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Rescue of p53 Blockage by the A_2A Adenosine Receptor via a Novel Interacting Protein, Translin-Associated Protein X

Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei, Taiwan (C.-N.S., H.-C.C., J.C., Y.-W.L., H.-L.L., H.-M.C., Y.C.); Institute of Neuroscience, National Yang-Ming University, Taipei, Taiwan (H.-C.C., J.C., Y.-W.L., Y.C.); Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (C.-N.S., Y.C.); and Department of Medical Technology, Yuanpei University of Science and Technology, Hsinchu, Taiwan (J.C.)

Received November 29, 2005; accepted April 14, 2006

	Abstract

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Blockage of the p53 tumor suppressor has been found to impair nerve growth factor (NGF)-induced neurite outgrowth in PC-12 cells. We report herein that such impairment could be rescued by stimulation of the A_2A adenosine receptor (A_2A-R), a G protein-coupled receptor implicated in neuronal plasticity. The A_2A-R-mediated rescue occurred in the presence of protein kinase C (PKC) inhibitors or protein kinase A (PKA) inhibitors and in a PKA-deficient PC-12 variant. Thus, neither PKA nor PKC was involved. In contrast, expression of a truncated A_2A-R mutant harboring the seventh transmembrane domain and its C terminus reduced the rescue effect of A_2A-R. Using the cytoplasmic tail of the A_2A-R as bait, a novel-A_2A-R-interacting protein [translin-associated protein X (TRAX)] was identified in a yeast two-hybrid screen. The authenticity of this interaction was verified by pull-down experiments, coimmunoprecipitation, and colocalization of these two molecules in the brain. It is noteworthy that reduction of TRAX using an antisense construct suppressed the rescue effect of A_2A-R, whereas overexpression of TRAX alone caused the same rescue effect as did A_2A-R activation. Results of [³H]thymidine and bromodeoxyuridine incorporation suggested that A_2A-R stimulation inhibited cell proliferation in a TRAX-dependent manner. Because the antimitotic activity is crucial for NGF function, the A_2A-R might exert its rescue effect through a TRAX-mediated antiproliferative signal. This antimitotic activity of the A_2A-R also enables a mitogenic factor (epidermal growth factor) to induce neurite outgrowth. We demonstrate that the A_2A-R modulates the differentiation ability of trophic factors through a novel interacting protein, TRAX.

Adenosine has been shown to play an essential role in modulating neuronal function via four adenosine receptors (Daval et al., 1991). The A_2A adenosine receptor (A_2A-R) belongs to the G protein-coupled receptor (GPCR) family and is involved in regulating neuronal plasticity and development (Weaver, 1993; Ribeiro, 1999; Cheng et al., 2002). We and others have previously demonstrated that in PC-12 cells, stimulation of the A_2A-R activates at least two major cellular signaling cascades: adenylyl cyclase/cAMP/protein kinase A (PKA) and the protein kinase C (PKC)-mediated pathways (Sobreviela et al., 1994; Chang et al., 1997; Huang et al., 2001; Charles et al., 2003). In addition, A_2A-R stimulation rescues the ability of PC-12 cells to proceed with NGF-evoked neurite outgrowth when the MAPK cascade is impaired (Cheng et al., 2002). In the present study, using two dominant-negative p53 mutants, we demonstrated that stimulation of the A_2A-R suppresses proliferation and rescues the differentiation process impaired by p53 inactivation. It is intriguing that neither the PKA-nor the PKC-mediated signaling pathway contributes to this rescue effect of the A_2A-R. Instead, a novel A_2A-R-interacting protein [the Translin-associated protein X (TRAX) (Aoki et al., 1997)] that binds to the C terminus of the A_2A-R might mediate the rescue effect of A_2A-R. TRAX was originally identified as a binding protein of Translin using a yeast two-hybrid system (Aoki et al., 1997). Translin is a protein that binds RNA and singlestranded DNA with potential functions in DNA rearrangement and repair, mitotic cell division, mRNA transport, and translational regulation (Aoki et al., 1995, 1997; Han et al., 1995; Wu et al., 1997; Hosaka et al., 2000; Ishida et al., 2002; Yang et al., 2003). TRAX is a 33-kDa protein whose amino acids have 28% identity to those of Translin (Aoki et al., 1997). Although the biological function of TRAX remains largely obscure, it forms complexes with Translin in neuronal dendrites and thus might be involved in dendritic RNA processing (Finkenstadt et al., 2000). TRAX has also been implicated in DNA repair via binding to the nuclear matrix protein, C1D, an activator of the DNA-dependent protein kinase important for DNA double-strand repair and V(D)J recombination (Erdemir et al., 2002). In the present study, we found that TRAX is a novel interacting protein of the A_2A-R and that overexpression of TRAX recovered the NGF-induced neurite outgrowth impaired by p53 inhibition in PC-12 cells. Moreover, down-regulation of endogenous TRAX using an antisense construct obliterated the rescue effect of A_2A-R. Taken together, our data suggest the involvement of TRAX in mediating the rescue effect of A_2A-R on neuronal differentiation in PC-12 cells.

	Materials and Methods

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Reagents. All reagents were purchased from Sigma Chemical (St. Louis, MO) except where otherwise specified. Forskolin (FK), CGS21680 (CGS), and 8-(3-chlorostyryl) caffeine (CSC) were obtained from Sigma/RBI (Natick, MA). Chelerythrine (CHE) and bisindolylmaleimide I (BIM) were from Calbiochem (San Diego, CA). DMEM, fetal bovine serum (FBS), and horse serum (HS) were purchased from Invitrogen (Carlsbad, CA). H89 was from BIOMOL Research Laboratories (Plymouth Meeting, PA). NGF was obtained from Alomone (Jerusalem, Israel).

Cell Culture. PC-12 cells were originally obtained from the American Type Culture Collection (Manassas, VA) and maintained in DMEM (HyClone, Logan, UT) supplemented with 5% FBS (HyClone) plus 10% HS (HyClone) in an incubation chamber gassed with 10% CO₂ and 90% air at 37°C. A123, a cAMP-dependent PKA-deficient variant of PC-12 cells (Ginty et al., 1991), was kindly provided by Dr. J. A. Wagner (Cornell University Medical College, Ithaca, NY). A123 cells were maintained in DMEM supplemented with 5% (v/v) HS and 10% (v/v) FBS. HEK 293T cells were maintained in DMEM supplemented with 10% (v/v) FBS and 2 mM glutamine in an incubation chamber supplied with 5% CO₂ and 95% air at 37°C.

