Zhang Pei, Greendorfer Jessica S, Jiao Jing, Kelpke Stacey C, Thompson John A
Department of Surgery and Biochemistry, The University of Alabama at Birmingham, 35294, USA.
Arch Biochem Biophys. 2006 Jun 1;450(1):50-62. doi: 10.1016/j.abb.2006.03.017. Epub 2006 Mar 30.
Ligand activation of fibroblast growth factor receptor-1 (FGFR-1) induces an angiogenic response following activation of multiple intracellular signaling substrates, including the Src family of nonreceptor tyrosine kinases (SFK). However, the direct association between FGFR-1 and SFK and the involvement of SFK in FGFR-1-dependent cell proliferation have been controversial. Structural variants of FGFR-1 are generated by alternative splicing which results in two major isoforms, containing either three (FGFR-1alpha) or two (FGFR-1beta) immunoglobulin-like domains in the extracellular region. To determine whether alternatively spliced FGFR-1 isoforms differentially activate SFK, we have examined FGF receptor-negative endothelial cells stably transfected with human cDNA encoding either FGFR-1alpha or FGFR-1beta. Transient activation of c-YES, the predominant SFK expressed in these endothelial cells, was restricted to FGFR-1beta transfectants following exposure to acidic fibroblast growth factor (FGF-1). Co-immunoprecipitation studies revealed that c-YES directly associated with FGFR-1beta. The Src homology (SH)2 domain (and not the SH3 domain) of c-YES was able to recognize tyrosine phosphorylated FGFR-1beta. FGFR-1beta-specific activation of c-YES was accompanied by its association with and activation of cortactin. FGF-1 treatment of both FGFR-1alpha and FGFR-1beta transfectants induced SFK-independent cellular proliferation and growth in low density cultures. At high density, under both anchorage-dependent and -independent conditions, FGF-1 failed to induce proliferation and growth of FGFR-1alpha transfectants. In contrast, FGF-1 induced proliferation, growth, and formation of cord-like structures in high density cultures of FGFR-1beta transfectants in an SFK-dependent manner. In vitro cord formation on Matrigel was restricted to FGFR-1beta transfectants in an SFK-dependent manner. Formation of vascular structures in vivo was limited to endothelial cells transfected with FGFR-1beta. Collectively, these results emphasize the roles of alternatively spliced FGFR-1 structural isoforms and activation of SFK as modulators of endothelial cell growth during the formation of neovascular structures.
成纤维细胞生长因子受体-1（FGFR-1）的配体激活在多个细胞内信号转导底物（包括非受体酪氨酸激酶的Src家族（SFK））激活后诱导血管生成反应。然而，FGFR-1与SFK之间的直接关联以及SFK在FGFR-1依赖性细胞增殖中的作用一直存在争议。FGFR-1的结构变体通过可变剪接产生，这导致两种主要的异构体，在细胞外区域含有三个（FGFR-1α）或两个（FGFR-1β）免疫球蛋白样结构域。为了确定可变剪接的FGFR-1异构体是否差异激活SFK，我们检测了稳定转染了编码FGFR-1α或FGFR-1β的人cDNA的FGF受体阴性内皮细胞。c-YES是这些内皮细胞中表达的主要SFK，其短暂激活仅限于暴露于酸性成纤维细胞生长因子（FGF-1）后的FGFR-1β转染子。免疫共沉淀研究表明，c-YES与FGFR-1β直接相关。c-YES的Src同源（SH）2结构域（而非SH3结构域）能够识别酪氨酸磷酸化的FGFR-1β。c-YES的FGFR-1β特异性激活伴随着它与皮层肌动蛋白的结合和激活。FGF-1处理FGFR-1α和FGFR-1β转染子在低密度培养中诱导了不依赖SFK的细胞增殖和生长。在高密度下，在贴壁依赖性和非依赖性条件下，FGF-1均未能诱导FGFR-1α转染子的增殖和生长。相反，FGF-1以依赖SFK的方式在FGFR-1β转染子的高密度培养中诱导增殖、生长和索状结构的形成。在基质胶上的体外索状结构形成以依赖SFK的方式仅限于FGFR-1β转染子。体内血管结构的形成仅限于转染了FGFR-1β的内皮细胞。总的来说，这些结果强调了可变剪接的FGFR-1结构异构体和SFK激活作为新生血管结构形成过程中内皮细胞生长调节剂的作用。
