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Vol. 19, Issue 4, 1304-1316, April 2008
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*IFOM, the FIRC Institute of Molecular Oncology Foundation, 20139 Milan, Italy; European Institute of Oncology, 20141 Milan, Italy; Department of Oncological Sciences, University of Turin, Institute for Cancer Research and Treatment, 10060 Candiolo, Torino, Italy; ||Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), Saitama 351-0198, Japan; ¶Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan; and #University of Milan, 20122 Milan, Italy
Submitted June 21, 2007;
Revised January 2, 2008;
Accepted January 9, 2008
Monitoring Editor: Sandra Schmid
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; McLysaght et al., 2002; Brosius, 2003). About 5% of the human genome is composed of duplicated segments that emerged during the past 35 million years of primate evolution, resulting in the generation of novel protein functions (Courseaux and Nahon, 2001; Taylor and Raes, 2004).
One of the cellular processes, whose regulation has became more and more complex and tightly regulated during evolution is endocytosis. Endocytosis serves to maintain cellular and organismal homeostasis by mediating the uptake of fluids, solutes, and signaling molecules and their receptors. Multiple mechanisms of endocytosis operate within a single cell (Conner and Schmid, 2003). Among them, two major categories are phagocytosis and pinocytosis, which mediate the uptake of particles or of fluid, respectively (Conner and Schmid, 2003).
Pinocytosis encompasses a variety of different membrane-entry routes and is invariably characterized by the relatively small size (50–150 nm) of the internalized particles. Large volumes of fluid are, instead, engulfed by extension, folding, and closure of plasma membranes (PM) through a process termed macropinocytosis (Swanson and Watts, 1995). Macropinocytosis displays many similarities to phagocytosis; the biochemical and cellular components of these endocytic mechanisms have been best characterized in hematopoietic cells, in processes such as phagocytosis by macrophages in response to Fc receptor stimulation, or macropinocytosis of the antigen-presenting dendritic cells (DCs; Swanson and Watts, 1995; Aderem and Underhill, 1999). In all these cases the dynamic assembly of actin filaments generates forces, which drive internalization (Cardelli, 2001; Amyere et al., 2002). In nonhematopoietic cells, the exact functional role, molecular components, and regulatory signaling pathways of macropinocytosis are only now beginning to be defined.
A robust body of evidence has established that dynamic actin structures, defined as dorsal or circular ruffles, are required for macropinocytosis (Buccione et al., 2004 and reference within). Consistent with this finding, is the fact that the de novo Arp2/3-dependent actin filament elongation, mediated by the WAVE family of proteins, is essential for the formation of dorsal membrane extensions needed to engulf fluids (Suetsugu et al., 2003; Innocenti et al., 2005). This pathway is under the control of the RhoGTPase, RAC, which mediates actin remodeling at the plasma membrane (PM) by controlling WASP family VE rprolin-homologous proteins (WAVEs) (Stradal et al., 2004). However, the sole activation of RAC is apparently insufficient to induce circular ruffles, underscoring the need for additional and coordinated signaling cascades (Lanzetti et al., 2004). In line with this, dynamin, a large GTPase best characterized for its ability to mediate vesicle scission during clathrin-dependent endocytosis, has been implicated in macropinocytosis through its binding to the actin regulatory protein, cortactin (McNiven et al., 2000). Finally, the concomitant occurrence of signals emanating from RAC, RAS-PI3K, and RAB5 was shown to be necessary for platelet-derived growth factor–mediated circular ruffles and macropinocytosis, providing evidence of cross-talk between signaling cascades controlled by different small GTPases (Lanzetti et al., 2004).
