探討腺嘌呤核苷二磷酸核糖化因子相似蛋白四D(ARL4D)之功能

ADP-ribosylation factor (Arf) family of small GTPase in mammals consists of 6 Arfs, 22 Arls (Arf-like), and 2 Sar proteins. The Arf family is best known for the role of Arfs and Arl1 as regulators for coat protein recruitment and phospholipid metabolism, but the biological functions of most Arls remain largely unknown. In this dissertation, a developmentally regulated member of Arl protein, ARL4D, was characterized. In interphase cells, ARL4D localizes to plasma membrane and nuclei and localization of ARL4D at the plasma membrane is GTP- and N-terminal myristoylation-dependent. In mitotic cells, ARL4D was associated predominantly with centrosomal region. We identified an ARL4D interacting protein, cytohesin-2/ARNO. Cytohesin-2/ARNO is a guanine nucleotide-exchange factor (GEF) for Arf and at the plasma membrane it can activate Arf6 to regulate actin reorganization and membrane ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of cytohesin-2/ARNO in a GTP-dependent manner. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or siRNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and modulate actin remodeling. We also show that GTP-binding-defective mutant ARL4D(T35N) localizes to mitochondria and causes dissipation of mitochondrial membrane potential, but not induce release of cytochrome c. Based on our data, we infer that ARL4D might regulate different cellular processes, including cytoskeleton reorganization and mitochondrial function.