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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

Received January 21, 2005; accepted March 18, 2005

	Abstract

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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.

The ADP-ribosylation factor (ARF) family of small GTPases has been implicated in the regulation of vesicle trafficking (Moss and Vaughan, 1998). These proteins regulate trafficking by shuttling between an active GTP-bound form and an inactive GDP-bound form. Of the six mammalian ARF isoforms (ARFs 1–6), ARF6 is of particular interest with regard to GPCR trafficking because it is localized to both plasma membrane and endosomes. Although it was thought that ARF6 existed at the plasma membrane in a GTP-bound form, and in endosomes in the GDP-bound form, recent findings indicate that GTP/GDP exchange for ARF6 occurs at the plasma membrane (Macia et al., 2004). ARF6 regulates cargo transport between plasma membrane and endosomes and is also implicated in cortical actin rearrangement at the plasma membrane, which is important for such processes as internalization, chemotaxis, and focal adhesion (Donaldson, 2003). Other proteins in turn regulate the activity of ARFs. ARF guanine nucleotide exchange factors (GEFs), such as the ARF6 GEF cytohesin 2 [also known as ARF nucleotide-binding site opener (ARNO)], activate ARFs by promoting the replacement of GDP by GTP on the ARF. On the other hand, ARF GTPase-activating proteins (GAPs), such as the ARF6 GAP GRK interacting protein 1 (GIT1), enhance the intrinsic GTPase activity of ARF6, resulting in the hydrolysis of ARF6-bound GTP to GDP (Vitale et al., 2000; Hawadle et al., 2002). Another ARF6 GAP that has recently been identified is centaurin ₁ (Hammonds-Odie et al., 1996; Venkateswarlu and Cullen, 1999). This protein, which is enriched in brain, has an N-terminal zinc finger motif as with other ARF GAPs, which is necessary for ARF GAP activity; centaurin ₁ also contains two PH domains required for the binding of the second-messenger phosphatidylinositol 3,4,5-trisphosphate (PIP₃), which is an important determinant of the recruitment of ARF6 to the plasma membrane (Venkateswarlu et al., 1999; Venkateswarlu and Cullen, 2000). We have shown recently that centaurin ₁ exerts GAP activity against ARF6 in vivo and can functionally switch off ARF6-activated processes, such as EGF-stimulated cortical actin rearrangement in HeLa cells (Venkateswarlu et al., 2004).

Because ARF6 has been implicated in the trafficking of GPCRs (Hunzicker-Dunn et al., 2002), it is thus possible that centaurin ₁ can also modify receptor internalization. Therefore, in the present study, we investigated the effect of centaurin ₁ on the widely studied and well characterized internalization of the ₂-adrenoceptor. We found that centaurin ₁ inhibits the agonist-induced internalization of the ₂-adrenoceptor and that this activity depends upon the ability of centaurin ₁ to act as a functional ARF6 GAP. Thus, the functional manipulation of ARF6 can modify GPCR trafficking in vivo, further supporting the importance of ARF6 as a regulator of receptor trafficking.

	Materials and Methods

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Materials. Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, LipofectAMINE 2000, and penicillin-streptomycin were obtained from Invitrogen (Paisley, Scotland, UK). [³H]CGP 12177 (specific activity, 1110–2220 GBq/mmol) was from PerkinElmer (Beaconsfield, Buckinghamshire, UK). The mouse monoclonal anti-HA antibody was from Cambridge Biosciences (Cambridge, UK), the colorimetric alkaline phosphatase substrate kit was from Bio-Rad (Hemel Hempstead, Hertfordshire, UK), and the enhanced chemiluminescence (ECL) kit, the anti-mouse horseradish peroxidase-conjugated antibody, and [³H]cyclic AMP (specific activity, 925 GBq/mmol) were from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). The anti-mouse IgG alkaline phosphatase conjugate antibody, mouse monoclonal anti-FLAG M2 antibody, and mouse monoclonal anti-FLAG M2-Cy3 antibody, were obtained from Sigma (Poole, Dorset, UK). The QuikChange II site-directed mutagenesis kit was purchased from Stratagene (Amesterdam, The Netherlands).