Transfection and Neuronal Differentiation. The expression constructs, which encode the dominant-negative mutants of p53 (R175H-p53 and R273H-p53) are described elsewhere (Bargonetti et al., 1992; Srivastava et al., 1993). The A_2A-R-truncated mutants were generated from pcDNA3-A_2A-R by PCR and were subcloned into a pcDNA3.1/V5-His vector. Primers for creating A_2A-R_253-410 were as follows: 5'-ACCATGTTCTGCTCCACGTGCCGG-3' and 5'-GGAAGGGGCAAACTCTGAAGA-3'. The antisense construct of TRAX (TRAX_AS, encoding the RNA containing nucleotides 451 to 870 in the antisense direction), was also produced by PCR amplification using the following primers: 5'-GCGGCCATGCAGTTGACATTTACA-3' and 5'-GCGGCGAGAAATGCCTCTTCCTG-3'. Cells were transfected using LipofectAMINE 2000 (Invitrogen) following the manufacturer's protocol. For analyzing neuronal differentiation, cells were transiently transfected with the indicated construct(s) along with 1/10 of the molar amount of an EGFP-expressing construct (Clontech, Mountain View, CA), and treated with the indicated reagent(s) for 3 days. Transfected cells were marked as EGFP-expressing cells under a fluorescent microscope with a blue filter. Cells containing neurites of at least two cell-body diameters in length were scored as neurite-bearing cells. Transfected cells that grew neurites were normalized to the number of total transfected cells and are presented as the percentage of neurite-bearing cells. In each experiment, at least 100 transfected cells were counted. HEK239T cells were transfected with the desired construct(s) at 3 x 10⁶ cells in 100-mm culture plates using LipofectAMINE 2000 as described above.

Flow Cytometry and Cell Sorting. PC-12 cells were transfected with the construct(s) of desired proteins plus hrGFP at a molar ratio of 10:1. One day after transfection, cells were harvested by centrifugation, resuspended in DMEM to a final density of 5 x 10⁶ cells/ml, and filtered through a cell strainer cap (Falcon Plastics, Oxnard, CA) to remove cell aggregates. Flow cytometry and sorting of hrGFP-positive cells were carried out using a FACSVantage instrument (BD Biosciences, San Jose, CA) with a 530 ± 15-nm bandpass filter as cells traversed the beam of an argon ion laser (488 nm, 100 mW). Data acquisition and analysis were performed with CellQuest software (BD Biosciences). Sorted cells were harvested and plated for further treatments.

cAMP Assay. Transfected and sorted PC-12 cells were plated at the density of 5 x 10⁵ cells/well (on 12-well plates) and incubated with the indicated reagent(s) for the desired period of time. Cells were washed twice with ice-cold Ca²⁺-free Locke's solution (150 mM NaCl, 5.6 mM KCl, 5 mM glucose, 1 mM MgCl₂, and 10 mM HEPES, adjusted to pH 7.4). Cellular cAMP was extracted by adding 0.3 ml of 0.1 N HCl to each well and incubating this for 10 min on ice. The cAMP content was assayed using the ¹²⁵I-cAMP assay system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Production of the Polyclonal Anti-TRAX Antibody. Oligopeptides (TRAX_273-290, corresponding to amino acids 273290 of mouse TRAX) were purchased from Genosys (The Woodlands, TX) and conjugated to bovine serum albumin (BSA; Sigma) using m-maleimidobenzoyl-N-hydroxysuccinimide ester. To remove the potentially existing anti-BSA IgG, the anti-TRAX antiserum was preabsorbed with 3% BSA in phosphate-buffered saline at 4°C overnight. For double immunohistochemical staining, the anti-TRAX antibody was biotinylated as described elsewhere (Lee et al., 2003).

Immunohistochemistry and Brain Tissue Preparation. Seventy-two hours after transfection, PC-12 cells were fixed and stained with the desired primary antibody reconstituted in phosphate-buffered saline/2% goat serum at 4°C for 1416 h. Dilution of the anti-HA antibody (Invitrogen) with anti-TRAX was at 1:1000. After extensive washing, slides were incubated with the corresponding secondary antibody conjugated with Alexa red (for the A_2A-R-_V5) and FITC (for TRAX) at RT for 1 h, and analyzed with the aid of a laser confocal microscope (MRC-1000; Bio-Rad, Hercules, CA).

For brain tissues, 8-week-old male C57BL6 mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Animal experiments were performed in accordance with the National Institutes of Health Guidelines under protocols approved by the Animal Care and Use Committee of Academia Sinica. In brief, animals were deeply anesthetized with sodium pentobarbital (50 mg/kg), and intracardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Brains were carefully removed, fixed with 4% paraformaldehyde/0.1 M PB for 35 h, and then immersed in 30% sucrose in 0.1 M PB for 2 days. Double immunostaining of A_2A-R and TRAX was conducted as detailed elsewhere (Shindler and Roth, 1996) with modifications. In brief, brain sections (20 µm) were first stained with an anti-A_2A-R antibody (Lee et al., 2003) and visualized using a highly sensitive biotin-tyramide amplification system with Avidin-Alexa Fluor 568. The sections were then blocked sequentially using the avidin D blocking solution (Vector Laboratories, Burlingame, CA) and the biotin blocking solution (Vector Laboratories), incubated with the anti-TRAX antibody, and followed by a goat anti-rabbit IgG conjugated with Alexa Fluor 488. Patterns of double immunostaining were analyzed with the aid of a laser confocal microscope.

Western Blot Analysis. For the antisense experiments, cells were transfected with the control vector or the TRAX antisense construct (TRAX_AS) for 72 h. For analyzing the expression level of p21, cells were transiently transfected with the indicated construct along with 1/4 of the molar amount of an EGFP expression construct. Twenty-four hours after transfection, EGFP-positive cells were sorted and harvested using a FACSVantage (BD Biosciences) with a 530 ± 15-nm bandpass filter, plated in 35-mm dishes, and treated with the indicated reagent(s) for 3 days. Cells were then lysed using a lysis buffer containing 50 mM HEPES, pH 7.6, 150 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 10 mM NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1 µM pepstatin A, 1 mM Na₃VO₄, 1 mM dithiothreitol, and 100 nM okadaic acid. Equal amounts of sample were separated by SDS-PAGE (Laemmli, 1970) and electroblotted onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA) for the Western blot analyses as described previously (Cheng et al., 2002). In general, we used a 1:1000 dilution for the anti-TRAX and anti-actin antibodies, and 1:500 for the anti-p21 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL).

Coimmunoprecipitation. HEK 293T cells were transfected with the A_2A-R-_V5 cDNA along with other desired plasmids for 72 h and lysed with ice-cold radioimmunoprecipitation assay buffer (10 mM sodium phosphate, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 150 mM NaCl). The lysates were then incubated with anti-V5 monoclonal antibody (1 µl/ml lysate; Invitrogen) for 1 h. Protein A beads (200 µl/ml lysate) were added into the mixture and rotated gently at 4°C for 2 h. The immunoprecipitates were pelleted and washed three times with ice-cold radioimmunoprecipitation assay buffer. Lysates (input) and washed immunoprecipitates were mixed with SDS-PAGE sample buffer, boiled for 5 min, and separated on 10% SDS-PAGE, followed by Western blot analyses using the corresponding antibodies.

