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Previous Article | Next Article 
Molecular and Cellular Biology, December 2006, p. 8942-8952, Vol. 26, No. 23
0270-7306/06/$08.00+0 doi:10.1128/MCB.00305-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Meng-Qiu Dong,1,3,
Ingrid Niesman,1,
Hyacynth Gacula,1
Xiaojing Lou,1
Tianlin Ma,2
Joseph R. Testa,2
John R. Yates III,3 and
Marilyn G. Farquhar1*
Department of Cellular and Molecular Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093,1 Human Genetics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111,2 Department of Cell Biology, Scripps Research Institute, La Jolla, California 920373
Received 17 February 2006/ Returned for modification 28 March 2006/ Accepted 3 September 2006
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ABSTRACT |
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INTRODUCTION |
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The TrkA nerve growth factor (NGF) receptor activates several signaling pathways, including the Ras/mitogen-activated protein (MAP) kinase and phosphatidylinositol (PI) 3-kinase/Akt pathways, and is required for survival, differentiation, and maintenance of neurons (25, 41). Work to date indicates that the early events in TrkA trafficking are similar to those for the EGF receptor, i.e., the receptor dimerizes, autophosphorylates, and is internalized via clathrin-coated pits which pinch off from the cell membrane in a dynamin-dependent process to become clathrin-coated vesicles which subsequently uncoat to become early endosomes (4, 19). Clathrin-coated vesicles and uncoated vesicles containing TrkA were designated "signaling endosomes" by Mobley and coworkers because they were shown to be enriched for NGF-bound, phosphorylated TrkA (pTrkA) and downstream signaling molecules in their active form, including GTP-bound Ras, C-Raf, pMek, Rap1, and phosphorylated extracellular signal-related kinases 1 and 2 (pErk1/2) (14, 23, 45). Endocytosis appears to be required for TrkA signaling, as blocking clathrin-mediated endocytosis leads to decreased NGF-induced neuron-like differentiation of PC12 cells and increased death of sympathetic neurons (46, 47). After internalization of TrkA, NGF and pTrkA are transported to the cell body in retrograde transport vesicles, where they are assumed to function in long-distance signal transduction of growth factors (14, 19, 22, 23).
GIPC (GAIP-interacting protein, C terminus) was originally identified based on its ability to bind to the RGS (regulator of G protein signaling) protein GAIP (RGS19), a GAP- or GTPase-activating protein for heterotrimeric G proteins (15). We previously showed that endogenous GIPC binds to TrkA and colocalizes with pTrkA in endocytic vesicles and that overexpression of GIPC attenuates NGF-induced MAP kinase activation in PC12(615) cells (31). Overexpression of GIPC was subsequently shown to also attenuate MAP kinase signaling mediated by the ß1-adrenergic and insulin-like growth factor 1 (IGF-1) receptors (6, 24).
To obtain further information on the role of GIPC in TrkA signaling, we used mass spectrometry to identify GIPC-interacting proteins. We identified four GIPC-interacting proteins in PC12(615) cells: APPL and APPL2 (33, 35), striatin (10), and SG2NA (37). APPL was of greatest interest, as it was recently found to bind Rab5 on signaling endosomes and to serve as an intermediate in EGF signaling between the cell membrane and the nucleus (33). In this paper, we show that after NGF stimulation, endogenous GIPC and APPL translocate to endocytic vesicles and presumably bind to TrkA on signaling endosomes. APPL recruits GIPC to endocytic vesicles with TrkA, and both GIPC and APPL are required for optimal TrkA signaling and for the efficient transport of TrkA vesicles from the cell periphery to early endosomes in the juxtanuclear region.
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MATERIALS AND METHODS |
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Antibodies.