Plasmids. FLAG-₂-adrenoceptor/pcDNA3, HA-ARF6/pXS, HA-ARF6(T27N)/pXS, HA-ARF6(Q67L)/pXS and HA-ARF1(Q71L)/pXS were described previously (Radhakrishna and Donaldson, 1997). HA-ARF6(T44N)/pXS mutant was generated using the sequence specific mutagenic primers (sense, 5'-GTGACCACCATTCCCAATGTGGGTTTCAACGTG-3'; antisense, 5'-CACGTTGAAACCCACATTGGGAATGGTGGTCAC-3'), with HA-ARF6/pXS as template and with the use of the QuikChange II site-directed mutagenesis kit and by following the manufacturer's instructions. Centaurin ₁ WT, centaurin ₁ R49C and centaurin ₁ DM (double PH mutant; R149C/R273C) were excised from the pEGFP vector (Venkateswarlu et al., 1999, 2004) by EcoRI-SalI digestion and subcloned into the EcoRI-XhoI sites of pCMV3-Myc.

Cell Culture and Transient Transfection. HEK 293 cells were maintained in culture using DMEM supplemented with 10% (v/v) fetal calf serum, penicillin (100 units/ml), streptomycin (0.1 mg/ml), and 2 mM glutamine. Cells were transfected using LipofectAMINE 2000 according to the manufacturers' instructions using a total of 4 µg of plasmid DNA for ELISA and immunofluorescence (60-mm plates) and 6 µg of plasmid DNA for cyclic AMP assays (100-mm plates). After transfection, cells were split onto 24-well plates and used for experimentation 48 h after transfection.

Agonist-Induced Receptor Internalization. For transiently expressed ₂-adrenoceptor, the loss of surface receptor in response to agonist treatment was quantified by ELISA exactly as described previously using mouse monoclonal anti-FLAG M2 antibody and anti-mouse IgG alkaline phosphatase conjugate secondary antibody (Mundell et al., 2001). For endogenous ₂-adrenoceptor, cell surface loss of receptor was assessed using the radioligand [³H]CGP 12177. In brief, cells grown on 60-mm dishes (transfected with constructs 48 h previously) were incubated in the presence or absence of 10 µM isoproterenol for various lengths of time. Cells were then detached and washed three times with ice-cold phosphate-buffered saline (PBS), and the cells were resuspended in ice-cold binding buffer (20 mM HEPES, 1 mM MgCl₂, pH 7.4). Cells were then incubated with 10 nM [³H]CGP 12177 ± 10 µM propranolol (the latter was included to define specific binding, which was 70% of total binding) for 90 min at 4°C. Cells were then collected on filter paper using a Brandel cell harvester, and the filter disks were added to scintillant for scintillation counting.

Immunofluorescence and Confocal Imaging. Transiently transfected HEK 293 cells were seeded onto 0.1 mg/ml poly-L-lysine-coated 13-mm coverslips, using normal growth media. The next day, the medium was removed and cells incubated with monoclonal anti-FLAG M2-Cy3, diluted 1:200 in 1% (w/v) bovine serum albumin/DMEM for 1 h at 4°C. The cells were washed twice in PBS and stimulated with 0.5 µM isoproterenol at 37°C for the required length of time, then cells were fixed using 4% (w/v) paraformaldehyde in PBS for 30 min at room temperature. The cells were washed twice with PBS. The coverslips were mounted onto glass slides using Mowiol/DABCO as described previously (Venkateswarlu, 2003). Immunofluorescence staining of the cells was examined using a Leica TCS-NT confocal microscope (Leica, Wetzlar, Germany).

Gel Electrophoresis and Immunoblotting. Whole-cell lysates were prepared from transiently transfected HEK 293 cells. Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membrane, and immunoblotted using a mouse monoclonal anti-HA antibody as described previously (Venkateswarlu et al., 2004).

Cyclic AMP Assay. Medium was removed, and 0.5 ml of prewarmed fresh medium containing a 250 µM concentration of the phosphodiesterase inhibitor Ro 201724 was added to each well, followed immediately by isoproterenol (0.5 µM) or water (control). This was incubated for 0 to 30 min at 37°C, and the reaction was terminated by the addition of 20 µl of 100% (w/v) ice-cold trichloroacetic acid. The cyclic AMP content of the samples was analyzed with the use of a protein binding assay using [³H]cyclic AMP as described previously (Mundell et al., 1998).

Statistical Analysis. Unless indicated otherwise, data were analyzed by one-way ANOVA with Bonferroni post test. Differences were taken as statistically significant when p < 0.05.