DNA Synthesis Assay. For the [³H]thymidine incorporation assay, transfected and sorted cells were plated onto 12-well plates and incubated with the indicated reagents for 3 days. [³H]Thymidine (1 mCi/ml; PerkinElmer Life and Analytical Sciences, Boston, MA) was added to the growth medium and used to label cells for4 h at 37°C. After removing the labeling medium, cells were collected onto glass microfiber filters (Whatman, Ann Arbor, MI), washed twice with 5 ml of 0.15 M NaCl, and soaked with 5% trichloroacetic acid for 5 min. The membranes were then washed with 5% trichloroacetic acid, neutralized with 0.5 M NaOH, air-dried, and counted.

DNA synthesis was also determined using a bromodeoxyuridine (BrdU) labeling and detection kit (Roche, Mannheim, Germany) according to the manufacturer's protocol. In brief, PC-12 cells were transfected with the indicated plasmids plus the expression construct of EGFP, incubated for 1618 h, and treated with the indicated reagent(s) for 4 days. BrdU (10 mM) was then added to the culture medium for 1 h, followed by immunostaining using an anti-BrdU antibody and an anti-EGFP antibody (BD Bisociences) to identify the transfected cells. Cell immunostaining was observed and photographed using a Zeiss immunofluorescent microscope (Carl Zeiss Inc., Thornwood, NY). Totals of at least 100 cells were scored for each condition.

	Results

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p53 has been shown to mediate NGF-triggered cell growth arrest in PC-12 cells (Eizenberg et al., 1996; Hughes et al., 2000). In line with previous findings (Eizenberg et al., 1996), we found that overexpression of a dominant-negative p53 mutant (R175H-p53) led to suppression of NGF-evoked neurite outgrowth in PC-12 cells (Fig. 1A). It is noteworthy that activation of the A_2A-R using an A_2A-R-selective agonist (CGS) counteracted the impaired neurite outgrowth caused by R175H-p53 (Fig. 1, A and B). Expression of another dominant-negative p53 mutant (R273H-p53) that lacks DNA binding ability also inhibited NGF-evoked neurite outgrowth. Again, this blockage of neurite outgrowth could be rescued by A_2A-R stimulation (Fig. 1C). Pretreating PC-12 cells with an A_2A-R antagonist, CSC, abolished the rescue effect of CGS (Fig. 1D), indicating that CGS operates through the A_2A-R.

View larger version (43K): [in this window] [in a new window]   Fig. 1. Stimulation of the A2A-R specifically rescued the suppression of NGF-evoked neurite outgrowth caused by p53 blockage. A, PC-12 cells were transiently transfected with a GFP vector, a construct encoding p53-R175H, wild-type p53, or an empty vector as indicated. One day after transfection, cells were incubated with normal growth medium containing NGF (100 ng/ml), or NGF plus CGS (1 µM), as indicated for 72 h. Representative merged pictures (right) are phase-contrast pictures (left) overlaid with images from fluorescence microscopy (middle). B, NGF-evoked neurite-bearing cells transfected with the indicated plasmids treated with NGF or NGF plus CGS were quantified. C, PC-12 cells were transiently transfected with a control vector or with a vector encoding a p53-R273H mutant along with a GFP vector as indicated. One day after transfection, cells were incubated with normal growth medium containing NGF or NGF plus CGS for 72 h. D, PC-12 cells were transiently transfected with a control vector or with a vector encoding p53-R175H along with a GFP vector as indicated. One day after transfection, cells were pretreated with an A2A-R-specific antagonist (CSC, 10 µM) for 30 min before further treatment with NGF or NGF plus CGS for 3 days. E, expression of the C terminus of the A2A-R suppressed its rescue effect. PC-12 cells were transfected with the indicated cD-NAs along with a GFP vector. One day after transfection, cells were treated with NGF (100 ng/ml) or NGF plus CGS (1 µM) for 72 h. NGF-evoked neurite-bearing cells were quantified. Data are presented as the mean ± S.E.M. values from at least three independent experiments. b, specific comparison between cells treated with or without CGS in each group (p < 0.001; two-way ANOVA). c, specific comparison between cells treated with or without CSC in each group (p < 0.001; two-way ANOVA). e, specific comparison between cells transfected with the indicated plasmid or a control vector (p < 0.001; two-way ANOVA). f, specific comparison between cells transfected with A2A-R253-410 or a control vector (p < 0.001; two-way ANOVA).

To elucidate the signaling pathway underlying the rescue effect of the A_2A-R, we first measured whether activation of the A_2A-R elevated the cellular cAMP content under the conditions tested. Transfected cells were sorted by the expression of hrGFP as described and stimulated with NGF and/or CGS. As shown in Fig. 2, regardless of the absence or presence of NGF, activation of the A_2A-R using CGS enhanced the cellular cAMP content at both the early stage (3 h) and the late stage (3 days) of NGF-induced differentiation. Consistent with the previously described desensitization process upon chronic stimulation (Chern et al., 1993), in all conditions tested, the cAMP level evoked by a 3-h incubation of CGS was much higher than that evoked by a 3-day incubation. We were surprised to find that p53 blockage using an R273H-p53 mutant reduced the A_2A-R-evoked cAMP response by approximately 50%. The mechanism by which p53 blockage suppresses the cAMP response evoked by A_2A-R stimulation remains unclear.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Stimulation of the A2A-R elevated the intracellular cAMP content in NGF-treated, R273H-p53-expressing PC-12 cells. Cells were transfected with an expression construct of R273H-p53 cells or an empty vector plus an expression vector of hrGFP. Twenty-four hours after transfection, cells were sorted by the expression of hrGFP and stimulated with the indicated reagent(s) for 3 h (closed bars) or 3 days (shaded bars). Intracellular cAMP levels were measured as described under Materials and Methods. Values represent the mean ± S.E.M. of nine determinations and are expressed as percentages of the CGS-induced cAMP response in the cells transfected with an empty vector (37.0 ± 3.3 nmol/106 cells). Statistical significance was evaluated by one-way analysis of variance (ANOVA). a, p < 0.05 compared with cells in the control, vector-transfected group. b, p < 0.05 compared with the cAMP level after3hof incubation under the same treatment conditions.

