title: Centaurin-alpha 1, an ADP-ribosylation factor 6 GTPase activating protein, inhibits beta 2-adrenoceptor internalization.
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 alpha(1) as a GTPase activating protein (GAP) for ARF6. In the current study, we characterized the effects of centaurin alpha(1) on the agonist-induced internalization of the beta(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 alpha(1) strongly inhibited the isoproterenol-induced internalization of beta(2)-adrenoceptor. On the other hand, expression of functionally inactive versions of centaurin alpha(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 alpha(1), rendering it unable to translocate to the cell membrane, were unable to inhibit beta(2)-adrenoceptor internalization. In addition, a constitutively active version of ARF6, ARF6Q67L, reversed the ability of centaurin alpha(1) to inhibit beta(2)-adrenoceptor internalization. Finally, expression of centaurin alpha(1) also inhibited the agonist-induced internalization of beta(2)-adrenoceptor endogenously expressed in HEK 293 cells, whereas the R49C and DM mutant versions of centaurin alpha(1) had no effect. Together, these data indicate that by acting as an ARF6 GAP, centaurin alpha(1) is able to switch off ARF6 and so inhibit its ability to mediate beta(2)-adrenoceptor internalization. Thus, ARF6 GAPs, such as centaurin alpha(1), are likely to play a crucial role in GPCR trafficking by modulating the activity of ARF6.
authors.affiliation: Department of Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
authors.forename: Joanna
authors.initials: J
authors.lastname: Lawrence
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Mundell
authors.forename: Hongruo
authors.initials: H
authors.lastname: Yun
authors.forename: Eamonn
authors.initials: E
authors.lastname: Kelly
authors.forename: Kanamarlapudi
authors.initials: K
authors.lastname: Venkateswarlu
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors* / Q000502:physiology*; D048868:Adaptor Proteins, Signal Transducing; D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*
chemical_list: C100956:ADAP1 protein, human; D000089982:ADP-Ribosylation Factor 6; D048868:Adaptor Proteins, Signal Transducing; D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D009419:Nerve Tissue Proteins; D018343:Receptors, Adrenergic, beta-2; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human