RT Journal Article
SR Electronic
T1 Centaurin-α<sub>1</sub>, an ADP-Ribosylation Factor 6 GTPase Activating Protein, Inhibits β<sub>2</sub>-Adrenoceptor Internalization
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1822
OP 1828
DO 10.1124/mol.105.011338
VO 67
IS 6
A1 Joanna Lawrence
A1 Stuart J. Mundell
A1 Hongruo Yun
A1 Eamonn Kelly
A1 Kanamarlapudi Venkateswarlu
YR 2005
UL http://molpharm.aspetjournals.org/content/67/6/1822.abstract
AB 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.