To determine whether the cAMP-dependent kinase (PKA) mediates the rescue effect of the A_2A-R, we set out to determine the role of PKA. As shown in Table 1, neither of two PKA inhibitors (H89 and PKI) attenuated the rescue effect of A_2A-R. We also used a PKA-deficient PC-12 variant (A123) to verify the functional role of PKA in the action of A_2A-R. Expression of the p53-R175H mutant in A123 cells resulted in blockage of NGF-induced neurite outgrowth as occurs in parental PC-12 cells (Table 1). Most importantly, A_2A-R stimulation retained the ability to rescue the impaired neurite outgrowth caused by p53 blockage in A123 cells (Table-1). We next examined the involvement of PKC using two PKC inhibitors (CHE and BIM), because A_2A-R stimulation has been shown to activate PKCs in PC-12 cells (Lai et al., 1997; Huang et al., 2001). Similar to the effect observed with PKA inhibitors, neither CHE nor BIM alone could suppress the rescue effect of the A_2A-R on neurite outgrowth caused by p53 blockage (Table 2). Taken together, the rescue effect of the A_2A-R was both PKA- and PKC-independent.

Note that stimulation of the A_2A-R causes only a low and transient cAMP response as a result of desensitization of the cAMP system (Chern et al., 1993). In contrast, direct stimulation of adenylyl cyclase by FK evoked sustained and high levels of cAMP. The elevation in cAMP induced by FK (10 µM) and CGS (1 µM) was 10.9 ± 0.4 and 2.1 ± 0.3 nmol/10⁶ cells (mean ± S.E.; with 20 min of incubation at RT), respectively, in PC-12 cells. It is noteworthy that FK treatment (10 µM) also overcame the inhibitory effect of p53 blockage on neurite outgrowth and that it was inhibited by PKI (Table 1). Thus, in contrast to the PKA-independent pathway used by A_2A-R stimulation, FK rescued p53 blockage via a PKA-dependent pathway.

Results described above suggest that A_2A-R stimulation might rescue p53 blockage through a previously uncharacterized pathway. Studies of other GPCRs have demonstrated that in addition to G proteins, GPCRs might transmit their signals via specific interacting proteins that bind to the C terminus and/or other cytosolic domains of a GPCR (Xia et al., 2003; Nickols et al., 2004). Because the C-terminal domain of the A_2A-R is relatively long and might exert novel function(s) (Gsandtner et al., 2005; Milojevic et al., 2006), we set out to determine whether the C terminus of the A_2A-R is involved in its rescue effect. A truncated A_2A-R mutant (A_2A-R_253-410), comprising the seventh transmembrane domain and the entire C terminus of the A_2A-R, was generated. As shown in Fig. 1E, expression of A_2A-R_253-410 reduced the rescue effect of A_2A-R, supporting our hypothesis that the A_2A-R might exert its rescue effect through direct interaction with a novel protein at its C terminus. Note that expression of A_2A-R_253-410 itself moderately reduced the extent of neurite outgrowth in the presence of CGS plus NGF. The C terminus of the A_2A-R might therefore contribute to its modulatory effect on NGF-induced neurite outgrowth as reported previously (Cheng et al., 2002).

View larger version (33K): [in this window] [in a new window]   Fig. 3. The A2A-R interacted with TRAX at its C terminal domain. A and B, TRAX bound the striatal A2A-R in GST pull-down assays. Striatum membrane fractions (0.4 mg) were solubilized using Nonidet P-40 (1%) and incubated with GST (1 mg) or GST-TRAX (1 mg) for 60 min at 37°C. The interacting proteins were then pulled-down using 50 µl of glutathione-Sepharose beads. The blots were probed with an anti-A2A-R antibody which recognized the C terminus of the A2A-R (A2A-Rc; Lee et al., 2003; A) and the A2A-R preadsorbed with the peptide antigen (A2A-Rabs;B)as indicated. C-E, the A2A-R bound TRAX in HEK293 cells. HEK 293T cells were transfected with A2A-R-V5 cDNA along with HA-TRAX cDNA (C) or AMPK cDNA (E) for 72 h. The A2A-R-V5 and associated proteins were immunoprecipitated using the V5 antibody and analyzed for the presence of HA-TRAX or an irrelevant protein (AMPK) by Western blotting with an anti-HA or anti-AMPK antibody, respectively. Whole cell lysates (50 µg) were run as input controls as indicated. D, the amount of TRAX bound to the A2A-R in the absence or presence of CGS21680 (CGS) was quantified and expressed as percentages of TRAX binding in the absence of CGS. Data were generated by quantitative computed densitometry of autoradiograms from three to five independent experiments using the image analysis software package ImageQuant v.3.15 (GE Healthcare). Values represent the mean ± S.E.M. Statistical significance was evaluated by Student's t test. IP, immunoprecipitation; IB, immunoblot. The asterisk (*) indicates a nonspecific band of HEK293 cells recognized by the anti-HA antibody.

To further demonstrate the interaction between the A_2A-R and TRAX, recombinant GST and GST-TRAX proteins were purified using glutathione-Sepharose beads and were incubated with the membrane extraction of the rat striatum, which contains high levels of the endogenous A_2A-R (Lee et al., 2003). As shown in Fig. 3A, GST-TRAX, but not GST, successfully pulled down the striatal A_2A-R. The addition of excess peptide antigen (amino acids 394410 of the A_2A-R) conjugated to an irrelevant protein (ovalbumin) resulted in the complete disappearance of the immunoreactive bands (Fig. 3B), confirming the specificity of the observed A_2A-R protein. The veracity of interaction was also confirmed by coimmunoprecipitation assays in HEK293 cells transfected with the A_2A-R (A_2A-R-_V5) and TRAX (_HA-TRAX) cDNAs. Immunoprecipitation of the A_2A-R protein using the V5 antibody pulled down TRAX (Fig. 3C) but not an irrelevant protein (AMPK; Fig. 3E). The transfected _HA-TRAX appeared to be slightly larger (Fig. 3C) than the endogenous TRAX (33 kDa; Fig. 4A) as a result of the presence of an HA tag at its N terminus and the 20-amino acid linker region between the HA tag and the TRAX cDNA. Colocalization of the A_2A-R-_V5 and _HA-TRAX could also be detected (Supplemental Materials, Fig. S2). Activation of the A_2A-R using CGS for 2 h did not alter the amount of TRAX coimmunoprecipitated with the A_2A-R (Fig. 3, C and D), suggesting that interactions between the A_2A-R and TRAX were independent of the receptor's activity.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Colocalization of the A2A-R and TRAX in vivo. A, characterization of the anti-TRAX antibody. An equal amount (50 µg) of cytosolic protein from the mouse cerebellum was loaded into each lane for Western blot analysis using the anti-TRAX antibody. The arrow indicates the TRAX band. The addition of the antigen peptide completely removed the immunoreactive band. B, double immunostaining of the A2A-R and TRAX in various regions of the brain. TRAX was visualized by the Alexa Fluor 488 conjugated secondary antibody (green; left). The A2A-R was visualized by streptavidin Alexa Fluor 568 (middle). Merged images are shown in the right. Scale bars, 6 µm.

