Department of Protein Engineering, Genentech, Inc, South San Francisco, California 94080, USA.
J Biol Chem. 2010 Dec 17;285(51):40362-72. doi: 10.1074/jbc.M110.179721. Epub 2010 Oct 11.
Hepatocyte growth factor (HGF) binds to its target receptor tyrosine kinase, Met, as a single-chain form (pro-HGF) or as a cleaved two-chain disulfide-linked α/β-heterodimer. However, only two-chain HGF stimulates Met signaling. Proteolytic cleavage of the Arg(494)-Val(495) peptide bond in the zymogen-like pro-HGF results in allosteric activation of the serine protease-like β-chain (HGF β), which binds Met to initiate signaling. We use insights from the canonical trypsin-like serine protease activation mechanism to show that isolated peptides corresponding to the first 7-10 residues of the cleaved N terminus of the β-chain stimulate Met phosphorylation by pro-HGF to levels that are ∼25% of those stimulated by two-chain HGF. Biolayer interferometry data demonstrate that peptide VVNGIPTR (peptide V8) allosterically enhances pro-HGF β binding to Met, resulting in a K(D)(app) of 1.6 μm, only 8-fold weaker than the Met/HGF β-chain affinity. Most notably, in vitro cell stimulation with peptide V8 in the presence of pro-HGF leads to Akt phosphorylation, enhances cell survival, and facilitates cell migration between 75 and 100% of that found with two-chain HGF, thus revealing a novel approach for activation of Met signaling that bypasses proteolytic processing of pro-HGF. Peptide V8 is unable to enhance Met binding or signaling with HGF proteins having a mutated activation pocket (D672N). Furthermore, Gly substitution of the N-terminal Val residue in peptide V8 results in loss of all activity. Overall, these findings identify the activation pocket of the serine protease-like β-chain as a "hot spot" for allosteric regulation of pro-HGF and have broad implications for developing selective allosteric activators of serine proteases and pseudoproteases.
肝细胞生长因子 (HGF) 以单链形式（原 HGF）或切割的二硫键连接的α/β-异源二聚体形式结合其靶受体酪氨酸激酶 Met。然而，只有二聚体 HGF 刺激 Met 信号转导。原 HGF 中的精氨酸 (494)-缬氨酸 (495) 肽键的蛋白水解切割导致酶原样β-链（HGF β）的别构激活，该β-链与 Met 结合以启动信号转导。我们利用经典的胰蛋白酶样丝氨酸蛋白酶激活机制的见解表明，与β-链切割 N 端的前 7-10 个残基相对应的分离肽刺激原 HGF 诱导 Met 磷酸化的水平达到二聚体 HGF 刺激水平的约 25%。生物层干涉数据表明，肽 VVNGIPTR（肽 V8）别构增强了原 HGF β 与 Met 的结合，导致 K(D)(app)为 1.6 μm，仅比 Met/HGF β-链亲和力弱 8 倍。值得注意的是，在存在原 HGF 的情况下，用肽 V8 体外刺激细胞导致 Akt 磷酸化，增强细胞存活，并促进细胞迁移，与二聚体 HGF 相比提高了 75%至 100%，从而揭示了一种绕过原 HGF 蛋白水解加工的 Met 信号转导激活的新方法。肽 V8 不能增强具有突变激活口袋（D672N）的 HGF 蛋白与 Met 的结合或信号转导。此外，肽 V8 中 N 端 Val 残基的 Gly 取代导致失去所有活性。总体而言，这些发现确定了丝氨酸蛋白酶样β-链的激活口袋是原 HGF 别构调节的“热点”，并对开发丝氨酸蛋白酶和假丝氨酸蛋白酶的选择性别构激活剂具有广泛的意义。
