Elenius K, Choi C J, Paul S, Santiestevan E, Nishi E, Klagsbrun M
Department of Surgery, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Oncogene. 1999 Apr 22;18(16):2607-15. doi: 10.1038/sj.onc.1202612.
Receptor tyrosine kinases regulate cell behavior by activating specific signal transduction cascades. Epidermal growth factor (EGF) receptor tyrosine kinases include ErbB1, ErbB2, ErbB3 and ErbB4. ErbB4 is a tyrosine kinase receptor that binds neuregulins (NRG) and several other EGF family members. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis identified two isoforms of ErbB4 that differed in their cytoplasmic domain sequences. Specifically, RT-PCR using primers flanking the putative phosphatidyl inositol 3-kinase (PI3-K) binding site of ErbB4 generated two specific bands when human and mouse heart and kidney tissues were analysed. Cloning and sequencing of these RT-PCR products revealed that one of the ErbB4 isoforms (ErbB4 CYT-2) lacked a 16 amino acid sequence including a putative PI3-K binding site, that was present in the other isoform (ErbB4 CYT-1). RT-PCR analysis of mouse tissues suggested that the expression of ErbB4 CYT-1 and ErbB4 CYT-2 was tissue-specific. Heart, breast and abdominal aorta expressed predominantly ErbB4 CYT-1 whereas neural tissues and kidney expressed predominantly ErbB4 CYT-2. To ascertain whether the absence of the putative PI3-K binding site in ErbB4 CYT-2 also resulted in the loss of PI3-K activity, NIH3T3 cell lines overexpressing ErbB4 CYT-1 or ErbB4 CYT-2 were produced. NRG-1 bound to and stimulated equivalent tyrosine phosphorylation of both isoforms. However, unlike ErbB4 CYT-1, the ErbB4 CYT-2 isoform was unable to bind the p85 subunit of PI3-K and to stimulate PI3-K activity in these cells. Furthermore, tyrosine phosphorylation of p85 or association of PI3-K activity with phosphotyrosine was not induced in NRG-1 treated cells expressing ErbB4 CYT-2, indicating that this isoform was incapable of activating PI3-K even indirectly. It was concluded that a novel naturally occurring ErbB4 isoform exists with a deletion of the cytoplasmic domain sequence required for the activation of the PI3-K intracellular signal transduction pathway and that this is the only PI3-K binding site in ErbB4.
受体酪氨酸激酶通过激活特定的信号转导级联反应来调节细胞行为。表皮生长因子（EGF）受体酪氨酸激酶包括ErbB1、ErbB2、ErbB3和ErbB4。ErbB4是一种酪氨酸激酶受体，可结合神经调节蛋白（NRG）和其他几种EGF家族成员。逆转录聚合酶链反应（RT-PCR）分析鉴定出ErbB4的两种异构体，它们的胞质结构域序列不同。具体而言，当分析人和小鼠的心脏和肾脏组织时，使用位于ErbB4假定的磷脂酰肌醇3激酶（PI3-K）结合位点两侧的引物进行RT-PCR产生了两条特异性条带。对这些RT-PCR产物进行克隆和测序后发现，其中一种ErbB4异构体（ErbB4 CYT-2）缺少一个包含假定PI3-K结合位点的16个氨基酸序列，而另一种异构体（ErbB4 CYT-1）则含有该序列。对小鼠组织的RT-PCR分析表明，ErbB4 CYT-1和ErbB4 CYT-2的表达具有组织特异性。心脏、乳腺和腹主动脉主要表达ErbB4 CYT-1，而神经组织和肾脏主要表达ErbB4 CYT-2。为了确定ErbB4 CYT-2中假定的PI3-K结合位点的缺失是否也导致PI3-K活性丧失，构建了过表达ErbB4 CYT-1或ErbB4 CYT-2的NIH3T3细胞系。NRG-1与两种异构体结合并刺激其等效的酪氨酸磷酸化。然而，与ErbB4 CYT-1不同，ErbB4 CYT-2异构体无法结合PI3-K的p85亚基，也无法在这些细胞中刺激PI3-K活性。此外，在表达ErbB4 CYT-2的NRG-1处理细胞中，未诱导p85的酪氨酸磷酸化或PI3-K活性与磷酸酪氨酸的结合，这表明该异构体即使间接也无法激活PI3-K。得出的结论是，存在一种新的天然存在的ErbB4异构体，其缺失激活PI3-K细胞内信号转导途径所需的胞质结构域序列，并且这是ErbB4中唯一的PI3-K结合位点。