In PC-12 cells, colocalization of the A_2A-R and TRAX was also evident in the presence of a p53 mutant (R273H-p53; Fig. 5A). Most importantly, transient expression of TRAX rescued the NGF-evoked neurite outgrowth impaired by p53 inhibition (Fig. 5B). Stimulation of the A_2A-R by CGS in TRAX-overexpressing cells did not further enhance NGF-induced neurite outgrowth, suggesting that A_2A-R stimulation and TRAX might use the same pathway to rescue defective neurite outgrowth. To verify whether the rescue effect of the A_2A-R on neurite outgrowth was mediated by TRAX, an antisense construct of TRAX (designated TRAX_AS) was created. Transient transfection of the antisense construct into PC-12 cells significantly diminished the expression of endogenous TRAX and markedly reduced the rescue effect of A_2A-R (Fig. 5C). Note that down-regulation of TRAX did not alter NGF-induced neurite outgrowth. TRAX is thus involved in the action of the A_2A-R but not that of NGF.

View larger version (26K): [in this window] [in a new window]   Fig. 5. TRAX mediated the rescue effect of the A2A-R on damaged NGF-induced neurite outgrowth caused by p53 blockage. A, PC-12 cells were transfected with constructs encoding A2A-R-V5, TRAX, and p53-R273H for 3 days. The A2A-R-V5 was visualized using streptavidin Alexa Fluor 568 (red). TRAX was visualized using the FITC secondary antibody (green). Arrows indicate the positions of colocalization. B, cells were transfected with the indicated plasmid(s) along with a GFP vector. C, cells were transfected with an antisense construct of TRAX (TRAXAS) along with a GFP vector. One day after transfection, cells were treated with NGF (100 ng/ml) or NGF plus CGS (1 µM) for 3 days. Neurite-bearing cells were then quantified. Data represent the mean ± S.E.M. values from at least three independent experiments. b, p < 0.001 compared with the corresponding non-CGS-treated sample (two-way ANOVA). e, specific comparison between cells transfected with the indicated p53 mutant or the control vector (p < 0.001; two-way ANOVA). g, specific comparison between cells transfected with the corresponding TRAX construct or the control vector (p < 0.001; two-way ANOVA). The insert is a representative picture which demonstrates the expression levels of TRAX (upper) and the internal control (actin, lower) in PC-12 cells transfected with an antisense construct of TRAX (TRAXAS) or an empty vector (V) by Western blot analysis.

View larger version (14K): [in this window] [in a new window]   Fig. 6. A2A-R stimulation restored NGF's ability to suppress proliferation in the presence of R273H-p53 ([3H]thymidine incorporation assay). PC-12 cells were transfected with an expression construct of R273H-p53 or an empty vector plus an expression vector of hrGFP. Twenty-four hours after transfection, cells were sorted by the expression of hrGFP and stimulated with the indicated reagent(s) for 3 days. [3H]Thymidine incorporation was performed as described under Materials and Methods. Values represent the mean ± S.E.M. from five to seven independent experiments and are expressed as percentages of [3H]thymidine incorporation of the control, nontreated cells in each group (17,909 ± 2933 cpm/1 x 104 cells for cells transfected with an empty vector and 12,738 ± 3260 cpm/1 x 104 cells for cells transfected with an expression construct of R273H-p53). Statistical significance was evaluated by one-way analysis of variance (ANOVA). a, specific comparison between cells transfected with an empty vector or R273H-p53 (p < 0.05). b, specific comparison between cells treated with NGF alone or NGF plus CGS (p < 0.05).

We next performed the BrdU incorporation assay to verify whether modulation of proliferation was involved in the rescue effect of A_2A-R. As shown in Fig. 7 and Table 3, blocking the p53-mediated pathway using a p53 mutant (R273H-p53) reversed NGF-reduced DNA synthesis. Overexpression of TRAX caused a reduction in BrdU incorporation in a p53-independent manner. Furthermore, down-regulation of endogenous TRAX by overexpression of an antisense TRAX construct abolished the decrease in DNA synthesis mediated by A_2A-R activation but not by NGF treatment in either the absence or presence of p53 blockage. TRAX therefore seems to mediate the A_2A-R rescue effect by suppressing proliferation in a p53-independent manner.

View larger version (16K): [in this window] [in a new window]   Fig. 7. A2A-R stimulation restored the ability of NGF to suppress proliferation in the presence of R273Hp53 (BrdU incorporation analysis). PC-12 cells were transiently transfected with a control or a p53-R273H vector along with a GFP reporter. One day after transfection, cells were treated with NGF (100 ng/ml) or NGF plus CGS (1 µM) for 4 days. Cells were then labeled with BrdU. Transfected cells were identified by GFP expression visualized using the FITC secondary antibody (green). BrdU incorporation was visualized using streptavidin Alexa Fluor 568 (red). Arrows indicate transfected cells positive for BrdU incorporation. To precisely visualize the BrdU incorporation in the nucleus, pictures were taken with the focus set at the mid-nuclear levels. Neurite outgrowth appeared at the bottom layer of the cells and, therefore, largely cannot be seen in the pictures. Note that approximately 40% of the NGF-treated and R273H-p53-expressing cells exhibited enlarged cell bodies (marked by arrowheads) and short neurites (with diameters of less than two cell bodies) which were not considered neurite-bearing cells in our study. Representative images from three independent experiments are shown.

It has been suggested that the antimitotic activity of NGF is required for differentiation (Mark and Storm, 1997). The above findings demonstrate that A_2A-R stimulation and TRAX overexpression might cause cessation of proliferation in PC-12 cells. We next examined whether A_2A-R activation might turn a mitogenic factor (e.g., EGF) into a differentiating factor through inhibiting proliferation. In PC-12 cells, both NGF and EGF stimulation led to a similar signal transduction pathway (Morooka and Nishida, 1998). Nevertheless, unlike NGF, EGF promoted PC-12 proliferation without evident morphological alterations (Huff et al., 1981; Maher, 1988). As shown in Fig. 8A, in combination with EGF, both an A_2A-R-selective agonist (CGS) and FK induced neurite outgrowth. It is noteworthy that down-regulation of TRAX using an antisense approach suppressed the neurite-inducing effect of CGS but not that of FK. In addition, overexpression of TRAX induced neurite outgrowth (Fig. 8B) and suppressed DNA synthesis (Fig. 8C) in the presence of EGF. These results support our hypothesis that stimulation of the A_2A-R causes a reduction in proliferation and subsequently enables EGF to trigger neuronal differentiation through a TRAX-dependent pathway.