	Results

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Agonist-Induced Internalization of Transiently Expressed ₂-Adrenoceptor Is Dependent on Functional ARF6. In initial experiments, both the concentration and time dependence of agonist-induced ₂-adrenoceptor internalization in HEK 293 cells transiently transfected with receptor was assessed by ELISA. When added to the cells for 30 min, the ₂-adrenoceptor agonist isoproterenol induced receptor internalization with an EC₅₀ of 0.3 µM, with a maximum 35% internalization being observed at 1 to 10 µM isoproterenol. Addition of 0.5 µM isoproterenol to the cells led to time-dependent internalization of ₂-adrenoceptor (percentage surface receptor loss was 23.5 ± 4.1, 29.3 ± 3.3, and 27.5 ± 2.6 at 15, 30, and 60 min after addition of agonist, respectively; values are means ± S.E.M., n = 3–4 in each case) with maximum ₂-adrenoceptor internalization observed 10 to 20 min after agonist addition. Confocal imaging of cells confirmed the agonist-dependent internalization of ₂-adrenoceptor (see Fig. 1B, left). Unless otherwise indicated, a standard agonist treatment protocol of 0.5 µM isoproterenol for 30 min was used in further experiments. We next sought to determine the effects of ARF6 on agonist-induced ₂-adrenoceptor internalization. Cells were transiently transfected with ₂-adrenoceptor and either pcDNA3 as control, wild-type ARF6 (ARF6 WT), or a constitutively inactive mutant form of ARF6 (ARF6 T27N). Whereas ARF6 WT had no effect on agonist-stimulated internalization, ₂-adrenoceptor internalization was inhibited by ARF6 T27N (Fig. 1A). Confocal imaging of ₂-adrenoceptor localization (Fig. 1B) showed that whereas in control cells or those transfected with ARF6 WT, agonist treatment led to the marked accumulation of receptor in endosome-like structures in the cell cytoplasm, this accumulation was greatly reduced when ARF6 T27N was cotransfected into the cells. Western blotting confirmed that both ARF6 WT and ARF6 T27N are expressed after transient transfection (Fig. 1C). Another constitutively inactive mutant form of ARF6 (ARF6 T44N; Macia et al., 2004) was also able to inhibit ₂-adrenoceptor internalization (internalization after 0.5 µM isoproterenol for 30 min was 47 ± 6% in pcDNA3-transfected cells, and 18 ± 4% in ARF6 T44N-transfected cells, p < 0.05; values are means ± S.E.M., n = 6).

View larger version (24K): [in this window] [in a new window]   Fig. 1. ARF6 is required for the agonist-induced internalization of transiently expressed 2-adrenoceptor. HEK 293 cells were transiently transfected with FLAG-tagged 2-adrenoceptor, as well as either pcDNA3, wild-type ARF6 (ARF6 WT), or a dominant-negative mutant form of ARF6 (ARF6 T27N), and used for internalization assays 48 h later. A, cell surface receptor loss in response to 0.5 µM isoproterenol for 30 min assayed by ELISA. Coexpression of ARF6 T27N significantly reduced agonist-induced internalization of 2-adrenoceptor (*, p < 0.01; values are means ± S.E.M., n = 4–12). B, representative confocal images showing agonist-induced 2-adrenoceptor (2-AR) redistribution in the presence of pcDNA3 (left), ARF6 WT (middle), or ARF6 T27N (right) after addition of 0.5 µM isoproterenol to cells for 30 min. Receptor immunofluorescence was undertaken with a monoclonal anti-FLAG M2-Cy3 antibody as described under Materials and Methods. C, Western blot with anti-HA antibody to illustrate the expression of the ARF6 constructs in HEK 293 cells after transient expression.

Agonist-Induced Internalization of Transiently Expressed ₂-Adrenoceptor Is Inhibited by Centaurin _1.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Centaurin 1 inhibits the agonist-induced internalization of transiently expressed 2-adrenoceptor. HEK 293 cells were transiently transfected with FLAG-tagged 2-adrenoceptor and Myc-tagged wild-type centaurin 1 (centaurin 1 WT), or in some cases with centaurin 1 R49C or centaurin 1 DM, and used for internalization assays 48 h later. A, time course of effect of centaurin 1 on 2 -adrenoceptor internalization. Cell surface receptor was assayed by ELISA after stimulation with 0.5 µM isoproterenol for the time points indicated. The internalization of 2-adrenoceptor was markedly reduced in the presence of centaurin 1 (p < 0.0001 by 2-way ANOVA; values are means ± S.E.M., n = 3). B, effect of centaurin 1 mutants on the 2-adrenoceptor internalization. Cells transfected with indicated constructs were treated with 0.5 µM isoproterenol for 30 min and cell surface loss of receptor assayed by ELISA. Neither centaurin 1 R49C nor centaurin 1 DM had any effect on agonist-induced 2-adrenoceptor internalization. On the other hand, centaurin 1 WT inhibited cell surface loss (*, p < 0.05; values are means ± S.E.M., n = 5–10). C, representative confocal images showing agonist-induced 2-adrenoceptor (2-AR) redistribution the presence of in pcDNA3, centaurin 1 WT, centaurin 1 R49C, or centaurin 1 DM after addition of 0.5 µM isoproterenol to cells for 30 min. Receptor immunofluorescence was undertaken with a monoclonal anti-FLAG M2-Cy3 antibody as described under Materials and Methods. Note the lack of agonist-induced 2-adrenoceptor internalization in centaurin 1 WT-transfected cells.