Rabbit anti-GIPC serum (15) was affinity purified on glutathione S-transferase (GST)-GIPC immobilized on polyvinylidene difluoride membranes. Rabbit anti-APPL serum was characterized previously (35). Rabbit antistriatin and anti-SG2NA sera (11, 36) were provided by Francis Castets and Ariane Monneron. Affinity-purified, mouse anti-pan-Trk monoclonal antibody (MAb) (B-3) and rabbit anti-Trk immunoglobulin G (IgG) (C-14), raised against the highly conserved C-terminal region of TrkA, and rabbit anti-Erk (C-14) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MAP kinase (Erk1/2) MAbs were purchased from Zymed Laboratories (San Francisco, CA). Rabbit antirat TrkA (RTA) serum and affinity-purified anti-TrkA MAb 5C3 IgG were provided by Louis Reichardt (University of California, San Francisco, CA) and H. Uri Saragovi (Lady Davis Institute-Jewish General Hospital, Montreal, Quebec, Canada). Rabbit anti-pErk (phospho-p44/p42) MAP kinase (Thr202/Tyr204), anti-pAkt(Ser473), and anti pTrkA(Tyr490) IgG were purchased from Cell Signaling Technology (Beverly, MA). Mouse antiactin, rabbit anti-FLAG, and mouse anti-FLAG (M2) IgGs and anti-FLAG M2-agarose beads were from Sigma-Aldrich (St. Louis, MO). Mouse anti-protein kinase B 
/Akt and anti-EEA1 IgG were purchased from Transduction Laboratories, BD Biosciences (San Diego, CA). Affinity-purified mouse antihemagglutinin (anti-HA) (HA.11) IgG was from Covance (Berkeley, CA). Goat anti-rabbit and goat anti-mouse Alexa-594 or -488 F(ab')2 were from Molecular Probes (Eugene, OR). Goat anti-rabbit and goat anti-mouse Alexa Fluor 680 or IRDye 800 F(ab')2 were from Li-Cor Biosciences (Lincoln, NE).
Immunoprecipitation. Cells were lysed on ice for 30 min in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 50 mM NaF, 1 mM sodium orthovanadate, and protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO]). The insoluble fraction was removed by centrifugation (10,000 x g for 30 min at 4°C), and the protein concentration of the supernatant was determined by the Bradford assay (6a) (Bio-Rad Laboratories, Hercules, CA). Cell lysates (3 to 4 mg protein) were incubated at 4°C with anti-FLAG M2 beads for 5 h or with polyclonal antibodies for 5 h to overnight, followed by incubation with protein A-Sepharose beads (Sigma-Aldrich) for 1 h. Beads were then washed extensively with lysis buffer and boiled in sodium dodecyl sulfate sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Mass spectrometry and database searching. Immunoprecipitations were carried out as described above with preimmune or anti-GIPC serum, and bead-bound proteins were resolved by SDS-PAGE and stained with silver (GelCode SilverSNAP stain kit; Pierce Biotechnology, Rockford, IL). Selected silver-stained protein bands were excised from the gel and digested with trypsin in gel (39). Peptides were loaded onto a reverse-phase microcapillary high-pressure liquid chromatography column (100 µm inside diameter packed with 7 cm of 5-µm C18 reverse-phase resin from Vydac) and eluted into a Finnigan LCQ ion trap mass spectrometer with a linear gradient of 100% RP-A buffer (0.1% formic acid, 5% acetonitrile) to 40% RP-A plus 60% RP-B buffer (0.1% formic acid, 80% acetonitrile) over 30 min at 200 nl/min flow speed. The tandem mass spectrometric data were searched against the human, mouse, and rat protein databases from http://www.ncbi.nlm.nih.gov/ by using the SEQUEST program (18), and the results were filtered and sorted using DTASelect (43).
Immunoblotting. Proteins separated by SDS-PAGE were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking with Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk, the membranes were incubated with primary antibodies at room temperature for 1 h or at 4°C overnight, followed by incubation for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories, Hercules, CA) or sheep anti-mouse IgG (Amersham Biosciences, Piscataway, NJ). Detection was accomplished with SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). Infrared imaging with two-color detection and quantification of Western blots was performed according to the manufacturer's protocols using an Odyssey imaging system (Li-Cor Biosciences, Lincoln, NE).
Plasmid construction and RNA interference.
Construction of His-tagged, full-length mouse GIPC expression plasmid (pcDNA3-mGIPC-FLAG) and the PDZ domain mutant [pcDNA3-mGIPC (L142G143>AE)-FLAG] was described previously (31). The HA-tagged APPL construct in the pENTR vector (Invitrogen) was subcloned from APPL cDNA constructs (35) and used to generate HA-tagged APPL
C (a mutant lacking the four C-terminal amino acids) by PCR with a reverse primer lacking the coding sequence for the last four residues. pENTR-HA-APPL and pENTR-HA-APPLC were then recombined separately into a baculovirus destination vector, pDEST10, to generate HA/His6-APPL and HA/His6-APPLC by use of a baculoviral expression system from Invitrogen. Knockdown of GIPC protein expression was performed in PC12(615) cells by use of a duplex small interfering RNA (siRNA) (sense sequence, 5-AGAGGUGGAAGUAUUCAAGdTdT, where dT is deoxyribosylthymine) purchased from Dharmacon, Inc. (Chicago, IL). Knockdown of APPL1 was achieved using siGenome SMARTpool reagent (Dharmacon, Inc.). A negative-control scrambled siRNA (silencer no. 1) was purchased from Ambion (Austin, TX). Transfection of PC12(615) cells was performed using Dharmafect4 transfection reagent according to the manufacturer's protocol (Dharmacon, Inc.), with 60 nM siRNA concentration in the medium, 0.8 µg/µl siRNA-to-lipid ratio, and cell density of 
100 cells/mm2 surface area.
