Degl'Innocenti Debora, Arighi Elena, Popsueva Anna, Sangregorio Romina, Alberti Luisella, Rizzetti Maria Grazia, Ferrario Cristina, Sariola Hannu, Pierotti Marco A, Borrello Maria Grazia
Department of Experimental Oncology, Research Unit #3, Istituto Nazionale Tumori, Via G. Venezian, 1 20133 Milan, Italy.
Oncogene. 2004 Sep 23;23(44):7297-309. doi: 10.1038/sj.onc.1207862.
The receptor tyrosine kinase RET is alternatively spliced to yield two main isoforms, RET9 and RET51, which differ in their carboxyl terminal. Activated RET induces different biological responses such as morphological transformation, neurite outgrowth, proliferation, cell migration and branching. The two isoforms have been suggested to have separate intracellular signaling pathways and different roles in mouse development. Here we show that both isoforms are able to induce cell scattering of SK-N-MC neuroepithelioma cell line and branching tubule formation in MDCK cell line. However, the Y1062F mutation, which abrogates the transforming activity of both activated RET isoforms in NIH3T3 cells, does not abolish scattering and branching morphogenesis of RET51, whereas impairs these biological effects of RET9. The GDNF-induced biological effects of RET51 are inhibited by the simultaneous abrogation of both Tyr1062 and Tyr1096 docking sites. Thus, Tyr1096 may substitute the functions of Tyr1062. GRB2 is the only known adaptor protein binding to Tyr1096. Dominant-negative GRB2 expressed in MDCK cells together with RET9 or RET51 significantly reduces branching. Therefore, GRB2 is necessary for RET-mediated branching of MDCK cells.
受体酪氨酸激酶RET可通过可变剪接产生两种主要异构体，即RET9和RET51，它们的羧基末端有所不同。激活的RET可诱导不同的生物学反应，如形态转化、神经突生长、增殖、细胞迁移和分支。有研究表明，这两种异构体在小鼠发育过程中具有不同的细胞内信号通路和不同作用。在此我们发现，这两种异构体均能诱导SK-N-MC神经上皮瘤细胞系发生细胞散射，并在MDCK细胞系中形成分支小管。然而，Y1062F突变可消除激活的RET异构体在NIH3T3细胞中的转化活性，但不会消除RET51的散射和分支形态发生，而会损害RET9的这些生物学效应。同时消除Tyr1062和Tyr1096对接位点可抑制GDNF诱导的RET51生物学效应。因此，Tyr1096可能替代Tyr1062的功能。GRB2是唯一已知的与Tyr1096结合的衔接蛋白。在MDCK细胞中与RET9或RET51共同表达的显性负性GRB2可显著减少分支。因此，GRB2是RET介导的MDCK细胞分支所必需的。
