Cholesterol and phosphoinositides increase affinity of the epidermal growth factor receptor

The epidermal growth factor receptor (EGF-R) has been purified from human epidermoid carcinoma A431 cells by affinity chromatography in a single step using a monoclonal antibody (528) which competes with EGF for receptor binding. The purified EGF-R exhibits EGF inducible tyrosine kinase and autophosphorylation activity. Steady-state binding of EGF to the purified receptor revealed the presence of one class of binding sites exhibiting an apparent dissociation constant (Kd) of approx. 2 nM. When Angiotensin II was used as a receptor tyrosine kinase substrate the specific activity of the EGF induced kinase was 87 nmol/min per mg and the Km of the reaction was about 2 mM. Reconstitution of the EGF receptors into lipid vesicles was achieved by octylglucoside dialysis. Reconstitution of the receptor into pure dioleoylphosphatidylcholine (DOPC) vesicles had no effect on the EGF-binding properties in comparison to receptors in Triton X-100 micelles. Binding of EGF to the reconstituted receptor with ATP and Angiotensin II incorporated into the vesicles resulted in a five fold stimulation of the receptor kinase activity. The introduction of cholesterol, ranging from 10% to 50% (w/w), into DOPC vesicles resulted in an increase of the affinity of the receptor for its ligand. The Kd for EGF decreased from 1.8 nM in pure DOPC vesicles to 0.3 mM in DOPC/cholesterol (1:1 (w/w)) vesicles. With the introduction of small amounts (2% (w/w)) of phosphatidylinositol lipids into DOPC vesicles the Kd changed from 1.8 nM to 0.2 nM with phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-biphosphate (PtdIns4,5-P2) and to 0.1 nM in the case of phosphatidylinositol 4 phosphate (PtdIns4-P). No change in affinity was found when equal amounts of phosphatidylserine (PS) or phosphatidic acid (PA) were used.