Estrada L, Caron E, Gorski J L
Department of Human Genetics, The University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Hum Mol Genet. 2001 Mar 1;10(5):485-95. doi: 10.1093/hmg/10.5.485.
FGD1, the gene responsible for the inherited disease faciogenital dysplasia, encodes a guanine nucleotide exchange factor (GEF) that specifically activates the p21 GTPase Cdc42. In order, FGD1 is composed of a proline-rich N-terminal region, adjacent GEF and pleckstrin homology (PH) domains, a FYVE-finger domain and a second C-terminal PH domain (PH2), structural motifs involved in signaling and subcellular localization. Fgd1, the mouse FGD1 ortholog, is expressed in regions of active bone formation within osteoblasts and in the osteoblast-like cell line MC3T3-E1, a finding consistent with its role in skeletal formation. Here, we use subcellular fractionation studies to show that endogenous Fgd1 protein is localized in the cytosolic and Golgi and plasma membrane fractions of mouse calvarial cells. Immunocytochemical studies performed with osteoblast-like MC3T3-E1 cells and other mammalian cell lines confirm the localization of Fgd1 and show that the proline-rich N-terminal region is necessary and sufficient for Fgd1 subcellular localization to the plasma membrane and Golgi complex. In contrast, the FYVE-finger and PH2 domains do not appear to direct the localization of Fgd1 or the activation of Cdc42. In addition, microinjection studies indicate that the N-terminal Fgd1 domain inhibits filopodia formation, suggesting that this region down-regulates GEF function. These results characterize the function of the Fgd1 domains for both protein localization and Cdc42 activation and indicate that the Fgd1 Cdc42GEF protein is involved in the regulation of Cdc42 activity at the subcortical actin cytoskeleton and Golgi complex.
FGD1是导致遗传性疾病面生殖器发育异常的基因，它编码一种鸟嘌呤核苷酸交换因子（GEF），该因子特异性激活p21 GTP酶Cdc42。FGD1依次由富含脯氨酸的N端区域、相邻的GEF和普列克底物蛋白同源（PH）结构域、一个FYVE指结构域和第二个C端PH结构域（PH2）组成，这些结构基序参与信号传导和亚细胞定位。小鼠FGD1直系同源基因Fgd1在成骨细胞内活跃的骨形成区域以及成骨细胞样细胞系MC3T3-E1中表达，这一发现与其在骨骼形成中的作用一致。在此，我们通过亚细胞分级分离研究表明，内源性Fgd1蛋白定位于小鼠颅骨细胞的胞质、高尔基体和质膜部分。用成骨细胞样MC3T3-E1细胞和其他哺乳动物细胞系进行的免疫细胞化学研究证实了Fgd1的定位，并表明富含脯氨酸的N端区域对于Fgd1亚细胞定位于质膜和高尔基体复合体是必要且充分的。相比之下，FYVE指结构域和PH2结构域似乎并不指导Fgd1的定位或Cdc42的激活。此外，显微注射研究表明，Fgd1的N端结构域抑制丝状伪足的形成，这表明该区域下调了GEF功能。这些结果描述了Fgd1结构域在蛋白质定位和Cdc42激活方面的功能，并表明Fgd1 Cdc42GEF蛋白参与了在皮质下肌动蛋白细胞骨架和高尔基体复合体处对Cdc42活性的调节。
