Centaurin-α₁, an ADP-Ribosylation Factor 6 GTPase Activating Protein, Inhibits β₂-Adrenoceptor Internalization

Abstract

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.

ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling

“…Immunofluorescence-Intracellular distribution of cell surface HLHCGR was assessed by immunofluorescence as described (24). Briefly, cells serum starved and treated with inhibitors as mentioned above were incubated with an antiMyc mouse monoclonal antibody for 1 h at 4°C, stimulated with HCG 10 IU/ml in presence or absence of the inhibitors at 37°C for 30 min.…”

“…Receptor Internalization Assay-Internalization of HLHCGR was assessed by ELISA as previously described (24). Briefly, HEK293 cells transiently transfected with Myc-tagged HLH-CGR were serum starved for 2 h and then incubated without or with HCG 10 IU/ml for 30 min at 37°C.…”

“…Where indicated, cells were incubated with the inhibitors for indicated time at 37°C prior to stimulation with HCG. cAMP levels were then determined as previously described (24).…”

“…NM23-H1 H118C was generated by using QuickChange site-directed mutagenesis kit (Stratagene). GST-GGA3 protein binding domain (PBD) and GFP-cytohesin 2 plasmids described previously (23)(24)(25). EPS15 (EGFR pathway substrate clone 15) dominant negative (DN) mutant excised from pEGFP plasmid by EcoRI digestion and subcloned into the EcoRI site of pCMV-FLAG vector (26).…”

Luteinizing hormone/chorionic gonadotrophin receptor overexpressed in granulosa cells from polycystic ovary syndrome ovaries is functionally active

“…The reaction was terminated by adding 4% (w/v) ice-cold trichloroacetic acid and the cAMP content of the samples was analysed by a cAMP binding protein assay as described previously (Lawrence et al, 2005).…”