Stoica G E, Kuo A, Aigner A, Sunitha I, Souttou B, Malerczyk C, Caughey D J, Wen D, Karavanov A, Riegel A T, Wellstein A
Lombardi Cancer Center, Georgetown University, Washington, DC 20007, USA.
J Biol Chem. 2001 May 18;276(20):16772-9. doi: 10.1074/jbc.M010660200. Epub 2001 Feb 8.
Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.
多效生长因子（PTN）是一种分泌型生长因子，可诱导神经突生长，对成纤维细胞、上皮细胞和内皮细胞具有促有丝分裂作用。在肿瘤生长过程中，PTN可作为血管生成因子，促进肿瘤侵袭和转移。为了鉴定PTN的受体，我们以固定化的PTN蛋白为诱饵，筛选了一个噬菌体展示人cDNA文库。从中我们分离出一个噬菌体插入片段，它与孤儿受体酪氨酸激酶间变性淋巴瘤激酶（ALK）细胞外结构域（ECD）中的一段氨基酸序列同源。与PTN一样，ALK在神经系统围产期发育期间高表达，在成体中下调。在这里，我们在无细胞试验以及完整细胞的放射性配体受体结合研究中表明，PTN与ALK ECD结合，其表观解离常数（Kd）为32±9皮摩尔。过量的PTN、ALK ECD以及抗PTN和抗ECD抗体均可抑制这种受体结合。添加到表达ALK的细胞中的PTN可诱导ALK以及下游效应分子IRS-1、Shc、磷脂酶C-γ和磷脂酰肌醇3激酶的磷酸化。此外，PTN对培养的不同细胞系的生长刺激作用与ALK mRNA的内源性表达一致，并且ALK过表达可增强PTN的作用。由此我们得出结论，ALK是一种转导PTN介导信号的受体，并提出PTN-ALK轴在发育和疾病过程中可能发挥重要作用。