This concept seems to apply to other GTPases that govern actin membrane protrusions and trafficking at the PM, such as RAB34 (Sun et al., 2003) and, in particular, ARF6 (Donaldson, 2002). ARF6 is localized at the PM, where it participates with the regulation of membrane trafficking and actin remodeling (Schafer et al., 2000; Donaldson, 2002; D'Souza-Schorey and Chavrier, 2006). Notably, ARF6-containing, specialized endosomes have been identified (Donaldson, 2002), and, in some cases, these vesicles can fuse with RAB5-associated endosomes (Naslavsky et al., 2003), supporting the idea of cross-talk between different entry routes and various GTPases controlling the fate of internalized proteins. In this scenario, regulators of the biochemical activity and effectors of GTPases, particularly of RAB5 and ARF6, are likely to play a critical role. Among these regulators are the ARF effectors of the GGA family (Bonifacino, 2004), and the vast and poorly characterized family of TBC (Tre-2, Bub2, Cdc16) domain–containing proteins (Bernards, 2003).
GGAs are ubiquitously expressed adaptor proteins, involved in ARF-dependent sorting of cargos, such as the mannose-6-phosphate receptor, between the trans-Golgi network (TGN) and endosomes (Bonifacino, 2004). More recently GGA3, one of the family members, has also been implicated in plasma membrane receptor trafficking and down-regulation (Bonifacino, 2004; Puertollano and Bonifacino, 2004).
TBC domains are found in more than 80 mammalian proteins. In some cases, TBC-containing proteins act as GAPs (GTPase-activating proteins) for RABs, with the TBC domain encoding the catalytic activity (Cuif et al., 1999; Lanzetti et al., 2000; Haas et al., 2005; Miinea et al., 2005; Zhang et al., 2005). It is not clear, however, whether all TBC-containing proteins display similar activity. Within the superfamily of human TBC-containing proteins, three distinct proteins belong to a distinct subfamily: RNTRE (USP6NL; Matoskova et al., 1996), USP6 (also known as Tre17 or Tre2; Richardson and Zon, 1995), and TBC1D3. RNTRE regulates clathrin-dependent endocytosis and macropinocytosis by acting as a GAP on RAB5 (Lanzetti et al., 2000, 2004), and possibly on other RABs (Haas et al., 2005). Conversely, USP6, whose TBC domain is apparently devoid of GAP activity, is involved in the control of plasma membrane–endosomal trafficking in an ARF6-dependent manner (Martinu et al., 2004). Little is known, instead, about TBC1D3. Because the TBC1D3 gene originated during primate speciation (Paulding et al., 2003), we reasoned that studies of its function might provide a unique opportunity to understand how the machinery of membrane traffic and endocytosis contributes to evolution. Thus, the present studies were undertaken to understand the biological function of TBC1D3.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), HA-ARF6-T27N, HA-ARF6-Q67L and GFP-PLC
(pleckstrin homology [PH] domain) domain were from Dr. P. Chavrier. HA-RNTRE and glutathione S-transferase (GST)-RNTRE TBC domains used in this article were previously generated and described (Lanzetti et al., 2000). Details of the engineering strategies and sequences of the oligonucleotides used are available upon request. All fragments obtained by PCR were sequence verified.
Tissue Culture
HeLa cells were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine calf serum (FCS), 100 µg/ml streptomycin, 100 µg/ml penicillin, and 2 mM glutamine. Phoenix cells were grown in DMEM supplemented with 10% bovine North American serum, 100 µg/ml streptomycin, 100 µg/ml penicillin, and 2 mM glutamine. COS-7 cells were grown in DMEM supplemented with 10% bovine South American serum, 100 µg/ml streptomycin, 100 µg/ml penicillin, and 2 mM glutamine. Transfections were performed using either calcium phosphate, FUGENE (Invitrogen), or LipofectAMINE (Invitrogen) reagents, according to manufacturer's instructions. HeLa cells were transfected, for biochemical studies, using the LipofectAMINE reagent, and for immunofluorescence, using FUGENE 6. Phoenix cells were transfected using the calcium phosphate procedure. For actin analysis HeLa cells were seed on gelatin-coated coverslips and after 24 h, cells were processed for immunofluorescence (IF) as described below.