View larger version (15K): [in this window] [in a new window]   Fig. 8. A2A-R stimulation enabled a mitogenic factor, EGF, to induce neurite outgrowth and to suppress DNA synthesis through a TRAX-dependent pathway. PC-12 cells were transfected with either an empty vector, an antisense construct of TRAX (TRAXAS), or a TRAX expression construct, along with a GFP vector. One day after transfection, cells were treated with normal growth medium containing no additive, CGS (1 µM), or FK (10 µM) in the presence or absence of EGF (10 ng/ml) for 4 days as indicated. (A and B) Data are expressed as the percentage of neuritebearing cells, and represent the mean values from three different fields. C, cells were labeled with BrdU. Data (mean ± S.E.M.) are expressed as the percentage of transfected cells incorporating BrdU from three different fields. Transfected cells were identified by GFP expression. Three independent experiments showed similar results. b, specific comparison between cells treated with or without CGS (p < 0.05; two-way ANOVA). c, specific comparison between cells treated with or without FK (p < 0.05; two-way ANOVA). e, specific comparison between cells transfected with a control construct or a TRAX expression construct (p < 0.05; two-way ANOVA).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Many studies have shown that GPCR-interacting molecules participate in the desensitization, trafficking, targeting, signaling, fine-tuning, and allosteric regulation of GPCRs (Heuss and Gerber, 2000; Brady and Limbird, 2002; Presland, 2004). Ample evidence suggests that these interacting molecules have diverse functions and affect the action of GPCRs through direct interaction. Compared with other GPCRs, the C terminus of the A_2A-R is relatively long. An actin-binding protein was previously shown to interact with the C terminus of the A_2A-R and to contribute to the rapid desensitization process (Burgueno et al., 2003). A de-ubiquitination enzyme (Usp4) was shown to interact with the A_2A-R. This interaction allows the A_2A-R to remain in the de-ubiquitinated state and to enhance the receptor's expression on the cell surface (Milojevic et al., 2006). In addition, the C terminus of the A_2A-R binds to a nucleotide exchange factor for ARF6 (ARNO) which mediates the sustained, G protein-independent activation of mitogen-activated protein kinase by the A_2A-R (Gsandtner et al., 2005). This is of particular interest because ARNO and ARF6 have been implicated in axonal elongation and branching (Hernandez-Deviez et al., 2004). These findings, together with our data presented herein, suggest that the C terminus of the A_2A-R seems to form complexes with multiple proteins (designated A_2A-R signalosome) and to mediate G protein-independent actions of the A_2A-R. We therefore cannot exclude the possibility that, in addition to hijacking TRAX from its association with the A_2A-R, expression of the A_2A-R C terminus might also interfere with other A_2A-R-interacting proteins (such as ARNO) that contribute to the the rescue effect of A_2A-R (Fig. 1E). Nevertheless, multiple lines of evidence clearly demonstrate that TRAX plays a crucial role in the rescue effect of A_2A-R. First, overexpression of TRAX rescued the reduced neurite outgrowth caused by p53 blockage as did A_2A-R stimulation (Fig. 5B). Second, down-regulation of endogenous TRAX suppressed the ability of A_2A-R to rescue the p53 blockage (Fig. 5C). TRAX therefore seemed to mediate the rescue effect of A_2A-R by inhibiting the proliferation of PC-12 cells (Table 3; Figs. 6, 7, 8).

Accumulating evidence suggests that NGF evokes neuronal differentiation in PC-12 cells through multiple processes, including the MAPK cascade and the p53/p21 pathway (Pang et al., 1995; Hughes et al., 2000). Blocking either pathway hinders NGF-induced differentiation. Damage occurring at the MAPK cascade or the p53/p21 pathway with NGF treatment can be rescued by A_2A-R stimulation through distinct mechanisms. When the NGF-evoked MAPK pathway is blocked, A_2A-R stimulation rescues the NGF-induced neurite outgrowth via a PKA/cAMP response element-binding protein-dependent pathway (Cheng et al., 2002). In contrast, when the NGF-induced p53 pathway is suppressed, damaged neurite outgrowth is compensated for by A_2A-R stimulation through a PKA-independent pathway (Fig. 9). Expression of a dominant-negative MAPK mutant (Seth et al., 1992; Cheng et al., 2002) did not jeopardize the ability of the A_2A-R to rescue the blockage of neurite outgrowth caused by p53 damage (data not shown), suggesting that MAPK is unlikely to be involved. Because long-term CGS treatment led to a sustained but low level of cAMP elevation (Fig. 2), the role of cAMP is therefore interesting. Using various kinase inhibitors, we demonstrated that PKA is not involved (Table 1). Another important cAMP effector, Epac (Bos, 2003), has recently been shown to convert a PKA-mediated proliferative signal into a differentiation signal in PC-12 cells (Kiermayer et al., 2005). In addition, activation of the A_2A-R during hypoxia or by a prokaryotic nucleoside has been shown to induce neurite outgrowth via a cAMP-dependent pathway (Charles et al., 2003; O'Driscoll and Gorman, 2005). Although TRAX seems to play the major role in mediating the rescue effect of A_2A-R on p53 blockage (Fig. 5; Table 3), we cannot completely rule out the potential involvement of Epac. In the future, it will be of great interest to assess whether Epac is involved in the TRAX-mediated suppression of proliferation by A_2A-R stimulation.

View larger version (16K): [in this window] [in a new window]   Fig. 9. Schematic representation of signaling pathways that mediate the action of A2A-R action in rescuing NGF-induced neurite outgrowth abolished by blockage of the p53-mediated proliferation suppression.

TRAX was originally identified as a Translin-interacting protein (Aoki et al., 1997). Binding with TRAX greatly reduces the ability of Translin to bind to RNA but not to DNA (Chennathukuzhi et al., 2001). Both TRAX and Translin have been found in centrosomes and are believed to play critical roles in cell cycle controls (Castro et al., 2000; Ishida et al., 2002). Indeed, down-regulation of TRAX using the siRNA approach was shown to reduce the growth rate in HeLa cells (Yang et al., 2004), suggesting that TRAX plays a central role in proliferation. We were surprised to find that overexpression of TRAX in PC-12 caused a reduction in proliferation (Table 3). Cell-type specificity might have contributed to such a discrepancy. Recent studies have indicated that TRAX might work in concert with other interacting molecules. For example, TRAX interacts specifically with the nuclear matrix protein, C1D, an activator of DNA-dependent protein kinase (Erdemir et al., 2002) and an upstream activator of p53 (Rothbarth et al., 1999). In addition, four other interacting proteins of TRAX (i.e., snaxip1, MEA-2, Akap9, and Sun-1) that are located in the cytoplasmic fractions have been reported (Bray and Hecht, 2002). In the present study, we identified the A_2A-R, a membrane protein, as a novel TRAX-interacting protein. The mechanism employed by TRAX to suppress proliferation upon A_2A-R activation is currently unclear. Because p21 is a major downstream target of p53 for cell cycle regulation, we performed experiments to determine whether the rescue effect of A_2A-R/TRAX is mediated by p21. In agreement with a previous report (Yan and Ziff, 1995), NGF treatment elevated p21 (Supplemental Materials, Fig. S6). Blocking p53 signaling using R273H-p53 markedly reduced the p21 expression level under all conditions tested. The ability of the A_2A-R to regulate proliferation thus seems to be independent of the p53/p21 pathway. Results of a recent study employing Translin-null mice suggest that post-transcriptional stabilization is important for the function of TRAX in proliferation (Yang et al., 2004). Because a de-ubiquitination enzyme exists in the A_2A-R signalosome and TRAX is a ubiquitinated protein (Yang et al., 2004; Milojevic et al., 2006), it will be of great interest to examine whether A_2A-R stimulation regulates the ubiquitination/expression of TRAX and subsequently affects proliferation. Another interesting aspect is that Translin and TRAX were found to bind to a nonprotein-coding RNA (BC1) and to form ribonucleoprotein particles (Muramatsu et al., 1998). These ribonucleoprotein particles are translocated to neuronal dendrites and might play a modulating role in local translation within dendrites in response to activity-dependent regulation (Kobayashi et al., 1998). The interaction between TRAX and the A_2A-R might thus provide a novel link that allows a membrane protein to transmit signals to the nucleus or to the translational machinery upon extracellular stimuli.

