Sastry L, Lin W, Wong W T, Di Fiore P P, Scoppa C A, King C R
Oncologix Inc., Gaithersburg, Maryland 20878, USA.
Oncogene. 1995 Sep 21;11(6):1107-12.
Grb2 is an adaptor protein that links receptor and cytoplasmic tyrosine kinases to the Ras signalling pathway by binding the Ras-specific guanine nucleotide exchange factor, Sos1, through its SH3 domains. The Grb2-SH3 domain binding has been localized to the carboxy-terminal two hundred amino acids of Sos1 (Sos1-c). By using real time biospecific interaction analysis (BIAcore), we studied the kinetic parameters and binding affinity of the Grb2-Sos1-c interaction. The binding of Grb2 to Sos1-c is a high affinity interaction with a moderate association rate (9.45 x 10(4) per M per s), a slow dissociation rate (13.8 x 10(-5) s), and an affinity constant of 1.48 nM. BIAcore measurements on isolated N-terminal and C-terminal SH3 domains (NSH3 and CSH3) further indicate that the high affinity Grb2-Sos1-c interaction is primarily mediated through the NSH3 domain (Kd = 1.68 nM). The CSH3 domain shows substantially reduced binding to Sos1-c in these measurements. Inhibition studies with BIAcore using proline rich peptides derived from the C-terminus of Sos1 show that there is a single major binding site for Grb2 in Sos1. This binding site is contained within the peptide N20, which corresponds to amino acids 1143-1162 of Sos1. This peptide completely blocks the Grb2-Sos1-c and NSH3-Sos1-c interactions with IC50 values of 8 microM and 4 microM respectively. The discrete interaction between the NSH3 domain and the N20 peptide may be amenable for drug discovery through screening or peptidomimetic approaches.
Grb2是一种衔接蛋白，它通过其SH3结构域与Ras特异性鸟嘌呤核苷酸交换因子Sos1结合，从而将受体和细胞质酪氨酸激酶连接到Ras信号通路。Grb2-SH3结构域的结合已定位到Sos1的羧基末端两百个氨基酸（Sos1-c）。通过实时生物特异性相互作用分析（BIAcore），我们研究了Grb2-Sos1-c相互作用的动力学参数和结合亲和力。Grb2与Sos1-c的结合是一种高亲和力相互作用，具有中等的缔合速率（9.45×10⁴/M·s）、缓慢的解离速率（13.8×10⁻⁵/s）和1.48 nM的亲和常数。对分离的N端和C端SH3结构域（NSH3和CSH3）进行的BIAcore测量进一步表明，高亲和力的Grb2-Sos1-c相互作用主要通过NSH3结构域介导（Kd = 1.68 nM）。在这些测量中，CSH3结构域与Sos1-c的结合显著降低。使用源自Sos1 C末端的富含脯氨酸的肽进行的BIAcore抑制研究表明，Sos1中存在一个Grb2的主要结合位点。该结合位点包含在肽N20内，其对应于Sos1的1143-1162位氨基酸。该肽分别以8 μM和4 μM的IC50值完全阻断Grb2-Sos1-c和NSH3-Sos1-c的相互作用。NSH3结构域与N20肽之间离散的相互作用可能适合通过筛选或拟肽方法进行药物发现。