Pulldown and Coimmunoprecipitation Experiments
HeLa cells were lysed in a buffer containing 1% Triton X-100 (Pierce, Rockford, IL), 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, and 5 mM EGTA, adding the Calbiochem protease inhibitor cocktail (ID-539134; La Jolla, CA) and phosphatase inhibitors (10 mM sodium orthovanadate and 20 mM sodium pyrophosphate). Bovine serum albumin was added to the lysates at 0.5% final concentration, and lysates were left rocking on ice for 15 min. For pulldown experiments, 40 µg of GST-fusion proteins attached to the glutathione-Sepharose beads were incubated in the presence of 1 mg of total cell lysate for 1 h at 4°C rocking. After three washes in lysis buffer, bound proteins were resolved by SDS-PAGE and visualized with the appropriate antibodies. For coimmunoprecipitation studies, cells were lysed and cell lysates were used immediately, without freeze/thawing. Immunoprecipitations and coimmunoprecipitation experiments were performed for 1 h in the presence of the appropriate antibody, and immune complexes were recovered by adsorption to protein G-Sepharose beads (Zymed, South San Francisco, CA), and samples were subjected to one additional round of immunopurification. After three washes in lysis buffer, bound proteins were resolved by SDS-PAGE and visualized with the appropriate antibodies. [35S]methionine-labeled protein of full-length and deletion mutants of TBC1D3, used in the experiment in Figure 7C, was synthesized by in vitro transcription–translation using a commercial kit (Promega, Madison, WI).
Antibodies
Rabbit polyclonal antibodies against human TBC1D3 were raised using as immunogen, the peptide 501–549 of human TBC1D3 protein, produced as a GST fusion protein by the IFOM-IEO-Campus Antibody Facility in collaboration with Eurogentec (Serain, Belgium). Before immunization, purified proteins were eluted from GSH beads (glutathione-Sepharose 4B purchased from Amersham Pharmacia), subjected to overnight dialysis in PBS buffer, and kept at –80°C. Rabbit polyclonal antibodies anti-HA tag (HA.11) and mouse monoclonal antibodies anti-c-MYC (9E10) were from Eurogentec. Mouse monoclonal anti-HA tag antibodies (16B12) were from BabCO (Richmond, CA). Mouse monoclonal anti-FLAG M2 were from Sigma (St. Louis, MO). Anti-ARF6 (3A-1) and anti-RAB5A (S-19) rabbit polyclonal antibodies and mouse monoclonal antibodies anti-GFP (B-2) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Immunofluorescence Staining
Cells grown on coverslips were fixed freshly prepared 4% paraformaldehyde for 10 min at room temperature or 30 min on ice and permeabilized with Ca2/Mg2-free phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 1% bovine serum albumin for 5 min at room temperature. Coverslips were then incubated for 1 h with the appropriate primary antibody, washed extensively, and then incubated with the secondary antibodies for 30 min. Coverslips were mounted in Fluoromount (Fisher Scientific, Pittsburg, PA) containing 1 mg/ml para-phenylenediamine. F-actin was detected by staining with rhodamine-conjugated phalloidin (Sigma) at a concentration of 6.7 U/ml. Indirect IF was analyzed under an AX-70 Provis (Olympus, Melville, NY) fluorescence microscope equipped with a b/w cooled CCD camera (c5985; Hamamatsu, Bridgewater, NJ), or with a Leica TCS SP2 AOBS confocal microscope (Deerfield, IL) equipped with 405-, 488-, 543-, and 633-nm laser lines.
GAP Assay
GAP assays using GST-TBC1D3 or GST-RNTRE TBC domains or immunoprecipitated TBC1D3 or RNTRE proteins were performed using 0.2 µM substrate [
-32P]GTP-loaded Rab proteins and catalytic concentrations of GST-TBC1D3 (60 nM) and GST-RNTRE TBC domain (40 nM) or the immunoprecipitated full-length proteins as previously described (Lanzetti et al., 2000).
Dextran Internalization Assay
Cells were plated on glass coverslips, without gelatin coating, 1 d after cells were transfected with different constructs. Twenty-four hours after the transfection, cells were serum-starved for 16 h and then incubated with 1 mg/ml rhodamine-conjugated dextran, 70,000 MW (Molecular Probes, Eugene, OR; D-1818) for 30 min at 37°C, washed four times with ice-cold PBS, fixed in 4% paraformaldehyde (in PBS) for 10 min, washed four times with PBS, and processed for confocal analysis, as described above. Each assay was done in triplicate and at least 100 cells were counted in each experiment.
