Epidermal growth factor binding induces a conformational change in the external domain of its receptor.

Abstract

To study the properties of the extracellular epidermal growth factor (EGF) binding domain of the human EGF receptor, we have infected insect cells with a suitably engineered baculovirus vector containing the cDNA encoding the entire ectodomain of the parent molecule. This resulted in a correctly folded, stable, 110 kd protein which possessed an EGF binding affinity of 200 nM. The protein was routinely purified in milligram amounts from 1 litre insect cell cultures using a series of three standard chromatographic steps. The properties of the ectodomain were studied before and after the addition of different EGF ligands, using both circular dichroism and fluorescence spectroscopic techniques. A secondary structural analysis of the far UV CD spectrum of the ectodomain indicated significant proportions of alpha‐helix and beta‐sheet in agreement with a published model of the EGF receptor. The ligand additions to the receptor showed differences in both the near‐ and far‐UV CD spectra, and were similar for each ligand used, suggesting similar conformational differences between uncomplexed and complexed receptor. Steady‐state fluorescence measurements indicated that the tryptophan residues present in the ectodomain are buried and that the solvent‐accessible tryptophans in the ligands become buried on binding the receptor. The rotational correlation times measured by fluorescence anisotropy decay for the receptor‐ligand complexes were decreased from 6 to 2.5 ns in each case. This may indicate a perturbation of the tryptophan environment of the receptor on ligand binding. Ultracentrifugation studies showed that no aggregation occurred on ligand addition, so this could not explain the observed differences from CD or fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Analogs of human epidermal growth factor which partially inhibit the growth factor‐dependent protein‐tyrosine kinase activity of the epidermal growth factor receptor

“…Obviously, physicochemical studies are necessary to determine the nature of the conformational change(s). Studies examining the structure of the complexes formed between the EGF analogs and the external domain of the EGF receptor as described by Greenfield et al [19] for wild-type EGF may provide evidence of altered receptor conformations. Our findings also suggest that the design of growth-inhibitory EGF analogs may be feasible.…”

Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor

“…This result shows the power of the CD spectroscopy to generate highly reproducible and sensitive ellipticity measurements in order to observe conformational changes upon protein-protein interaction. This change in secondary structure in the RED-NGF complex is on the same order of magnitude, i.e., 4-8070 in ellipticity, as that in the EGF receptor extracellular domain upon binding of EGF (Greenfield et al, 1989). The extent of a conformational change necessary for transduction of ligand binding information from outside the cell to second messengers inside the cell is unknown.…”

“…A &turn structure is the only other derived structure (Compton & Johnson, 1986) that resembles that of the RED. This spectrum and structural interpretation are markedly different than for that observed for the epidermal growth factor (EGF) receptor extracellular domain, which has significant amounts of regular secondary structure (Greenfield et al, 1989). The spectrum of 0-NGF has a minimum at about 208 nm with a secondary structure that is largely 0-sheet (Williams et al, 1982;McDonald et al, 1991;Timm & Neet, 1992).…”

Epidermal growth factor receptor double targeting by a tyrosine kinase inhibitor (Iressa) and a monoclonal antibody (Cetuximab). Impact on cell growth and molecular factors

“…Thus, subsequent to its ubiquitination, EGFR is subject to lysosomal degradation (Citri et al, 2002). It has been reported that the binding of the natural ligand to EGFR results in a conformational change in the external domain of the receptor (Greenfield et al, 1989), which could be crucial to the ligandinduced internalisation of the receptor (Opresko et al, 1995). There is thus a ligand-controlled turnover in the expression of EGFR, which could be deregulated in the combined presence of C225 and ZD1839.…”