Nuclear trafficking of FGFR1: a role for the transmembrane domain.
Nuclear trafficking of FGFR1: a role for the transmembrane domain.
复制标题
FGFR1 的核运输：跨膜结构域的作用。
DOI：
10.1002/jcb.10476
复制
发表时间：
2003
影响因子：
4
通讯作者：
Stachowiak,MichalK
中科院分区：
文献类型：
--
作者：
Myers,JasonM;Martins,GabrielG;Ostrowski,Jacek;Stachowiak,MichalK
成纤维细胞生长因子家族中的几个成员缺乏信号肽(SP)序列，并且仅以微量存在于细胞外。然而，这些蛋白质含有核定位信号(NLS)，并积聚在细胞核中。我们的研究表明，全长的成纤维细胞生长因子受体1(FGFR1)与成纤维细胞生长因子-2(FGF2)平行地聚集在核内部。我们检验了非典型跨膜区(TM)在FGFR1转运到核内部的过程中起作用的假设。由于FGFR1由于其SP而注定要与质膜进行结构性融合，因此该受体如何进入细胞核尚不清楚。序列分析证实，FGFR1具有一个非典型的TM，其中含有短片段的疏水氨基酸(A.A.)被极地A.A.打断。β-Sheet是FGFR1TM的预测构象，与包括FGFR4TM在内的其他单个TM酪氨酸激酶受体的α-螺旋构象相反。在共聚焦显微镜下，通过将FGFR1与增强型绿色荧光蛋白(EGFP)融合，通过共聚焦显微镜研究了受体在活细胞中的转运，并通过亚细胞分离和免疫印迹证实了这一点。FGFR1-EGFP的核进入与核分裂无关，并在快速增殖的人TE671细胞、增殖较慢的胶质瘤SF763和有丝分裂后的牛肾上腺髓质细胞(BAMC)中观察到。相反，嵌合的FGFR1/R4-EGFP，其中FGFR1的TM被FGFR4的TM替换，与膜(Golgi-ER、胞浆和核)相连，但不存在于细胞核和胞浆中。FGFR1GFP-Δ突变体，具有疏水性TMA.A.用极性的A.A.取代，表现出与膜的结合减少，胞质/核积累增加，TM亲水性增加。FGFR1TM(TM−)-EGFP(TM缺失)存在于高尔基体小泡、胞浆和细胞核内部，表明FGFR1TM不具有NLS功能。为了检测胞质FGFR1是否提供核FGFR1的来源，将FGFR1(SP−)导入细胞(去掉SP)，导致胞浆、无膜、蛋白质在胞浆和细胞核内积聚。我们的结果表明，与细胞膜的不稳定结合是导致FGFR1释放到胞浆中的原因，而胞浆中的FGFR1是核受体的来源。J.细胞。生物化学。88：1273-1291,2003。©2003 Wiley-Liss公司
Several members of the fibroblast growth factor (FGF) family lack signal peptide (SP) sequences and are present only in trace amounts outside the cell. However, these proteins contain nuclear localization signals (NLS) and accumulate in the cell nucleus. Our studies have shown that full length FGF receptor 1 (FGFR1) accumulates within the nuclear interior in parallel with FGF‐2. We tested the hypothesis that an atypical transmembrane domain (TM) plays a role in FGFR1 trafficking into the nuclear interior. With FGFR1 destined for constitutive fusion with the plasma membrane due to its SP, how the receptor may enter the nucleus is unclear. Sequence analysis identified that FGFR1 has an atypical TM containing short stretches of hydrophobic amino acids (a.a.) interrupted by polar a.a. The β‐sheet is the predicted conformation of the FGFR1 TM, in contrast to the α‐helical conformation of other single TM tyrosine kinase receptors, including FGFR4. Receptor trafficking in live cells was studied by confocal microscopy via C‐terminal FGFR1 fusions to enhanced green fluorescent protein (EGFP) and confirmed by subcellular fractionation and Western immunoblotting. Nuclear entry of FGFR1–EGFP was independent of karyokinessis, and was observed in rapidly proliferating human TE671 cells, in slower proliferating glioma SF763 and post‐mitotic bovine adrenal medullary cells (BAMC). In contrast, a chimeric FGFR1/R4‐EGFP, where the TM of FGFR1 was replaced with that of FGFR4, was associated with membranes (golgi‐ER, plasma, and nuclear), but was absent from the nucleus and cytosol. FGFR1Δ‐EGFP mutants, with hydrophobic TM a.a. replaced with polar a.a., showed reduced association with membranes and increased cytosolic/nuclear accumulation with an increase in TM hydrophilicity. FGFR1(TM−)‐EGFP (TM deleted), was detected in the golgi‐ER vesicles, cytosol, and nuclear interior; thus demonstrating that the FGFR1 TM does not function as a NLS. To test whether cytosolic FGFR1 provides a source of nuclear FGFR1, cells were transfected with FGFR1(SP−) (SP was deleted), resulting in cytosolic, non‐membrane, protein accumulation in the cytosol and the cell nucleus. Our results indicate that an unstable association with cellular membranes is responsible for the release of FGFR1 into the cytosol and cytosolic FGFR1 constitutes the source of the nuclear receptor. J. Cell. Biochem. 88: 1273–1291, 2003. © 2003 Wiley‐Liss, Inc.