Dextran Internalization Assay for Fluorescence-activated Cell Sorting Analysis
HeLa cells were plated in six-well plates, without gelatin coating. For data presented in Figure 3 cells were transfected twice with siRNA TBC1D3-specific or scrambled control oligos. Twenty-four hours after the second transfection, cells were serum-starved for 16 h and then incubated with 1 mg/ml fluorescein-conjugated dextran, 70,000 MW (Molecular Probes; D1822) for 30 min at 37°C; at the same time cells were stimulated with different doses of epidermal growth factor (EGF; 1, 10, and 30 ng/ml). Cells were then harvested, washed four times with ice-cold PBS, and fixed in 1% formaldehyde for 15 min on ice. After two final washes, cells were analyzed on a FACSCan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using Cell Quest software. For data presented in Figure 8 cells were transfected twice with small interfering RNA (siRNA) GGA3-specific or scrambled control oligos, and 24 h after the second transfection, cells were transfected with FLAG-TBC1D3. Twenty-four hours after transfection cells were serum-starved for 16 h and then incubated with 1 mg/ml fluorescein-conjugated dextran, 70,000 MW (Molecular Probes; D1822) for 30 min at 37°C and processed for FACSCan analysis as described above.
Gene Silencing
TBC1D3 and GGA3 gene silencing in HeLa cells was obtained by transfecting siRNA oligos for TBC1D3 or GGA3 (Dharmacon, Boulder, CO) specific target sequence. HeLa cells were transfected according to suppliers' instructions, in six-well plates, with oligofectamine (Invitrogen). HeLa cells were subjected to double transfection with 200 nM siRNA oligos, and silencing was verified at various time points by immunoblot analysis with the specific antibodies against TBC1D3 or GGA3. As control, scrambled oligos were used. Each experiment was performed using two independent siRNA oligos. Oligonucleotide sequences are available upon request.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), is due to a duplication of the RNTRE locus. Accordingly, all TBCID3-coding exons are identical in length to those of RNTRE, with the exception of the last one, which also contains the 3' untranslated regions (UTRs; Figure 1B and Figure S1 in Supplementary Material). Gene structure conservation is a common feature among ortholog and paralog genes, and it has been proposed to represent a record of key events in evolution, even when protein sequence similarity is low (Batzoglou et al., 2000; Betts et al., 2001; Koonin, 2005). This applies to TBC1D3, which despite having originated only after the initial primate radiation (
60 million years ago; Paulding et al., 2003), accumulated, in a remarkably short period of time, a considerable number of mutations, accounting for the relatively low degree of similarity between the TBC1D3 and RNTRE gene products (Figure S2 in Supplementary Material). This also suggests that TBC1D3 has been subject to positive evolutionary pressure, likely resulting in the acquisition of novel functions with respect to RNTRE.
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99% of identity over >10 kb) contain ORFs potentially coding for full-length proteins (data not shown) and display a complex pattern of expression in several tissues (Hodzic et al., 2006). In addition specific patterns of expression of these paralogs were observed in different normal human tissues and altered in several prostate tumors in comparison to healthy prostate tissue (Hodzic et al., 2006). Conversely, the remaining two paralogs display in-frame stop codons and frameshifts caused by insertion/deletion of single nucleotides in the genomic sequence. In-depth bioinformatics analysis excluded that the redundancy of the TBC1D3 locus is due to an artifact resulting from a wrong assembly of the human genome (Figure S1 in Supplementary Material).
Duplication of large regions with high identity is not unusual throughout the human genome (Bailey et al., 2001). The duplication of the TBC1D3 locus, however, seems peculiar in that it occurred at least nine times, in regions that are not always contiguous on chromosome 17 and with a degree of sequence conservation that reflects its recent evolutionary origin. Thus, TBC1D3 encode for a TBC-domain–containing protein of particular interest because it is present only in primates and underwent remarkable gene duplication very recently in evolution giving raise to a novel, highly redundant gene that was susceptible to develop novel functions.