Protein purification and in vitro binding assay.
GST and GST-GIPC were expressed in Escherichia coli and purified on glutathione-Sepharose 4B beads (Amersham). HA/His6-tagged APPL or APPLC was expressed with baculovirus in Sf9 cells and purified on Ni-nitrilotriacetic acid agarose (QIAGEN, Valencia, CA). For the binding assay, 1.5 µg GST or GST-GIPC was incubated with 2 µg HA/His6-APPL and 1 µl glutathione-Sepharose in 300 µl binding buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 0.5% NP-40) for 5 h at 4°C. For the experiments involving cell lysates, 3 µg GST or GST-GIPC was incubated with 500 µl lysate prepared from Sf9 cells expressing HA/His6-APPL or HA/His6-APPLC for 2 h at 4°C. Beads were spun down and washed extensively in the binding buffer and boiled in Laemmli sample buffer. Bead-bound proteins were separated by SDS-PAGE.
Endocytosis assay for TrkA. PC12(615) cells grown on poly-L-lysine-coated coverslips (BD Biotech, San Diego, CA) were serum starved at 37°C in Dulbecco's modified Eagle's medium (high glucose) for 2 h; incubated on ice with NGF (50 to 100 ng/ml; Roche), 5C3 IgG (1:10,000), or RTA serum (1:500) diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 0.5 to 1 h; washed with ice-cold PBS containing 0.1% BSA (3x); and shifted to fresh medium at 37°C for various times. The cells were then fixed and processed for immunofluorescence.
Immunofluorescence. PC12(615) cells were fixed with 2% paraformaldehyde in PBS on ice for 30 min, permeabilized with 0.1% Triton X-100 containing 1% BSA, and incubated with primary antibodies for 1 h and goat anti-rabbit Alexa-594 or anti-mouse Alexa-488 F(ab')2 for 1 h. Fluorescence images were taken with a Zeiss Axiophot or AxioImager M1 (Carl Zeiss, Thornwood, NY) equipped with a digital ORCA-ER camera (Hamamatsu, Bridgewater, NJ) and processed with Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA). Fluorescence images of double-labeled samples were quantified as described previously (17). Cell membrane areas were traced for early time points (0 and 5 min), or entire cells were traced at 30-min time points and selected using Adobe Photoshop 5.0. For semiquantitative analysis of cell membrane-associated TrkA, images were calculated as follows. Only pixels with gray-scale intensities between 75 and 255 were included, and the numbers of total pixels for TrkA that overlapped with GIPC, APPL, or EEA1 (yellow pixels) were measured using NIH image (National Institutes of Health, Bethesda, MD).
Neurite outgrowth assay. PC12(615) cells were transfected with FLAG-tagged GIPC constructs, treated with 50 ng/ml NGF for 16 h to induce growth of neurites, and stained with anti-FLAG MAb and goat anti-mouse Alexa-488 F(ab')2. Transfected cells and nontransfected controls were assayed for neurite outgrowth as previously described (26). In each experiment, at least 50 transfected cells from several randomly selected fields were classified as differentiated or undifferentiated. A differentiated cell was defined as one that had grown a neurite of twice the cell body length or longer (26). The differentiation rate was expressed as the average percentage (±standard error) of differentiated cells from six experiments.
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RESULTS |
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(30), contains a pleckstrin homology domain, a phosphotyrosine binding domain, and a leucine zipper motif and can interact with Akt and PI 3-kinase (35). APPL2, previously named DIP13ß, is 50% identical to APPL. Striatin and SG2NA are 66% identical, sharing an N-terminal coiled-coil region, a Ca2+-calmodulin binding domain, and C-terminal WD repeats (10, 11, 37). Each of these four proteins has been reported to act as a scaffold linking signal transduction and vesicular trafficking (3, 32). APPL is a known marker of signaling endosomes and was shown to regulate trafficking through its interaction with Rab5 (32, 33).
