The ADP-ribosylation factor (ARF) 6 small GTPase regulates vesicle trafficking and cytoskeletal actin reorganization. The GTPase-activating proteins (GAPs) catalyze the formation of inactive ARF6 GDP . Centaurin-␣ 1 contains an ARF GAP and two pleckstrin homology (PH) domains, which bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). Here, we show that centaurin-␣ 1 specifically inhibits in vivo GTP loading of ARF6 and redistribution of ARF6 from the endosomal compartment to the plasma membrane, which are indicative of its activation. Centaurin-␣ 1 also inhibited cortical actin formation in a PIP 3 -dependent manner. Moreover, the constitutively active mutant of ARF6, but not that of ARF1, reverses the inhibition of cortical actin formation by centaurin-␣ 1 . An artificially plasma membrane-targeted centaurin-␣ 1 bypasses the requirement of PIP 3 for its involvement in ARF6 inactivation, suggesting that PIP 3 is required for recruitment of centaurin-␣ 1 to the plasma membrane but not for its activity. Together, these data suggest that centaurin-␣ 1 negatively regulates ARF6 activity by functioning as an in vivo PIP 3 -dependent ARF6 GAP. Phosphatidylinositol (PI)1 3-kinases phosphorylate the 3Ј position of the inositol ring of PI and its derivatives. The 3-phosphorylated PIs such as PIP 3 function as second messengers in the regulation of many cellular functions, including cell migration and vesicle transport (1). PIP 3 is localized in the cytosolic leaflet of the plasma membrane and acts as a site-specific signal for recruitment and/or activation of cytosolic proteins required for the formation of functional complexes at the plasma membrane. A large number of down stream targets have been identified for this lipid and used to characterize agonist activated PI 3-kinase associated cellular pathways. These include ARF regulators such as cytohesins, ARAP3 (ARF GAP, Rho GAP, ankyrin repeat, PH protein 3) and centaurin-␣ 1 (2).ARF family of small GTPases regulate vesicle trafficking by shuttling between an inactive GDP-and an active GTP-bound form (3). Among the known six-mammalian ARF isoforms (ARFs 1-6), ARF1 and ARF6 are the most distantly related and the best characterized. ARF1 localizes to the cytosol in GDP-bound form and to the Golgi membrane in GTP-bound form and regulates transport from the Golgi complex. In contrast, ARF6 localizes to endosomes in GDP-bound form and to the plasma membrane in GTP-bound form and regulates transport between these two organelles and cortical actin re-arrangements at the plasma membrane, which are vital for many cellular functions such as endocytosis, chemotaxis, and focal adhesion (4). ARF guanine-nucleotide exchange factors (GEFs) activate ARFs by catalyzing the release of ARF-bound GDP and permitting the subsequent binding of GTP. In contrast, ARF GAPs stimulate intrinsic ARF GTPase activity, resulting in the hydrolysis of ARF-bound GTP to GDP. Cytohesin 1-3 ARF GEFs recruit to the plasma membrane in agonist stimulated cells by binding PIP 3 and then...

The small GTP-binding protein ADP ribosylation factor 6 (ARF6) has recently been implicated in the internalization of G proteincoupled receptors (GPCRs), although its precise molecular mechanism in this process remains unclear. We have recently identified centaurin ␣ 1 as a GTPase activating protein (GAP) for ARF6. In the current study, we characterized the effects of centaurin ␣ 1 on the agonist-induced internalization of the ␤ 2 -adrenoceptor transiently expressed in human embryonic kidney (HEK) 293 cells. Using an enzyme-linked immunosorbent assay as well as confocal imaging of cells, we found that expression of centaurin ␣ 1 strongly inhibited the isoproterenolinduced internalization of ␤ 2 -adrenoceptor. On the other hand, expression of functionally inactive versions of centaurin ␣ 1 , including an R49C mutant, which has no catalytic activity, and a double pleckstrin homology (PH) mutant (DM; R148C/ R273C), which has mutations in both the PH domains of centaurin ␣ 1 , rendering it unable to translocate to the cell membrane, were unable to inhibit ␤ 2 -adrenoceptor internalization. In addition, a constitutively active version of ARF6, ARF6Q67L, reversed the ability of centaurin ␣ 1 to inhibit ␤ 2 -adrenoceptor internalization. Finally, expression of centaurin ␣ 1 also inhibited the agonist-induced internalization of ␤ 2 -adrenoceptor endogenously expressed in HEK 293 cells, whereas the R49C and DM mutant versions of centaurin ␣ 1 had no effect. Together, these data indicate that by acting as an ARF6 GAP, centaurin ␣ 1 is able to switch off ARF6 and so inhibit its ability to mediate ␤ 2 -adrenoceptor internalization. Thus, ARF6 GAPs, such as centaurin ␣ 1 , are likely to play a crucial role in GPCR trafficking by modulating the activity of ARF6.