The TBC Domain of TBC1D3 Displays No Apparent GAP Activity
TBC1D3 possesses an N-terminal TBC domain, which displays closest similarity to the TBC domains of USP6 and RNTRE (Figure 2A). The TBC domain of the latter protein was shown to be a Rab-GAP, with in vitro specificity for RAB5 (Lanzetti et al., 2000) and RAB30 and RAB41 (Haas et al., 2005). Conversely, no GAP activity could be detected for the TBC domain of USP6 (Bizimungu et al., 2003). Consistent with this, the TBC domain of USP6 lacks conserved residues (Figure 2A) that are essential for catalytic activity (Pan et al., 2006; Bos et al., 2007). The same critical residues are also absent in TBC1D3, suggesting that it might not be a GAP. Despite this, TBC1D3 has been reported to possess a modest, but measurable RAB5-specific GAP activity (Pei et al., 2002). We analyzed the ability of TBC1D3 to stimulate the intrinsic GTPase activity of a collection of 13 different RABs (Figure 2B), using either native, immunoprecipitated, full-length TBC1D3 or its isolated and recombinantly produced TBC domain. In all cases, we could not detect any Rab-GAP activity (Figure 2, B–D). In particular, no activity on RAB5 was detected, at variance with a previous report (Pei et al., 2002). The reason for this discrepancy is not clear. We note, however, that in the mentioned study (Pei et al., 2002), a 10-fold molar excess of TBC1D3 (PRC17 in that study) with respect to RAB5 was used, whereas we used catalytic concentrations of the protein.
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TBC1D3 Induces Dorsal Ruffling and Is Required for Optimal EGF-mediated Macropinocytosis
To gain insight into the biological function of TBC1D3, we initially performed overexpression experiments. Ectopic expression of TBC1D3 led to alterations of the actin cytoskeleton at the dorsal surface of HeLa cells (Figure 3A) and of a variety other cell types (not shown), with the formation of ruffle-like structures where TBC1D3 and F-actin colocalized (Figure 3A, merge). These protrusions are typically associated with the process of macropinocytosis, which can easily be monitored after the incorporation of the classical macropinocytic tracer 70-kDa rhodamine-labeled dextran (Dharmawardhane et al., 2000; Lanzetti et al., 2004). After 30 min of incubation little or no dextran internalization was observed in untransfected, serum-starved control cells. Conversely, about 50% of TBC1D3-overexpressing cells, displayed internalized, dextran-filled vesicles (Figure 3B and Table 1).
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; Bos et al., 2007). In TBC domains, these residues (corresponding to aa 150 and 187 of RNTRE, respectively) are highly conserved and have been shown to be essential for GAP activity (Lanzetti et al., 2000; Pan et al., 2006). In TBC1D3, these residues are not conserved (Figure 2A). However, two arginine residues at positions 147 and 154 might substitute for the "arginine finger" function (Figure 2A), exploiting an alternative mechanism of action that might render the "glutamine finger" dispensable for the catalytic process. Thus, we engineered a TBC1D3 mutant, harboring a double Arg
Ala substitution in these two positions. This mutant was capable of inducing macropinocytosis with an efficiency similar to that of WT TBC1D3 (Figure 3B and Table 1), further suggesting that a putative Rab-GAP function of TBC1D3 is not responsible for this effect.