Accumulating evidence suggests that p53 is critical for embryonic development. Mice expressing no p53 exhibit a high frequency of developmental abnormalities, including early embryonic death, neural tube defects, and craniofacial malformations (Armstrong et al., 1995). Moreover, p53 has been implicated in neuronal apoptosis caused by various stresses (Herzog et al., 1998; Jordan et al., 2003) and is believed to contribute to the synaptic dysfunction of neurodegenerative diseases (Gilman et al., 2003; Jordan et al., 2003). Suppression of the p53-mediated pathway has been reported under various conditions. For example, in Huntington's disease, mutant Huntingtin aggregates recruit p53 and subsequently alter the activities of the p53-regulated promoters p21 and MDR-1 (Steffan et al., 2000; Bae et al., 2005). Furthermore, addictive drugs were found to elevate the expression of murine double minute clone 2 (MDM2), a negative regulator of p53, suggesting the involvement of p53/Mdm2 in the development of drug addition (Jiang et al., 2003). Most importantly, germline and somatic mutations that inactivate the transactivating function of p53 have been well documented in a wide spectrum of tumor types (Malkin, 2001). Results of the present study suggest that stimulation of the A_2A-R might rescue the function of neurotrophins through TRAX upon p53 inactivation. Another intriguing finding reported herein is that A_2A-R stimulation converted a mitogenic factor (EGF) into a differentiating factor via suppression of proliferation in a TRAX-dependent manner (Fig. 7; Table 3). Note that expression of the A_2A-R is found in many areas of the brain (Lee et al., 2003) and has previously been implicated in neuronal development as a result of its dynamic expression during embryogenesis (Weaver, 1993). The role of the A_2A-R in modulating and compensating neuronal functions in vivo therefore might be more important than previously recognized.

In summary, we provide evidence to demonstrate that the C terminus of the A_2A-R interacts with TRAX to suppress proliferation in PC-12 cells. These findings add additional dimensions to the multiple signaling pathways used by GPCRs to transmit signals and modulate functions. Results of the present study also provide important insights into the cross-talk between trophic factors (differentiating or mitogenic factors) and the A_2A-R in neuronal trauma and neurological diseases.

	Acknowledgements

 
We are grateful to Dr. F. F. Wang (Department of Biochemistry, Yang Ming University, Taipei, Taiwan) for the dominant-negative p53 mutants and Dr. Sheau-Yann Shieh (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan) for valuable discussion. We also thank Dr. Pauline Yen, Dan Chamberlin, and Christine Shieh for editing the manuscript, and Yi-Chih Wu and Hua-an Tseng for construct preparations.

	Footnotes

 
This work was supported by grants from the National Science Council (NSC93-2320-B-001-009 and NSC94-2320-B-001-030) and Academia Sinica, Taipei, Taiwan.

C.-N.S., H.-C.C., and J.C. contributed equally to this work.

ABBREVIATIONS: NGF, nerve growth factor; A_2A-R, A_2A adenosine receptor; GPCR, G protein-coupled receptor; PKA, protein kinase A; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; TRAX, translin-associated protein X; FK, forskolin; CGS, CGS21680 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine; CSC, 8-(3-chlorostyryl) caffeine; CHE, chelerythrine; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HS, horse serum; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; HEK, human embryonic kidney; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PB, phosphate buffer; PAGE, polyacrylamide gel electrophoresis; BrdU, bromodeoxyuridine; BIM, bisindolylmaleimide I; AMPK, AMP-activated protein kinase; HA, hemagglutinin; ARNO, ADP-ribosylation factor nucleotide site opener; EGF, epidermal growth factor; ANOVA, analysis of variance; EGFP, enhanced green fluorescent protein; hrGFP, Vitality humanized GFP; PKI, protein kinase inhibitor.

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Yijuang Chern, Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529, Taiwan. E-mail: bmychern{at}ibms.sinica.edu.tw

	References

Top Abstract Materials and Methods Results Discussion References

 
Aoki K, Ishida R, and Kasai M (1997) Isolation and characterization of a cDNA encoding a Translin-like protein, TRAX. FEBS Lett 401: 109-112.[CrossRef][Medline]

Aoki K, Suzuki K, Sugano T, Tasaka T, Nakahara K, Kuge O, Omori A, and Kasai M (1995) A novel gene, Translin, encodes a recombination hotspot binding protein associated with chromosomal translocations. Nat Genet 10: 167-174.[Medline]

Armstrong JF, Kaufman MH, Harrison DJ, and Clarke AR (1995) High-frequency developmental abnormalities in p53-deficient mice. Curr Biol 5: 931-936.[CrossRef][Medline]

Bae BI, Xu H, Igarashi S, Fujimuro M, Agrawal N, Taya Y, Hayward SD, Moran TH, Montell C, Ross CA, et al. (2005) p53 mediates cellular dysfunction and behavioral abnormalities in Huntington's disease. Neuron 47: 29-41.[CrossRef][Medline]

Bargonetti J, Reynisdottir I, Friedman PN, and Prives C (1992) Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53. Genes Dev 6: 1886-1898.[Abstract/Free Full Text]

Bos JL (2003) Epac: a new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol 4: 733-738.[CrossRef][Medline]

Brady AE and Limbird LE (2002) G protein-coupled receptor interacting proteins: emerging roles in localization and signal transduction. Cell Signal 14: 297-309.[CrossRef][Medline]

Bray JDCV and Hecht NB (2002) Identification and characterization of cDNAs encoding four novel proteins that interact with translin associated factor-X. Genomics 79: 799-808.[CrossRef][Medline]

