Carraway K L, Cerione R A
Department of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
J Biol Chem. 1993 Nov 15;268(32):23860-7.
We have examined the perturbation of epidermal growth factor (EGF) receptor-receptor interactions by a monoclonal antibody (13A9) that binds to the receptor extracellular domain. While 13A9 did not inhibit EGF binding, it inhibited energy transfer between fluorescent-labeled EGF molecules bound to receptors in membranes from human A431 cells by 70-100%. This antibody also inhibited EGF-stimulated receptor dimerization in membranes as assessed by chemical cross-linking and Fab fragments of the antibody strongly inhibited the EGF-stimulated dimerization of solubilized receptors when assessed by velocity sedimentation. However, under conditions where 13A9 inhibited receptor-receptor interactions within the plasma membranes, the antibody had no effect on EGF-stimulated receptor autophosphorylation or tyrosine kinase activity toward an exogenous substrate. Moreover, although the antibody significantly inhibited receptor dimerization in A431 cells, it had no effect on EGF-stimulated changes in cytosolic free [Ca2+] or 125I-EGF uptake in these cells, or on EGF-stimulated DNA synthesis in Swiss 3T3 cells. We conclude that the dimerization of the EGF receptors in a membrane environment is not required for full activation of tyrosine kinase activity and that inhibition of the dimerization of a large fraction of EGF receptors in cells does not necessarily inhibit several EGF-mediated cellular responses.
我们研究了一种与表皮生长因子（EGF）受体胞外结构域结合的单克隆抗体（13A9）对EGF受体-受体相互作用的干扰。虽然13A9不抑制EGF结合，但它能将结合于人A431细胞膜上受体的荧光标记EGF分子间的能量转移抑制70%-100%。通过化学交联评估，该抗体还抑制了膜中EGF刺激的受体二聚化，并且当通过速度沉降评估时，抗体的Fab片段强烈抑制了可溶性受体的EGF刺激的二聚化。然而，在13A9抑制质膜内受体-受体相互作用的条件下，该抗体对EGF刺激的受体自身磷酸化或对外源底物的酪氨酸激酶活性没有影响。此外，尽管该抗体显著抑制了A431细胞中的受体二聚化，但它对这些细胞中EGF刺激的胞质游离[Ca2+]变化或125I-EGF摄取没有影响，对瑞士3T3细胞中EGF刺激的DNA合成也没有影响。我们得出结论，膜环境中EGF受体的二聚化对于酪氨酸激酶活性的完全激活不是必需的，并且抑制细胞中大部分EGF受体的二聚化不一定会抑制几种EGF介导的细胞反应。