From the above data, we concluded that overexpression of TBC1D3 is per se sufficient to elicit macropinosome formation in absence of stimulation, indicating that it may be part of a signaling cascade controlling this event. To assess whether TBC1D3 also is also needed for macropinocytosis under physiological conditions, we performed RNA interference of TBC1D3 in HeLa cells (Figure 3C) and measured the extent of macropinocytosis after stimulation with EGF, a known inducer of dextran incorporation (West et al., 1989). These experiments were performed using a FACS (fluorescent-activated cell sorter)-based analysis, to quantify the extent of dextran incorporation. EGF-stimulated, TBC1D3-knockdown HeLa cells showed a significant decrease in dextran internalization, compared with control cells both with respect to the total number of cell exhibiting macropinocytosis (Figure 3C) and to dextran uptake, as assessed by calculating the mean fluorescence intensity, by FACS analysis, of dextran-positive cells (Figure 3D). Notably and in agreement with previous published data (West et al., 1989), the levels of macropinocytosis in unstimulated cells were minimal and unaffected by ablation of TBC1D3. Together, these results suggest that TBC1D3 is physiologically required for optimal macropinocytosis in response to EGF stimulation. The observation that the TBC1D3 gene emerged during primate speciation, whereas macropinocytosis is conserved throughout the mammalian kingdom (Cardelli, 2001), supports the notion that, in higher eukaryotes TBC1D3 may have evolved to provide a novel function of optimization of the macropinocytic process.
TBC1D3 Mediates Macropinocytosis by Acting in a Pathway That Requires RAB5 and ARF6
ARF6 and RAB5 are GTPases known to control macropinocytosis (Roberts et al., 2000; Donaldson and Honda, 2005). The mechanisms underlying their action and their functional links in this process are, however, only partially defined. To identify the signaling pathways in which TBC1D3 might be involved, a molecular genetics approach was utilized, using dominant negative and activated mutants of these GTPases. Both dominant negative ARF6 (ARF6-T27N) and RAB5 (RAB5-S34N) efficiently inhibited TBC1D3-mediated internalization of rhodamine-dextran (Figure 4A and Table 1), indicating that TBC1D3 requires the activity of both ARF6 and RAB5.
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), induced dextran internalization, whereas a dominant negative ARF6, as expected, did not (Figure 4B and Table 1). Similarly, RAB5, but not a dominant negative RAB5, potently induced macropinocytosis (Figure 4B and Table 1). Additionally, RAB5-induced macropinocytosis was unaffected by a dominant negative ARF6, whereas a dominant negative RAB5 inhibited ARF6-mediated macropinocytosis (Figure 4C and Table 1). Thus, ARF6 acts upstream of RAB5 in mediating dextran internalization, at least under the conditions of ectopic expression used here.
On expression of ARF6-Q67L, TBC1D3 was recruited to and accumulated on enlarged vesicles (Figure 4D), which have been previously characterized as ARF6-positive endosomes (Caplan et al., 2002), suggesting that ARF6-dependent relocalization of TBC1D3 might be part of its mechanism of action. To confirm this, we investigated the colocalization of TBC1D3 with another marker of this compartment, phosphatidylinositol 4,5-bisphosphate (PIP2), which can be detected in living cells by the GFP-tagged PH domain of phospholipase C (Katan and Allen, 1999). On expression of ARF6-Q67L, TBC1D3 and GFP-PH-PLC- colocalized in vesicle-like structures (Figure 4E), providing evidence that TBC1D3 not only stimulates actin reorganization and macropinocytosis, but also cotraffics with ARF6 in specialized endosomal vesicles. Notably, ARF6 mediates the internalization of MHCI and of the IL-2 receptor 
-subunit, TAC (Donaldson, 2005). These cargos are subsequently delivered to RAB5-associated endosomes, supporting the existence of a hierarchical link between ARF6 and RAB5 and connecting endosome-dependent internalization to trafficking events (Naslavsky et al., 2003). Thus, collectively, these data support a possible mechanism of signaling whereby TBC1D3 acts in an ARF6-dependent pathway, requiring RAB5 activity, leading to macropinocytosis.
