Estival A, Monzat V, Miquel K, Gaubert F, Hollande E, Korc M, Vaysse N, Clemente F
INSERM U.151, CHU Rangueil, Université Paul Sabatier, Toulouse, France.
J Biol Chem. 1996 Mar 8;271(10):5663-70. doi: 10.1074/jbc.271.10.5663.
To evaluate possible functional differences between basic fibroblast growth factor (FGF) 2 isoforms we analyzed the effects of the 18-kDa FGF-2 which mainly localizes in the cytosol and that of the nuclear-targeted 22.5-kDa form on FGF receptors (FGFR) expression. These peptides were expressed at low amounts through a retroviral-infection system. Point mutated FGF-2 cDNAs under the control of the beta-actin promoter were used to infect a pancreatic cell line (AR4 2J) which does not produce FGF-2. Saturation and competition binding studies with 125I-FGF-2 revealed a 3-fold increase in both high and low affinity receptors in cells expressing the 22.5-kDa form and a 2-fold increase only in the high affinity receptors in cells producing the 18-kDa form. Kd values and molecular weights of the high affinity receptors were unaffected. Increasing cell densities or cell treatment with exogenous FGF-2 resulted in FGFR down-regulation as in control cells. Neutralizing anti-FGF-2 antibodies and suramin did not affect receptor density in control and in cells producing the 22.5-kDa form but further increased by 60 and 80%, respectively, the receptor level in cells synthesizing the 18-kDa form. These data suggest the involvement of the intracellular stored FGF-2 in FGFR up-regulation. Although all cells expressed FGFR-1, -2, and -3 mRNA only the FGFR-1 transcript was found increased, 6-fold in 22.5-kDa expressing cells and 3-fold in cell producing the shortest secreted isoform. The increase in FGFR-1 mRNA levels in the 22.5-kDa expressing cells was due to enhanced stability of the transcript. Confocal microscopy detected the presence of FGFR-1 at the cell surface whereas secretory isoforms of the receptor were not observed. Reverse transcriptase-polymerase chain reaction did not reveal significant differences in the expression of FGFR-1 variants. In the 22.5-kDa expressing cells exogenous FGF-2 evoked a stronger translocation of the calcium-phospholipid-dependent PKC. These results indicate that the transfected FGF-2 isoforms up-regulated FGFR-1 mRNA and protein. The 22.5-kDa form acted by increasing FGFR-1 mRNA stability enhancing cell responses to exogenous FGF-2.
为了评估碱性成纤维细胞生长因子（FGF）2亚型之间可能存在的功能差异，我们分析了主要定位于胞质溶胶的18 kDa FGF - 2以及靶向细胞核的22.5 kDa形式对FGF受体（FGFR）表达的影响。这些肽通过逆转录病毒感染系统低水平表达。使用在β - 肌动蛋白启动子控制下的点突变FGF - 2 cDNA感染不产生FGF - 2的胰腺细胞系（AR4 2J）。用125I - FGF - 2进行的饱和与竞争结合研究表明，表达22.5 kDa形式的细胞中高亲和力和低亲和力受体均增加了3倍，而产生18 kDa形式的细胞中仅高亲和力受体增加了2倍。高亲和力受体的解离常数（Kd）值和分子量未受影响。增加细胞密度或用外源性FGF - 2处理细胞会导致FGFR下调，如同对照细胞一样。中和抗FGF - 2抗体和苏拉明对对照细胞以及产生22.5 kDa形式的细胞中的受体密度没有影响，但分别使合成18 kDa形式的细胞中的受体水平进一步增加了60%和80%。这些数据表明细胞内储存的FGF - 2参与了FGFR的上调。尽管所有细胞都表达FGFR - 1、-2和-3 mRNA，但仅发现FGFR - 1转录本增加，在表达22.5 kDa的细胞中增加了6倍，在产生最短分泌亚型的细胞中增加了3倍。表达22.5 kDa的细胞中FGFR - 1 mRNA水平的增加是由于转录本稳定性增强。共聚焦显微镜检测到细胞表面存在FGFR - 1，而未观察到受体的分泌亚型。逆转录聚合酶链反应未揭示FGFR - 1变体表达的显著差异。在表达22.5 kDa的细胞中，外源性FGF - 2引起钙磷脂依赖性蛋白激酶C（PKC）更强的易位。这些结果表明转染的FGF - 2亚型上调了FGFR - 1 mRNA和蛋白。22.5 kDa形式通过增加FGFR - 1 mRNA稳定性起作用，增强了细胞对外源性FGF - 2的反应。
