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)

Immunocytochemical analysis of epidermal growth factor (EGF) receptor expression was carried out on frozen sections of 109 primary lung tumours resected at the Brompton Hospital from February 1984 to May 1985. Tumours with detectable levels of this proto-oncogene protein were significantly more frequent among squamous cell carcinomas than among other types of lung tumour. No truncated EGF receptors were detected in the tumours using two monoclonal antibodies (Mabs) directed against different portions of the receptor (EGFR1 and F4). Mab F4 is the first antibody to the EGF receptor to show reactivity in paraffin sections. Southern blot analysis of a subset of the tumours detected amplification of the EGF receptor gene in squamous cell carcinomas but not in adenocarcinomas. The one carcinosarcoma examined had a re-arranged and amplified EGF receptor gene. Measurement of EGF receptor expression in lung tumours can be of diagnostic value and may prove to be useful in the development of antibody-directed therapy.

The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X‐ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155‐kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(‐9) M in intact infected insect cells which falls to 2 X 10(‐7) M upon detergent solubilization. The recombinant protein exhibits an EGF‐stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large‐scale production.

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