TBC1D3 Interacts and Colocalize with GGA3 at the Plasma Membrane and in ARFQ67L-induced Enlarged Endosomes
One obvious, possible mechanism of action for TBC1D3 would be to induce ARF6 activation, similarly to what has been reported for USP6 (Martinu et al., 2004). Thus, we analyzed ARF6-GTP levels in cells overexpressing TBC1D3, or EFA6 or ARF6-Q67L as controls. We used an assay based on the ability of a -ear–containing fragment of GGA3, an ARF-binding protein, to bind to ARF6 in a GTP-dependent manner (Dell'Angelica et al., 2000; Martinu et al., 2004). No significant activation of ARF6 in TBC1D3-overexpressing cells could be detected, under conditions in which expression of EFA6 induced a significant increase in ARF6-GTP levels (Figure 5A). Surprisingly, however, the GGA3 bait interacted with TBC1D3. The strength of this interaction was not dissimilar to that between the -ear of GGA3 and ARF6-GTP (ARF6-Q67L; Figure 5B), at least under the conditions of our pulldown assay. Furthermore, coimmunoprecipitation experiments indicated that the association between ARF6 and GGA3 and between TBC1D3 and GGA3 also occurs in vivo, supporting the physiological relevance of this interaction (Figure 5, C and D). Next, we tested whether TBC1D3 and ARF6 bind GGA3 on difference surfaces. GGA3, like all other GGAs, binds to ARFs through its GAT domain (Takatsu et al., 2002). Conversely, the association of GGA3 to TBC1D3 is mediated by its VHS domain and is thus independent from the GAT domain (Figure 5E). These results also suggest that the possibility that the GGA3 may simultaneously bind to ARF6 and TBC1D3. Accordingly, ectopically coexpressed ARF6 and TBC1D3 associated with immobilized GST-GGA3 construct encompassing both the VHS and the GAT domain, whereas only TBC1D3, but not ARF6 bound to the immobilized GST-VHS domain (Figure 5E), suggesting that TBC1D3, ARF6-GTP, and GGA3 may form a trimeric complex.
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). On ARF6-Q67L coexpression, a significant fraction (>50%, Figure S3, top panel, in Supplementary Material) of TBC1D3 was detectable in ARF6-Q67L-induced enlarged endosomes (Figure 6B, top panel). More importantly, also GGA3 was recruited to plasma membrane and accumulated and extensively colocalized with ARF6-positive enlarged endosomes (Figure 6B, middle panel, and Figure S3, in Supplementary Material), suggesting that GGA3 can cotraffic with ARF6. We next tested whether GGA3 localization was perturbed by TBC1D3. In cells concomitantly expressing TBC1D3 and GGA3, the latter protein was partially recruited to the PM and to vesicle-like structures at the cell periphery, where, in both cases, colocalized with TBC1D3 (Figure 6B, bottom panel, and Figure S3, in Supplementary Material), thus supporting the notion that the two proteins not only coimmunoprecipitate, but can be found together in defined subcellular compartments. This latter notion was further reinforced after expression of activated ARF6. Under these conditions, GGA3 and TBC1D3 displayed significant and extensive colocalization (more than 80% of total protein colocalized; Figure S3, in Supplementary Material) along the plasma membrane, and at the level of ARF6-induced enlarged endosomes (Figure 6C). In cell expressing GGA3, TBC1D3, and ARF6Q67L or ARF6Q67L and TBC1D3, endosomes appear somewhat larger than in cells expressing only ARF6Q67L, suggesting that TBC1D3 may have an effect on endosome morphology. However, no statistically significant endosome size difference could be revealed when a larger number of TBC1D3/GGA3 and control cells were analyzed. Thus, collectively these findings suggest that the ARF6 induces relocalization of TBC1D3 and GGA3 to cellular compartments, where they are likely to promote and facilitate the macropinocytic process.
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-helices are present, which however are poorly conserved in TBC1D3 and are not part of the core of the domain, as defined by SMART and PFAM.
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). Remarkably, individual removal of either TBC1D3 or GGA3 significantly impaired EFA6-induced dextran uptake (Figure 8, F–I).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several questions remain to be answered. First, where does TBC1D3 act? In the simplest scenario it might act at the PM where it might participate in actin remodeling and in the formation of dorsal ruffles associated with macropinocytosis. The facts that overexpression of TBC1D3 alone can induce both dorsal ruffling and macropinocytosis and that TBC1D3 colocalizes with F-actin in dynamic actin-rich structures at the PM, together with ARF6, support this possibility. In this straightforward setting, ARF6 and RAB5 might be functionally connected to TBC1D3 at the PM, because both proteins have been reported to function in that location in association with dynamic actin events leading to macropinocytosis (Schafer et al., 2000; Lanzetti et al., 2004).
