions in the beta-sheet structure (residues 19-31) of the amino-terminal domain were generated by site-directed mutagenesis. Affinity of the epidermal growth factor (EGF) receptor for each of these mutant hEGF analogues was measured by both radioreceptor competition binding and receptor tyrosine kinase stimulation assays. The relative binding affinities obtained by these two methods were generally in agreement for each hEGF species. The results indicate that hydrophobic residues on the exposed surface of the beta-sheet structure of the amino-terminal domain of hEGF have an important role in the formation of the active EGF-receptor complex. The substitution of hydrophobic amino acid residues, Val-19----Gly, Met-21----Thr, Ile-23----Thr, and Leu-26----Gly, resulted in decreased binding affinity, with the most severe reductions observed with the last two mutants. The mutations Ala-25----Val and Lys-28----Arg introduced amino acid residues resulting in slightly increased receptor binding affinity. Similar to previous results with acidic residues in this region [Engler, D.A., Matsunami, R.K., Campion, S.R., Stringer, C.D., Stevens, A., & Niyogi, S.K. (1988) J. Biol. Chem. 263, 12384-12390], removal of the positive charge in the Lys-28----Leu substitution had almost no effect on binding affinity, indicating the lack of any absolute requirement for ionic interactions at this site. Substitution of Tyr-22, which resulted in decreased receptor binding affinity, provides further indication of the importance of aromatic residues in this region of the molecule, as found earlier with Tyr-29 (cf. reference above).(ABSTRACT TRUNCATED AT 250 WORDS)

Seven site-specific mutants (including changes to other hydrophobic, charged, and heterocyclic amino acids) of leucine 47 of human epidermal growth factor (EGF) were generated by protein engineering and characterized for their activity in three assays: radioreceptor competition binding in membrane fractions, the stimulation of the EGF receptor's tyrosine kinase activity, and the stimulation of thymidine uptake in tissue culture cells. K1/2 (concentration required for half maximum response) values for each of the mutants are reported in the three assays. The results show that the native leucine residue is quite important for EGF activity. Substitutions are tolerated to different degrees, depending upon hydrophobicity and size of the side chain. Substitution with ionic residues led to the most drastic reduction in activity. One-dimensional nuclear magnetic resonance spectroscopy, at physiological pH, of several of the mutants did not detect any major structural perturbations which would account for the loss of activity. The results suggest that the side chain of leucine 47, because of its charge neutrality, size, and hydrophobicity, is highly important, although not absolutely essential for the interaction of EGF with its receptor. A striking finding was the lower (compared with wild type) Vmax values of the mutants in the tyrosine kinase reaction, but these low Vmax mutants, in cell culture experiments, were able to stimulate at high concentrations a growth response equivalent to wild type EGF.

No 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 (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.

Three site-directed mutants of human epidermal growth factor, Leu-26+Gly, Leu-47-+Ala, and Be-23-+Thr, were examined for their ability to stimulate the protein-tyrosine kinase activity of the epidermal growth factor receptor. The receptor binding atiinities of the mutant growth factors were 20-to 50-fold lower, as compared to wild-type growth factor. At saturating concentrations of growth factor, the velocities of the phosphorylation of exogenously added substrate and receptor autophosphorylation were significantly lower with the mutant analogs, suggesting a partial 'uncoupling' of signal transduction. The mutant analogs were shown to compete directly with the binding of wild-type, resulting in a decrease in growth factor-stimulated kinase activity.Epidermal growth factor analog; Epidermal growth factor receptor kinase; Competitive inhibitors of epidermal growth factor