Burgueno J, Blake DJ, Benson MA, Tinsley CL, Esapa CT, Canela EI, Penela P, Mallol J, Mayor F Jr, Lluis C, et al. (2003) The adenosine A2A receptor interacts with the actin-binding protein -actinin. J Biol Chem 278: 37545-37552.[Abstract/Free Full Text]

Castro A, Peter M, Magnaghi-Jaulin L, Vigneron S, Loyaux D, Lorca T, and Labbe JC (2000) Part of Xenopus translin is localized in the centrosomes during mitosis. Biochem Biophys Res Commun 276: 515-523.[CrossRef][Medline]

Chang YH, Conti M, Lee YC, Lai HL, Ching YH, and Chern Y (1997) Activation of phosphodiesterase IV during desensitization of the A2A adenosine receptormediated cyclic AMP response in rat pheochromocytoma (PC12) cells. J Neurochem 69: 1300-1309.[Medline]

Charles MP, Adamski D, Kholler B, Pelletier L, Berger F, and Wion D (2003) Induction of neurite outgrowth in PC12 cells by the bacterial nucleoside N6-methyldeoxyadenosine is mediated through adenosine A2a receptors and via cAMP and MAPK signaling pathways. Biochem Biophys Res Commun 304: 795-800.[CrossRef][Medline]

Cheng HC, Shih HM, and Chern Y (2002) Essential role of cAMP-response elementbinding protein activation by A2A adenosine receptors in rescuing the nerve growth factor-induced neurite outgrowth impaired by blockage of the MAPK cascade. J Biol Chem 277: 33930-33942.[Abstract/Free Full Text]

Chennathukuzhi VM, Kurihara Y, Bray JD, and Hecht NB (2001) Trax (translinassociated factor X), a primarily cytoplasmic protein, inhibits the binding of TB-RBP (translin) to RNA. J Biol Chem 276: 13256-13263.[Abstract/Free Full Text]

Chern Y, Lai HL, Fong JC, and Liang Y (1993) Multiple mechanisms for desensitization of A2a adenosine receptor-mediated cAMP elevation in rat pheochromocytoma PC12 cells. Mol Pharmacol 44: 950-958.[Abstract]

Daval JL, Nehlig A, and Nicolas F (1991) Physiological and pharmacological properties of adenosine: therapeutic implications. Life Sci 49: 1435-1453.[CrossRef][Medline]

Eizenberg O, Faber-Elman A, Gottlieb E, Oren M, Rotter V, and Schwartz M (1996) p53 plays a regulatory role in differentiation and apoptosis of central nervous system-associated cells. Mol Cell Biol 16: 5178-5185.[Abstract]

el-Deiry WS, Harper JW, O'Connor PM, Velculescu VE, Canman CE, Jackman J, Pietenpol JA, Burrell M, Hill DE, and Wang Y (1994) WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 54: 1169-1174.[Abstract/Free Full Text]

Erdemir T, Bilican B, Oncel D, Goding CR, and Yavuzer U (2002) DNA damagedependent interaction of the nuclear matrix protein C1D with Translin-associated factor X (TRAX). J Cell Sci 115: 207-216.[Abstract/Free Full Text]

Erhardt JA and Pittman RN (1998) Ectopic p21(WAF1) expression induces differentiation-specific cell cycle changes in PC12 cells characteristic of nerve growth factor treatment. J Biol Chem 273: 23517-23523.[Abstract/Free Full Text]

Finkenstadt PM, Kang WS, Jeon M, Taira E, Tang W, and Baraban JM (2000) Somatodendritic localization of Translin, a component of the Translin/Trax RNA binding complex. J Neurochem 75: 1754-1762.[CrossRef][Medline]

Gilman CP, Chan SL, Guo Z, Zhu X, Greig N, and Mattson MP (2003) p53 is present in synapses where it mediates mitochondrial dysfunction and synaptic degeneration in response to DNA damage and oxidative and excitotoxic insults. Neuromolecular Med 3: 159-172.[CrossRef][Medline]

Ginty DD, Glowacka D, DeFranco C, and Wagner JA (1991) Nerve growth factor-induced neuronal differentiation after dominant repression of both type I and type II cAMP-dependent protein kinase activities. J Biol Chem 266: 15325-15333.[Abstract/Free Full Text]

Gsandtner I, Charalambous C, Stefan E, Ogris E, Freissmuth M, and Zezula J (2005) Heterotrimeric G protein-independent signaling of a G protein-coupled receptor. Direct binding of ARNO/cytohesin-2 to the carboxyl terminus of the A2A adenosine receptor is necessary for sustained activation of the ERK/MAP kinase pathway. J Biol Chem 280: 31898-31905.[Abstract/Free Full Text]

Han JR, Yiu GK, and Hecht NB (1995) Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules. Proc Natl Acad Sci USA 92: 9550-9554.[Abstract/Free Full Text]

Hernandez-Deviez DJ, Roth MG, Casanova JE, and Wilson JM (2004) ARNO and ARF6 regulate axonal elongation and branching through downstream activation of phosphatidylinositol 4-phosphate 5-kinase alpha. Mol Biol Cell 15: 111-120.[Abstract/Free Full Text]

Herzog KH, Chong MJ, Kapsetaki M, Morgan JI, and McKinnon PJ (1998) Requirement for Atm in ionizing radiation-induced cell death in the developing central nervous system. Science (Wash DC) 280: 1089-1091.[Abstract/Free Full Text]

Heuss C and Gerber U (2000) G-protein-independent signaling by G-protein-coupled receptors. Trends Neurosci 23: 469-475.[CrossRef][Medline]

Hosaka T, Kanoe H, Nakayama T, Murakami H, Yamamoto H, Nakamata T, Tsuboyama T, Oka M, Kasai M, Sasaki MS, et al. (2000) Translin binds to the sequences adjacent to the breakpoints of the TLS and CHOP genes in liposarcomas with translocation t(12;6). Oncogene 19: 5821-5825.[CrossRef][Medline]

Huang NK, Lin YW, Huang CL, Messing RO, and Chern Y (2001) Activation of protein kinase A and atypical protein kinase C by A_2A adenosine receptors antagonizes apoptosis due to serum deprivation in PC12 cells. J Biol Chem 276: 13838-13846.[Abstract/Free Full Text]

Huff K, End D, and Guroff G (1981) Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor. J Cell Biol 88: 189-198.[Abstract/Free Full Text]

Hughes AL, Gollapudi L, Sladek TL, and Neet KE (2000) Mediation of nerve growth factor-driven cell cycle arrest in PC12 cells by p53. Simultaneous differentiation and proliferation subsequent to p53 functional inactivation. J Biol Chem 275: 37829-37837.[Abstract/Free Full Text]

Ishida R, Okado H, Sato H, Shionoiri C, Aoki K, and Kasai M (2002) A role for the octameric ring protein, Translin, in mitotic cell division. FEBS Lett 525: 105-110.[CrossRef][Medline]

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