Zhou M, Felder S, Rubinstein M, Hurwitz D R, Ullrich A, Lax I, Schlessinger J
Department of Pharmacology, New York University Medical Center, New York 10016.
Biochemistry. 1993 Aug 17;32(32):8193-8. doi: 10.1021/bi00083a020.
We have tested one aspect of the allosteric dimerization model for the activation of EGF receptor (EGFR) by EGF: whether EGF binding favors dimerization of the receptor. For this to be true, EGF molecules must bind with higher affinity to dimeric receptors than to monomeric receptors. We have tested this directly in a defined system using the soluble, extracellular ligand binding domain of EGFR monomers (sEGFR) and sEGFR dimers stabilized by treatment with a covalent cross-linking agent. We describe real-time kinetic measurements of EGF binding to receptor monomers and dimers employing the method of total internal reflection (surface plasmon resonance). Our data show that sEGFR dimers bound EGF with 30-40-fold higher affinity [KD = (2-3) x 10(-8) M] than did sEGFR monomers. The enhanced binding affinity of sEGFR dimers resulted mainly from a reduced off-rate with k(off) = 0.001 s-1 for sEGFR dimers as compared to k(off) = 0.06 s-1 for sEGFR monomers. These measurements indicate that dimerization of sEGFR increases its affinity for EGF by prolonging the amount of time that EGF remains bound to the receptor. This provides evidence that EGF binding stabilizes receptor dimerization and provides further support for the allosteric dimerization model as a mechanism for ligand induced receptor activation.
我们已经对表皮生长因子（EGF）激活表皮生长因子受体（EGFR）的变构二聚化模型的一个方面进行了测试：即EGF结合是否有利于受体的二聚化。若要如此，EGF分子与二聚体受体的结合亲和力必须高于与单体受体的结合亲和力。我们已在一个特定系统中直接对此进行了测试，该系统使用了EGFR单体的可溶性细胞外配体结合结构域（sEGFR）以及经共价交联剂处理而稳定的sEGFR二聚体。我们描述了采用全内反射（表面等离子体共振）方法对EGF与受体单体和二聚体结合的实时动力学测量。我们的数据表明，sEGFR二聚体结合EGF的亲和力比sEGFR单体高30 - 40倍[解离常数KD =（2 - 3）×10^(-8) M]。sEGFR二聚体结合亲和力的增强主要源于解离速率的降低，sEGFR二聚体的解离速率常数k(off) = 0.001 s^(-1)，而sEGFR单体的解离速率常数k(off) = 0.06 s^(-1)。这些测量结果表明，sEGFR的二聚化通过延长EGF与受体结合的时间来增加其对EGF的亲和力。这为EGF结合稳定受体二聚化提供了证据，并为变构二聚化模型作为配体诱导受体激活的一种机制提供了进一步支持。