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APPL binds to GIPC through its PDZ-binding domain.
We noted that APPL and APPL2 have the same C-terminal PDZ-binding motif (-SEA) as GAIP (15), suggesting that, like GAIP, they might bind directly to GIPC. This proved to be the case, as GST-GIPC specifically pulled down HA/His6-APPL (Fig. 1C). Moreover, this interaction occurs through the PDZ-binding motif of APPL, because GST-GIPC did not pull down APPLC, a mutant lacking the four C-terminal amino acids (Fig. 1D). Furthermore, mutation of the PDZ domain of GIPC, which prevents its binding to GAIP but not to TrkA (31), also prevented the coimmunoprecipitation of APPL (Fig. 1E). These results indicate direct interaction between the PDZ domain of GIPC and the PDZ-binding motif of APPL.
TrkA, GIPC, and APPL meet on peripheral endosomes. To determine where in the cell GIPC, APPL, and TrkA meet, we followed their distribution in PC12(615) cells by immunofluorescence after activation of TrkA. We incubated serum-starved PC12(615) cells at 4°C with either NGF or anti-TrkA antibody (RTA or 5C3), shifted the cells to 37°C to initiate endocytosis, and analyzed TrkA internalization by immunofluorescence. We found that the dynamics of TrkA internalization were the same whether internalization was induced with NGF or TrkA antibody. Both RTA and 5C3 bind specifically to the ectodomain of TrkA and mimic NGF, compete with NGF for binding sites on TrkA, and induce TrkA internalization and signaling (12, 28). In cells incubated at 4°C as described above, TrkA was distributed uniformly along the cell membrane in dots or patches, presumably due to concentration of the receptor in specific microdomains of the plasma membrane (PM) (Fig. 2A and G). Endogenous GIPC (Fig. 2B) and APPL (Fig. 2H) were diffusely distributed in the cytoplasm or located on scattered vesicles in the cell bodies or cell processes, and there was no overlap in staining with TrkA (Fig. 2C and I). When the cells were shifted to 37°C for 5 min, TrkA became concentrated in clusters of endocytic vesicles located along the cell membrane at the tips of the cell processes (Fig. 2D and J). At the same time, there was a striking shift in distribution of both GIPC (Fig. 2E) and APPL (Fig. 2K) to the clustered, endocytic vesicles located at the tips of the cell processes, where they colocalized with TrkA (Fig. 2F and L).
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APPL, but not GIPC, traffics with TrkA to juxtanuclear EEA1 endosomes.
To determine if GIPC and APPL traffic with TrkA to early endosomes, we followed the distribution of TrkA, GIPC, APPL, and EEA1, an early endosome marker, 15 to 30 min after activation of TrkA. TrkA did not codistribute significantly with EEA1 in cells incubated at 4°C (0 min) (Fig. 5A) or 5 min after shifting the cells to 37°C but began to appear in larger EEA1-positive endosomes in the juxtanuclear region after 15 min and peaked there at 30 min (Fig. 5B). By contrast, colocalization of TrkA and GIPC was prominent at 5 min (Fig. 2F) and was reduced at 30 min (Fig. 5C) and colocalization of GIPC and EEA1 (Fig. 5E) was minimal at both 5 and 30 min. Quantification of overlapping pixels revealed that at 0 min only 1% of the surface-labeled TrkA was associated with EEA1 endosomes whereas by 30 min 42% overlapped with EEA1 on juxtanuclear endosomes (Fig. 3C). The quantification also confirmed that the overlap in distribution of TrkA and GIPC peaked at 5 min and had decreased by 15 min: 30% of the surface-labeled TrkA overlapped with GIPC at 5 min, but overlap was reduced to 15% at 15 min (Fig. 3A). In contrast to GIPC, colocalization of TrkA and APPL (Fig. 5D) and APPL and EEA1 (Fig. 5F) was striking at 30 min; 33% of the total surface-labeled TrkA overlapped with APPL on juxtanuclear endosomes at both 15 and 30 min (Fig. 3B).