View larger version (40K): [in this window] [in a new window]   Fig. 3. The inhibition of 2-adrenoceptor internalization by centaurin 1 is reversed by a constitutively active form of ARF6. HEK 293 cells were transiently transfected with FLAG-tagged 2-adrenoceptor and either pcDNA3 or wild-type centaurin 1 (centaurin 1 WT), and either constitutively active ARF6 (ARF6 Q67L) or constitutively active ARF1 (ARF1 Q71L) and used for internalization assays 48 h later. A, cell surface receptor loss in response to 0.5 µM isoproterenol for 30 min assayed by ELISA. Coexpression of ARF6 Q67L but not ARF1 Q71L reversed the centaurin 1-mediated inhibition of 2 -adrenoceptor internalization (internalization in the presence of centaurin 1 WT or centaurin 1 WT plus ARF1Q71L was significantly less than control (*, p < 0.005; values are means ± S.E.M., n = 5–10). B, representative confocal images showing agonist-induced 2-adrenoceptor (2-AR) redistribution in the presence of centaurin 1 WT, and either ARF6 Q67L or ARF1 Q71L. For stimulated cells, 0.5 µM isoproterenol was added to the cells for 30 min. Receptor immunofluorescence was undertaken with a monoclonal anti-FLAG M2-Cy3 antibody as described under Materials and Methods. After agonist stimulation, 2-adrenoceptor undergoes redistribution into intracellular compartments when expressed with centaurin 1 and ARF6 Q67L but largely remains at the cell membrane when expressed with ARF1 Q71L.

Centaurin ₁ Increases ₂-Adrenoceptor Mediated Cyclic AMP Production in HEK 293 Cells. We also assessed the ability of centaurin ₁ to modulate the functional coupling of ₂-adrenoceptor (Fig. 4). In cells transiently expressing ₂-adrenoceptor, basal cyclic AMP accumulation was not affected by expression of centaurin ₁ (basal values were 0.72 ± 0.05, 0.65 ± 0.06, 0.71 ± 0.07, and 0.69 ± 0.06 pmol of cyclic AMP/well for pcDNA3, centaurin ₁ WT, centaurin ₁ R49C, and centaurin ₁ DM transfected cells, respectively; values are means ± S.E.M., n = 4). In the absence of centaurin ₁, isoproterenol induced a time-dependent increase in cyclic AMP accumulation, which leveled off after 5 to 10 min (Fig. 4A). When centaurin ₁ was coexpressed, agonist-stimulated cyclic AMP accumulation was greater than in pcDNA3-transfected controls. The effect of centaurin ₁ was dependent upon the functional activity of the protein, because expression of neither the R49C nor the DM form of centaurin ₁ was able to enhance isoproterenol-stimulated cyclic AMP accumulation (Fig. 4B).

View larger version (16K): [in this window] [in a new window]   Fig. 4. Centaurin 1 enhances isoproterenol-stimulated cyclic AMP accumulation in cells transiently expressing 2-adrenoceptor. HEK 293 cells were transiently transfected with FLAG-tagged 2-adrenoceptor and either pcDNA3, centaurin 1 WT, centaurin 1 R49C, or centaurin 1 DM and used for cyclic AMP assays 48 h later. A, time course of 0.5 µM isoproterenol-stimulated cyclic AMP (cAMP) accumulation in cells transfected with either pcDNA3 (control) or wild-type centaurin 1 (centaurin 1 WT). Cyclic AMP accumulation was greater in the presence of centaurin 1 WT (p < 0.001, 2-way ANOVA; values are means ± S.E.M., n = 3–6). B, the mutant forms of centaurin 1, centaurin 1 R49C and centaurin 1 DM, have no effect on isoproterenol-stimulated cyclic AMP (cAMP) accumulation. Agonist-induced cyclic AMP accumulation in the presence of centaurin 1 WT was greater than control (*, p < 0.05; values are means ± S.E.M., n = 4).