More complex scenarios can, however, be envisioned. Endocytic events seem not only to be a consequence of actin remodeling at the PM, but also to be required for it. For instance, a complex interplay of endocytic routes is necessary to ensure proper localization of RAC and RHO in the formation of membrane protrusions (reviewed in Polo and Di Fiore, 2006). In addition, TBC1D3 might work, at least in part, in a vesicular/endosomal compartment, as witnessed by its localization in ARF6-induced endosomes and by its in vivo interaction with GGA3 in a vesicular compartment. Thus, TBC1D3 might also participate in endocytic/rerouting events required to properly localize the molecular machinery of ruffle formation and macropinocytosis.
A second important question relates to the biochemical function of TBC1D3. Our molecular genetics analysis indicated a hierarchical relationship TBC1D3-ARF6-RAB5. If the pathway were linear, then TBC1D3 might work as an activator of ARF6. However, we failed to detect any increase in ARF6-GTP in TBC1D3-overexpressing cells. Hierarchy does not necessarily mean, however, a linear relationship. This might be especially true in an endocytic/rerouting scenario (see above). In this framework, evidences provided in this work might help shed light on the function of TBC1D3. We demonstrate the existence of a TBC1D3:GGA3 and GGA3:ARF6-GTP complexes in vivo, both at the plasma membrane and ARF6-induced endosomes, and implicated them in the control of macropinocytosis; more importantly both TBC1D3 and GGA3 are required for ARF6-induced macropinocytosis. This body of evidence favors the possibility that TBC1D3 is involved in an effector function of ARF6, in which a putative GGA3:TBC1D3 dimeric effector is recruited to ARF6-GTP through GGA3 and acts downstream through TBC1D3 or through the GGA3:TBC1D3 complex. An alternative possibility is that TBC1D3 stabilizes an ARF6-GTP:GGA3 complex. In this case TBC1D3 might be required for macropinocytosis only in the presence of rate-limiting concentration of components of the complex (e.g., low ARF-GTP): an "optimizing" role is in line with the fact that macropinocytosis also occurs in species in which the TBC1D3 gene is not present.
A final question concerns the molecular links between ARF6 and RAB5, in the novel TBC1D3-controlled pathway described here. A recent set of observations indicated that GGAs can directly associate with the RAB5 activator complex Rabaptin-5:Rabex-5 in the trafficking between the trans-Golgi network and endosomes (reviewed in Kawasaki et al., 2005). Whether this interaction leads to activation of the GEF activity of the Rabaptin-5:Rabex-5 complex or regulates its proper localization or whether TBC1D3 participates to these events remains to be established.
An additional intriguing issue to point out arises from the observation that TBC1D3 is present in multiple copies on chromosome 17, in a region that also contains the CCL3 and CCL4 genes. CCL3 and CCL4 are chemokines, which are the natural ligands of the receptor utilized by the HIV virus to infect T-cells. Importantly, the copy number of these two genes varies in the human population as a consequence of segmental duplication events, correlating with HIV susceptibility (Gonzalez et al., 2005). A similar fluctuation in the population of TBC1D3 copy number may also occur, possibly accounting for variable individual rates of macropinocytosis. Notably, macropinocytosis can be utilized by viruses for infectious entry and/or postentry events (reviewed in Pelkmans, 2005). It will therefore be interesting to investigate whether TBC1D3 copy number impacts on viral infection. In more general terms, genomic regions embedded within segmental duplications are frequently enriched in genes associated with immunity and defense, likely contributing to the enhanced ability of humans to adapt to their environment (Bailey et al., 2002). This might provide one possible explanation for the positive selection and amplification of the TBC1D3 gene, which may have evolved to promote an efficient immune response via enhanced macropinocytosis.
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work. 
Address correspondence to: Stefano Confalonieri (stefano.confalonieri{at}ifom-ieo-campus.it) or Giorgio Scita (giorgio.scita{at}ifom-ieo-campus.it)
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