van Bergen en Henegouwen P M, Defize L H, de Kroon J, van Damme H, Verkleij A J, Boonstra J
Department of Molecular Cell Biology, University of Utrecht, Netherlands.
J Cell Biochem. 1989 Apr;39(4):455-65. doi: 10.1002/jcb.240390411.
Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X-100-insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady-state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF-binding sites: a high-affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 x 10(4) sites per cell) and a low-affinity site with a KD of 8.5 nM (1.9 x 10(6) sites per cell). Non-equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast-dissociating site, with a dissociation rate constant (k-1) of 1.1 x 10(-3) s-1 (1.0-1.3 x 10(6) sites per cell) and a slow-dissociating site, with a k-1 of 3.5 x 10(-5) s-1 (0.6-0.7 x 10(6) sites per cell). The cytoskeleton of A431 cells was isolated by Triton X-100 extraction. Scatchard analysis revealed that approximately 5% of the original number of receptors were associated with the cytoskeleton predominantly via high-affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast-dissociating component (k-1 = 2.0 x 10(-3) s-1) and a slow-dissociating component (k-1 = 9.1 x 10(-5) s-1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4 degrees C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton-associated receptors appeared to represent low-affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast-dissociating sites. These results indicate that at 4 degrees C EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.
最近，我们获得了支持表皮生长因子（EGF）受体与表皮样癌A431细胞的Triton X-100不溶性细胞骨架之间存在结构相互作用的证据。在此，我们对附着于细胞骨架的EGF受体的特性进行进一步分析。根据Scatchard方法分析的稳态EGF结合研究表明，A431细胞含有两类EGF结合位点：一类高亲和力位点，其表观解离常数（KD）为0.7 nM（每细胞7.5×10⁴个位点），另一类低亲和力位点，KD为8.5 nM（每细胞1.9×10⁶个位点）。非平衡结合研究揭示存在两个动力学上可区分的位点：一个快速解离位点，解离速率常数（k-1）为1.1×10⁻³ s⁻¹（每细胞1.0 - 1.3×10⁶个位点），以及一个缓慢解离位点，k-1为3.5×10⁻⁵ s⁻¹（每细胞0.6 - 0.7×10⁶个位点）。通过Triton X-100提取分离出A431细胞的细胞骨架。Scatchard分析表明，约5%的原始受体数量主要通过高亲和力位点（KD = 1.5 nM）与细胞骨架相关联。这类受体的进一步特征是存在一个快速解离成分（k-1 = 2.0×10⁻³ s⁻¹）和一个缓慢解离成分（k-1 = 9.1×10⁻⁵ s⁻¹）。细胞骨架中快速和缓慢位点之间的分布与完整细胞相似（65%为快速位点，35%为缓慢位点）。在4℃下于EGF存在的条件下将A431细胞孵育2小时，导致与细胞骨架相关联的EGF受体数量显著增加。这些新的与细胞骨架相关的受体似乎代表低亲和力结合位点（KD = 7 nM）。解离动力学也显示快速解离位点增加。这些结果表明，在4℃时，EGF诱导低亲和力、快速解离位点与A431细胞的细胞骨架结合。
