Berkers J A, van Bergen en Henegouwen P M, Boonstra J
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
J Biol Chem. 1991 Jan 15;266(2):922-7.
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 (Kd = 0.12 nM) and 25,000 low affinity receptors per cell (Kd = 9.2 nM). The kinetic analysis of EGF binding to high affinity receptors was performed with cells pretreated with the monoclonal antibody 2E9, 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 (kass) of 6.2 x 10(5) M-1.s-1 and a kinetic dissociation constant (kdis) 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 kass = 3.3 x 10(6) M-1.s-1 and kdis = 8.1 x 10(-3) s-1 and the type III or slow sites with kass = 3.2 x 10(4) M-1.s-1 and kdis = 1.6 x 10(-4) s-1. The regulatory mechanism which may determine the EGF binding characteristics is discussed.
研究了¹²⁵I标记的表皮生长因子（EGF）与HeLa细胞上受体的结合动力学。Scatchard分析显示，每个细胞存在22,000个高亲和力受体（Kd = 0.12 nM）和25,000个低亲和力受体（Kd = 9.2 nM）。用单克隆抗体2E9预处理细胞后，对EGF与高亲和力受体的结合进行动力学分析，该抗体可特异性阻止EGF与低亲和力受体结合。用佛波酯佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯预处理细胞后，进行EGF仅与低亲和力受体结合的研究，该佛波酯可诱导高亲和力受体转化为低亲和力受体。对EGF与HeLa细胞结合的动力学分析揭示了三种类型受体的存在。发现高亲和力受体由一种受体类型（I型）组成，其动力学缔合常数（kass）为6.2×10⁵ M⁻¹·s⁻¹，动力学解离常数（kdis）为3.5×10⁻⁴ s⁻¹。发现低亲和力受体由两个动力学可区分的位点组成：II型或快速位点，kass = 3.3×10⁶ M⁻¹·s⁻¹，kdis = 8.1×10⁻³ s⁻¹；III型或慢速位点，kass = 3.2×10⁴ M⁻¹·s⁻¹，kdis = 1.6×10⁻⁴ s⁻¹。讨论了可能决定EGF结合特性的调节机制。
