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Centaurin-₁, an ADP-Ribosylation Factor 6 GTPase Activating Protein, Inhibits ₂-Adrenoceptor Internalization

Department of Pharmacology, University of Bristol, School of Medical Sciences, University Walk, Bristol, United Kingdom

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 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 isoproterenol-induced 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.

Address correspondence to: Kanamarlapudi Venkateswarlu, Department of Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, United Kingdom. E-mail: k.venkateswarlu{at}bristol.ac.uk

This article has been cited by other articles:

				K. Venkateswarlu, K. G. Brandom, and H. Yun PI-3-kinase-dependent membrane recruitment of centaurin-{alpha}2 is essential for its effect on ARF6-mediated actin cytoskeleton reorganisation J. Cell Sci., March 1, 2007; 120(5): 792 - 801. [Abstract] [Full Text] [PDF]