Prudovsky I A, Savion N, LaVallee T M, Maciag T
Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.
J Biol Chem. 1996 Jun 14;271(24):14198-205. doi: 10.1074/jbc.271.24.14198.
The alternatively spliced fibroblast growth factor receptor (FGFR)-1 isoforms, FGFR-1alpha and FGFR-1beta, are characterized by the presence of either three or two Ig-like loops in the extracellular domain and are differentially expressed during embryonic development and tumor progression. We have previously shown that in cells irreversibly committed to DNA synthesis by FGF-1, approximately 15% of cell surface FGFR-1 traffics to a perinuclear locale as a structurally intact and functional tyrosine kinase (Prudovsky, I., Savion, N., Zhan, X., Friesel, R., Xu, J., Hou, J., McKeehan, W. L., and Maciag, T. (1994) J. Biol. Chem. 269, 31720-31724). In order to define the structural requirement for association of FGFR-1 with the nucleus, the expression and trafficking of FGFR-1 in FGFR-1alpha and FGFR-1beta L6 myoblast transfectants was studied. Although FGFR-1alpha was expressed as p145 and p125 forms, FGFR-1beta was expressed as p120 and p100 forms in the L6 myoblast transfectants. Tunicamycin and N-glyconase experiments suggest that these forms of FGFR-1alpha and FGFR-1beta are the result of differential glycosylation. However, only the p145 form of FGFR-1alpha and the p120 form of FGFR-1beta were able to bind FGF-1 and activate tyrosine phosphorylation. Pulse-chase analysis of FGFR-1 biosynthesis suggests that the p125 and p100 proteins are the precursor forms of p145 FGFR-1alpha and p120 FGFR-1beta, respectively. Because ligand-chase analysis demonstrated that FGFR-1beta L6 myoblast transfectants exhibited a reduced efficiency of nuclear translocation of exogenous FGF-1 when compared with FGFR-1alpha transfectants, the intracellular trafficking of the FGFR-1alpha and FGFR-1beta isoforms was studied using an in vitro kinase assay to amplify immunoprecipitated FGFR-1. Indeed, the appearance of the FGFR-1alpha but not FGFR-1beta isoform in the nuclear fraction of L6 myoblast transfectants suggests that the distal Ig-like loop in FGFR-1alpha mediates the differential nuclear association of FGFR-1alpha as a structurally intact and functional tyrosine kinase. Further, the FGFR-1beta L6 myoblast transfectants but not the FGFR-1alpha myoblast transfectants exhibited a pronounced morphologic change in response to exogenous FGF-1. Because this phenotype change involves the induction of a rounded cellular shape, it is possible that the FGFR-1alpha and FGFR-1beta may ultimately exhibit differential trafficking to adhesion sites.
选择性剪接的成纤维细胞生长因子受体（FGFR）-1亚型，即FGFR-1α和FGFR-1β，其特征在于细胞外结构域中存在三个或两个免疫球蛋白样环，并且在胚胎发育和肿瘤进展过程中差异表达。我们之前已经表明，在被FGF-1不可逆地诱导进入DNA合成的细胞中，约15%的细胞表面FGFR-1作为结构完整且具有功能的酪氨酸激酶转运至核周区域（普鲁多夫斯基，I.，萨维翁，N.，詹，X.，弗里塞尔，R.，徐，J.，侯，J.，麦基汉，W.L.，以及马西亚格，T.（1994年）《生物化学杂志》269，31720 - 31724）。为了确定FGFR-1与细胞核结合的结构要求，我们研究了FGFR-1α和FGFR-1β L6成肌细胞转染子中FGFR-1的表达和转运。尽管在L6成肌细胞转染子中FGFR-1α以p145和p125形式表达，但FGFR-1β以p120和p100形式表达。衣霉素和N-糖苷酶实验表明，这些FGFR-1α和FGFR-1β的形式是糖基化差异的结果。然而，只有FGFR-1α的p145形式和FGFR-1β的p120形式能够结合FGF-1并激活酪氨酸磷酸化。FGFR-1生物合成的脉冲追踪分析表明，p125和p100蛋白分别是p145 FGFR-1α和p120 FGFR-1β的前体形式。因为配体追踪分析表明，与FGFR-1α转染子相比，FGFR-1β L6成肌细胞转染子中外源FGF-1的核转位效率降低，所以我们使用体外激酶测定法来扩增免疫沉淀的FGFR-1，研究了FGFR-1α和FGFR-1β亚型的细胞内转运。实际上，FGFR-1α而非FGFR-1β亚型出现在L6成肌细胞转染子的核部分中，这表明FGFR-1α中的远端免疫球蛋白样环作为结构完整且具有功能的酪氨酸激酶介导了FGFR-1α的差异核结合。此外，FGFR-1β L6成肌细胞转染子而非FGFR-1α成肌细胞转染子对外源FGF-1表现出明显的形态变化。因为这种表型变化涉及诱导细胞呈圆形，所以FGFR-1α和FGFR-1β最终可能在向黏附位点的转运上表现出差异。