Centaurin ₁ Inhibits the Agonist-Induced Internalization of Endogenous ₂-Adrenoceptor in HEK 293 Cells.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Centaurin 1 inhibits the agonist-induced internalization of endogenous 2-adrenoceptor. HEK 293 cells were transiently transfected with Myc-tagged wild type centaurin 1 (centaurin 1 WT), or in some cases with centaurin 1 R49C or centaurin 1 DM, and used for internalization assays 48 h later. A, time course of effect of centaurin 1 endogenous on 2-adrenoceptor internalization. Cell surface receptor was assayed using [3H]CGP 12177 binding to intact cells in suspension, after stimulation with 0.5 µM isoproterenol for the time points indicated. The internalization of endogenous 2-adrenoceptor was markedly reduced in the presence of centaurin 1 (p < 0.0001 by 2-way ANOVA; values are means ± S.E.M., n = 3). B, effect of centaurin 1 mutants on endogenous 2-adrenoceptor (2-AR) internalization. Cells transfected with the indicated constructs were treated with 0.5 µM isoproterenol for 30 min and cell surface loss of receptor assayed with [3H]CGP 12177. Neither centaurin 1 R49C nor centaurin 1 DM had any effect on agonist-induced internalization of endogenous 2-adrenoceptor, whereas centaurin 1 WT inhibited cell surface loss (*, p < 0.05; values are means ± S.E.M., n = 3). C, centaurin 1 enhances cyclic AMP accumulation caused by endogenous 2-adrenoceptor activation in HEK 293 cells. Shown is the time course of 0.5 µM isoproterenol-stimulated cyclic AMP (cAMP) accumulation in cells transfected with either pcDNA3 (control) or wild-type centaurin 1 (centaurin 1 WT). Cyclic AMP accumulation was greater in the presence of centaurin 1 WT (p < 0.001, 2-way ANOVA; values are means ± S.E.M., n = 3).

	Discussion

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Using constitutively inactive mutant forms of ARF6, including the ARF6 T27N mutant used in the current study, an important role for ARF6 in the agonist-induced internalization of the ₂-adrenoceptor has previously been demonstrated (Claing et al., 2001). Furthermore, these authors showed that the ARF6 GEF, ARNO, was able to enhance agonist-stimulated ₂-adrenoceptor internalization. In the present study, we also found that inhibition of endogenous ARF6 activity with the GTP binding-defective ARF6 T27N mutant could significantly inhibit agonist-induced internalization of ₂-adrenoceptor. A recent study has questioned the effectiveness of ARF6 T27N as a dominant-negative mutant of ARF6 (Macia et al., 2004), suggesting that whereas ARF6 T27N could act as a dominant-negative mutant, its effectiveness was compromised by a tendency for the mutant protein to denature in vivo. Therefore, we found that the GTP binding defective mutant designed by these authors (ARF6 T44N) is a very effective inhibitor of ₂-adrenoceptor internalization. Together, our findings are in agreement with those of Claing et al. (2001) and Macia et al. (2004) and are further supported by a very recent study showing that the reduction of cellular ARF6 by small interfering RNA markedly reduces the internalization of transiently expressed ₂-adrenoceptor (Houndolo et al., 2005).

We found that expression of centaurin ₁ in HEK 293 cells strongly inhibited the agonist-induced internalization of ₂-adrenoceptor, whether endogenously expressed or transiently introduced into the cells. To determine whether this was caused by the ARF6 GAP activity of centaurin ₁, we employed two mutant forms of the protein. Centaurin ₁ R49C is mutated in the zinc finger domain, which is critical for the ARF6 GAP activity. Upon EGF stimulation of HeLa cells, this mutant translocates to the plasma membrane in a manner indistinguishable from that of centaurin ₁ WT, but it is unable to inhibit the ability of ARF6 to mediate the rearrangement of cortical actin in the cells. On the other hand, centaurin ₁ DM has the two PH domains mutated (R149C/R273C; Venkateswarlu et al., 1999), which are necessary for the PIP₃-mediated translocation of centaurin ₁ to the membrane in cells stimulated by PIP₃-generating agonists such as EGF. Although this mutant is unable to translocate, it is still effective as an ARF6 GAP if it is constitutively associated with the plasma membrane by the addition of a CAAX motif to the protein (Venkateswarlu et al., 2004). Like the R49C mutant, the DM mutant is unable to inhibit the EGF-stimulated rearrangement of cortical actin in cells. In the present experiments and in contrast to centaurin ₁ WT, neither the centaurin ₁ R49C nor the centaurin ₁ DM mutant was able to inhibit agonist-induced internalization of ₂-adrenoceptor. This provides very strong evidence that the ARF6 GAP activity and correct localization are essential for the ability of centaurin ₁ to regulate ARF6 and hence GPCR trafficking.

