Three classes of epidermal growth factor receptors on HeLa cells

The kinetics of ¹²⁵I-labeled epidermal growth factor (EGF) binding to receptors on HeLa cells were investigated. Scatchard analysis revealed the presence of 22,000 high affinity receptors (K(d) = 0.12 nM) and 25,000 low affinity receptors per cell (K(d) = 9.2 nM). The kinetic analysis of EGF binding to high affinity receptors was performed with cells pretreated with the monoclonal antibody 2F9, which prevents specifically EGF binding to low affinity receptors. The study of EGF binding to only low affinity receptors was performed with cells pretreated with the phorbol ester phorbol 12-myristate 13-acetate, which induces a conversion of high affinity receptors to low affinity receptors. This kinetic analysis of EGF binding to HeLa cells revealed the presence of three types of receptors. High affinity receptors were found to consist of one receptor type (type I) with a kinetic association constant (k(ass)) of 6.2 x 10⁵ M^-1·s^-1 and a kinetic dissociation constant (k(dis)) of 3.5 x 10^-4 s^-1. The low affinity receptors were found to consist of two kinetic distinguishable sites: type II or fast sites with k(ass) = 3.3 x 10⁶ M¹·s^-1 and k(dis)= 8.1 x 10^-3 s^-1 and the type III or slow sites with k(ass) = 3.2 x 10⁴ M¹·s ¹ and h(dis) = 1.6 x 10^-4 s^-1 The regulatory mechanism which may determine the EGF binding characteristics is discussed.