Abstract

The small GTP-binding protein ADP ribosylation factor 6 (ARF6) has recently been implicated in the internalization of G protein-coupled receptors (GPCRs), although its precise molecular mechanism in this process remains unclear. We have recently identified centaurin α₁ as a GTPase activating protein (GAP) for ARF6. In the current study, we characterized the effects of centaurin α₁ on the agonist-induced internalization of the β₂-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 α₁ strongly inhibited the isoproterenol-induced internalization of β₂-adrenoceptor. On the other hand, expression of functionally inactive versions of centaurin α₁, 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 α₁, rendering it unable to translocate to the cell membrane, were unable to inhibit β₂-adrenoceptor internalization. In addition, a constitutively active version of ARF6, ARF6Q67L, reversed the ability of centaurin α₁ to inhibit β₂-adrenoceptor internalization. Finally, expression of centaurin α₁ also inhibited the agonist-induced internalization of β₂-adrenoceptor endogenously expressed in HEK 293 cells, whereas the R49C and DM mutant versions of centaurin α₁ had no effect. Together, these data indicate that by acting as an ARF6 GAP, centaurin α₁ is able to switch off ARF6 and so inhibit its ability to mediate β₂-adrenoceptor internalization. Thus, ARF6 GAPs, such as centaurin α₁, are likely to play a crucial role in GPCR trafficking by modulating the activity of ARF6.