We have previously shown that the constitutively active form of ARF6, ARF6 Q67L (Venkateswarlu et al., 2004), is able to reverse the functional effects of centaurin ₁ by acting as a dominant-negative mutant for centaurin ₁. Thus, ARF6 Q67L prevented the inhibition of cortical actin formation by centaurin ₁ in EGF-stimulated cells. In the present experiments, we also found that ARF6 Q67L could fully reverse the effects of centaurin ₁ on ₂-adrenoceptor internalization. It is noteworthy that this effect was highly selective because a constitutively active form of ARF1, ARF1 Q71L, was unable to reverse the effects of centaurin ₁, just as this constitutively active mutant was unable to reverse the effects of centaurin ₁ on cortical actin rearrangement (Venkateswarlu et al., 2004). On its own, ARF6 Q67L moderately inhibited ₂-adrenoceptor internalization, which might be unexpected for a constitutively active ARF6 mutant. However, this effect on ₂-adrenoceptor internalization is the same as that reported previously for ARF6 Q67L (Claing et al., 2001) and may reflect the necessity of constant ARF6 GTP/GDP cycling for normal function.

Finally, we assessed the effects of centaurin ₁ on ₂-adrenoceptor signaling. Expression of centaurin ₁ enhanced the agonist-stimulated accumulation of cyclic AMP, an effect that would be consistent with the ability of centaurin ₁ to inhibit ₂-adrenoceptor internalization and thus prolong signaling. It is noteworthy that neither of the centaurin ₁ mutants was able to increase ₂-adrenoceptor-stimulated cyclic AMP accumulation, indicating that the effect of centaurin ₁ on ₂-adrenoceptor signaling is related to the ARF6 GAP activity of the protein. It is interesting that another ARF6 GAP, GIT1, a GRK2-interacting protein, also inhibits the agonist-induced internalization of ₂-adrenoceptor (Premont et al., 1998; Claing et al., 2001) as well as some other GPCRs (Claing et al., 2000). However, unlike centaurin ₁ in the present study, GIT1 has been reported to inhibit ₂-adrenoceptor-stimulated cyclic AMP accumulation. The reason for this difference is not clear at present, but it should be noted that GIT1 does interact with other cellular proteins (such as GRK2) that potentially could modulate ₂-adrenoceptor-stimulated cyclic AMP accumulation apart from ARF6 GAP activity.

In summary, the present results suggest an important role for centaurin ₁ as a regulator of ARF6 in the trafficking of endogenous GPCRs. In the future, it will be crucial to assess the role of endogenous centaurin ₁ in the regulation of ARF6 and GPCR trafficking using a small interfering RNA or related approach. It will also be important to determine exactly where in the internalization pathway ARF6 exerts its regulatory activity, although our results and others suggest that ARF6 is important in the very early steps of GPCR internalization.

	Footnotes

 
This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) and Medical Research Council. K.V. was the recipient of a BBSRC David Phillips Research Fellowship, and S.J.M. holds a British Heart Foundation Fellowship.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.011338.

ABBREVIATIONS: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; ARF, ADP-ribosylation factor; GEF, guanine nucleotide exchange factor; ARNO, ARF nucleotide-binding site opener; GAP, GTPase-activating protein; GIT1, ARF6-GAP-GRK interacting protein 1; PIP₃, phosphatidylinositol 3,4,5-trisphosphate; EGF, epidermal growth factor; DMEM, Dulbecco's modified Eagle's medium; [³H]CGP 12177, (–)-4(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one [5,7-³H]; HA, hemagglutinin; PH, pleckstrin homology; DM, double PH mutant; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; Ro 201724, 4-(3-butoxy-4-methoxybenzyl) imidazolidin-2-one; WT, wild-type; ₂-AR, ₂-adrenoceptor.

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

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