Epidermal growth factor-receptor-protein kinase interactions.

Area of Science:

• Cell biology
• Biochemistry
• Molecular oncology

Background:

• Epidermal growth factor (EGF) is a potent mitogen.
• EGF exerts its effects by binding to specific cell surface receptors.
• The precise biochemical events following EGF receptor activation are under investigation.

Purpose of the Study:

• To investigate the biochemical consequences of EGF binding to A-431 cell membranes.
• To characterize the EGF-stimulated phosphorylation system.
• To identify the molecular components involved in EGF-mediated signaling.

Main Methods:

• Preparation of membranes from A-431 human epidermoid carcinoma cells.
• In vitro binding assays using 125I-labeled EGF.
• Phosphorylation assays using [gamma-32P]ATP and various substrates (endogenous proteins, histone).
• Solubilization of membrane proteins using non-ionic detergents.
• Purification of the EGF receptor complex via affinity chromatography.
• SDS-gel electrophoresis for protein analysis.

Main Results:

• A-431 cell membranes specifically bind EGF and exhibit EGF-stimulated phosphorylation of endogenous proteins and histone.
• The phosphorylation is cyclic AMP- and cyclic GMP-independent.
• EGF binding rapidly activates a membrane-associated, cyclic AMP-independent protein kinase.
• The kinase activity and EGF binding are retained after detergent solubilization and affinity purification.
• A 150,000 molecular weight protein is identified as the major component, likely the EGF receptor and a phosphorylation substrate.
• The phosphorylation reaction is specific for tyrosine residues, similar to src protein kinase.

Conclusions:

• EGF binding to its receptor on A-431 cell membranes triggers a rapid, reversible activation of an intrinsic protein kinase.
• The 150,000 molecular weight protein is identified as the EGF receptor, which is also phosphorylated in an EGF-dependent manner.
• This EGF-stimulated tyrosine phosphorylation system is a key early event in EGF signal transduction.