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APPL is required for the recruitment of GIPC to TrkA endocytic vesicles. To find out if preventing interaction between GIPC and APPL is associated with impaired recruitment of GIPC or APPL or impaired trafficking of TrkA, we expressed a GIPC mutant [GIPC(LG>AE)] that cannot bind APPL and analyzed the distribution of TrkA and GIPC after NGF stimulation. We found that GIPC(LG>AE) did not affect TrkA endocytosis on a gross level (Fig. 4E and F). However, in contrast to endogenous GIPC (Fig. 2E) or GIPC-FLAG (Fig. 4B), the mutant failed to translocate to the cell membrane or to colocalize with TrkA (Fig. 4E and F) or APPL (Fig. 4G and H) after activation of TrkA. Quantification of the overlap between GIPC-FLAG or GIPC(LG>AE)-FLAG and APPL demonstrated that at 2 min of NGF uptake, 47% of cell membrane-associated GIPC-FLAG overlapped with endogenous APPL, whereas only 15% of the mutant overlapped with APPL (Fig. 3D). The lack of overlap in staining of TrkA and GIPC(LG>AE)-FLAG suggests that binding of GIPC to a protein containing a PDZ-binding motif, most likely APPL, is necessary to recruit GIPC to the cell membrane and for GIPC to bind to TrkA.
To verify that the recruitment of GIPC is dependent on its interaction with APPL, we examined the effects of knocking down APPL on translocation of GIPC to incoming TrkA vesicles. We found that in cells transfected with scrambled siRNA, GIPC's recruitment 5 min after NGF stimulation occurred normally (Fig. 6A and C); however, in cells transfected with APPL siRNA (60% knockdown [see Fig. 8]), GIPC was not translocated to the PM and failed to appear on vesicles with TrkA after NGF stimulation (Fig. 6B and D). Indeed, cell counts revealed that colocalization (yellow pixels) of TrkA and GIPC was evident (based on visual inspection) in 41% of cells transfected with control siRNA but in only 7% of cells treated with APPL siRNA. From these results, we conclude that interaction with APPL is required for the recruitment of GIPC to incoming vesicles carrying TrkA.
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4% of control (unstimulated) PC12(615) cells differentiated, i.e., grew neurites of at least twice the cell body length (Fig. 7A). In contrast, 50% of the nontransfected PC12(615) cells, 41% of the mock-transfected cells, and 37% of the cells transiently transfected with wild-type GIPC-FLAG differentiated after 16 h of NGF treatment (50 ng/ml). Transfection of GIPC(LG>AE)-FLAG caused a significant reduction (27%) in neurite outgrowth (P < 0.05 compared to the mock control). Therefore, disruption of GIPC's ability to bind the PDZ-binding motif of APPL impairs NGF-induced cell differentiation, indicating the importance of this interaction for NGF signaling.
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GIPC or APPL knockdown inhibits TrkA signaling.
To further characterize the role of GIPC-APPL interaction in TrkA signaling, we assessed the effects of knocking down GIPC or APPL on activation of TrkA and on the downstream Erk/MAP kinase and PI 3-kinase/Akt signaling pathways. We found that when GIPC was knocked down activation of TrkA was not affected but effects on TrkA signaling were evident, as pAkt and pErk1/2 were reduced by 20% compared to controls transfected with scrambled siRNA (Fig. 8A and B). Similar results were obtained after knocking down APPL. Activation of TrkA was not affected, but Erk/MAP kinase and PI 3-kinase/Akt signaling pathways were inhibited by 30 and 40%, respectively (Fig. 8A and B). These findings suggest that interaction between TrkA, GIPC, and APPL is required for efficient signaling.
GIPC knockdown slows TrkA internalization and trafficking. Since GIPC, through its interaction with myosin VI, can facilitate the translocation of endocytic vesicles from the cell periphery to the juxtanuclear region (1, 7), we investigated if the effects on signaling seen after GIPC and APPL knockdown could be attributed to changes in TrkA trafficking. In cells transfected with scrambled siRNA, TrkA was internalized normally and found in clusters of vesicles at the tips of the cell processes or just beneath the cell membrane after 5 min (Fig. 6E) and in juxtanuclear endosomes by 15 min after NGF stimulation (Fig. 6G). By contrast, in cells treated with GIPC siRNA in which GIPC expression was knocked down 80 to 90% (Fig. 8A) there was a striking slowing of the internalization and trafficking of TrkA. At 5 min, TrkA remained largely at the PM, with fewer vesicles that stained for TrkA seen inside the cell (compare Fig. 6F and E), and by 15 min TrkA remained in the peripheral cytoplasm and little enrichment in the juxtanuclear region was evident (compare Fig. 6H and G).
