Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells.

E Livneh,R Prywes,O Kashles,N Reiss,I Sasson,Y Mory,A Ullrich,J Schlessinger

doi:10.1016/s0021-9258(18)67114-5

Abstract

DNA sequences encoding the human epidermal growth factor (EGF) receptor and various EGF-receptor deletion mutants were transfected into chinese hamster ovary (CHO) cells devoid of endogenous EGF receptors. A functional human EGF-receptor is expressed on the surface of heterologous CHO cells with the following properties: it exhibits typical high affinity (10%; Kd = 3 X 10(-10) M) and low affinity (90%; Kd = 3 X 10(-9) M) binding sites for 125I-EGF; it is expressed as a polypeptide of 170,000 molecular weight with intrinsic protein tyrosine kinase activity. EGF stimulates the kinase activity leading to self-phosphorylation and to phosphorylation of exogenous substrate; 125I-EGF is rapidly internalized into the CHO cells by receptor mediated endocytosis and; EGF stimulates DNA synthesis in the cells expressing the human EGF-receptor. Deletion of 63 amino acids from the C-terminal end of EGF-receptor, which removes two autophosphorylation sites, abolishes the high affinity state of the receptor. Nevertheless, this receptor mutant is able to undergo endocytosis and to respond mitogenically to EGF to a similar extent as the "wild type" receptor. Further deletions from the cytoplasmic domain give rise to low affinity endocytosis-defective receptor mutants. Finally, deletion of the transmembrane domain of the human receptor yields an EGF-receptor ligand binding domain which is secreted from the cells.

Highlights

DNA sequences encoding the human epidermal and that their presence is regulated by the phosphorylation growth factor (EGF)receptor and various epidermal growth factor (EGF)-recep- of the EGF-receptor by the Caz+ andphospholipid sensitive tor deletion mutants were transfected into Chinese protein kinase C which binds to andis activated by the tumor hamster ovary (CHO) cells devoid of endogenous EGF receptors
We have examined the capacity of the EGF-receptor and its mutants tophosphorylate the exogenous substrate angiotensin-I which was previously shown to be a good substrate of protein-tyrosine kinases [23]
10”’ M (“high affinity” receptors)while the restof the receptors have a Kd = 3 X lo-’ M (“lowaffinity” receptors).2) The reconstituted receptor has an apparent molecular weight of 170,000.It possesses intrinsic protein-tyrosine kinaseactivity which is stimulated by EGF leading to self-phosphorylation and to phosphorylation of exogenous substrates

Summary

RESULTS

DNA sequences encoding the full size cDNA and the truncated forms of EGF-receptor were inserted intoan expression plasmid brought under the control of SV40 early promoter (Fig. 1A). PLSXC (construct 11) encodes an EGF-receptor with a deletion of 63 amino acids from the Cterminal end. This mutant is devoid of two autophosphorylation sites [18].pLAB71-2 (construct 111) encodes a receptor protein without most of the protein-tyrosine kinase domain with 98 cytoplasmic amino acids. PLANA8 (construct IV) encodes a receptor protein with the entire extracellular domain and a short cytoplasmic tail of nine amino acids. This mutant is devoid of Thr-654 which is the major protein kinase C phosphorylation site. Lines-The CX-17 cells transfected with the full-length EGFreceptor cDNA expressed a protein of molecular weight of 170,000 which comigrated with the EGF-receptor immunoprecipitated from A-431 cells(Fig. 2A, lane a). Thewild type receptor expressed by the CX-17 cells was immunoprecipi-

COOH mo mo

Angiotensin I ab cdefa bc abcdef

DISCUSSION

Cell line