Similar slowing in APPL trafficking was observed after GIPC knockdown (Fig. 6I to L). In cells transfected with control (scrambled) siRNA, APPL was recruited normally to the cell membrane after 5 min and colocalized with TrkA, and by 15 min it was concentrated in juxtanuclear endosomes (Fig. 6I). In cells transfected with GIPC siRNA, APPL was recruited to the cell membrane, colocalized with TrkA, but at 15 min remained largely at the cell membrane and was not seen on juxtanuclear endosomes in the majority of cells (Fig. 6J). However, by 30 min, some of the APPL eventually accumulated in the larger juxtanuclear endosomes (Fig. 6L).
We conclude that GIPC is required for efficient trafficking of TrkA and APPL to early endosomes. This effect is most likely due to its interactions with myosin VI. GIPC is known to bind myosin VI (20), and its interaction with myosin VI is required for efficient endocytosis of the transferrin receptor (20) as well as megalin, a member of the low-density lipoprotein receptor superfamily (38), and the glucose transporter GLUT1 (7).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper, we found that GIPC represents another marker for peripheral signaling endosomes, as TrkA, GIPC, and APPL codistribute in endocytic vesicles on or near the cell membrane shortly after NGF binds to TrkA. We have shown previously that GIPC binds to the juxtamembrane region of TrkA (31), and here we show that it binds to the C-terminal PDZ-binding motif of APPL. Thus, although GIPC is presumably capable of assembling TrkA and APPL into a multiprotein complex, it requires APPL to be efficiently recruited. APPL has recently been shown to bind directly to TrkA (29). The colocalization of GIPC, TrkA, and APPL on endosomes is restricted temporally to a short time window almost immediately following TrkA activation and spatially to the cell periphery. Expression of a GIPC PDZ mutant that cannot bind APPL or knockdown of APPL disrupted this scenario, as it interfered with recruitment of GIPC to the peripheral signaling endosomes and impaired TrkA signaling based on the inhibition of neurite outgrowth and Erk activation. Thus, our results suggest that GIPC is required for efficient internalization of TrkA and for optimal signaling from peripheral endosomes.
APPL has been shown to bind to Rab5 after EGF stimulation and to be distributed on a population of endosomes which were suggested to correspond to signaling endosomes (33). Miaczynska et al. (33) also found that in HeLa cells EGF treatment caused APPL to translocate from peripheral vesicles to the nucleus. In that work, localization of APPL and EGF was observed to occur only on peripheral vesicles and little colocalization of APPL and EEA1 was observed to occur, from which it was proposed that APPL vesicles constitute an EEA1-independent route for receptor trafficking. In PC12 cells, we found a rapid, transient increase of APPL staining on the cell periphery after NGF stimulation, followed by its appearance on EEA1 endosomes in the juxtanuclear region, suggesting that in PC12 cells APPL vesicles and EEA1 vesicles are not independent trafficking routes.
In addition to TrkA, GIPC can bind to a variety of membrane receptors, including several growth factor receptors, e.g., the TrkB (31), transforming growth factor ß III (5), and IGF-1 (6) receptors, and several G protein-coupled receptors, including the ß1-adrenergic (24), D2 dopamine (27), and LH (21) receptors. TrkA and TrkB bind to GIPC through their juxtamembrane regions (31), and the same may apply to the Xenopus laevis IGF-1 receptor. In the case of the IGF-1 and ß1-adrenergic receptors, overexpression of GIPC was also shown to reduce MAP kinase activation. At present, it is not known whether APPL is involved in promoting signaling via other growth factor and G protein-coupled receptors that bind GIPC.
In addition to binding to various receptors, GIPC can bind myosin VI in two-hybrid and immunoprecipitation assays (1, 7, 20). Myosin VI is a retrograde motor protein that associates with clathrin-coated pits or vesicles, modulates clathrin-mediated endocytosis (8), and partially colocalizes with GIPC in newly uncoated vesicles (1). GIPC/myosin VI complexes coordinately move within cells in an actin-dependent manner and facilitate the translocation of endocytic vesicles across the peripheral actin barrier (20). The ability of GIPC to form homodimers (7) and bind to APPL, striatin, and many receptor proteins suggests that GIPC could function to assemble protein complexes required for endocytosis and signal transduction.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grants CA100768 and DK17780 to M.G.F.
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FOOTNOTES |
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Published ahead of print on 2 October 2006.
These three authors contributed equally to this work.
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