J Biol Chem, Vol. 274, Issue 50, 35499-35504, December 10, 1999

Adenosine A_2A Receptor mRNA Regulation by Nerve Growth Factor Is TrkA-, Src-, and Ras-dependent via Extracellular Regulated Kinase and Stress-activated Protein Kinase/c-Jun NH₂-terminal Kinase^*

Renae L. Malek, Zhongzhen Nie^§, Vickram Ramkumar^§, and Norman H. Lee^¶

From the  Department of Molecular and Cellular Biology, The Institute for Genomic Research, Rockville, Maryland 20850 and the ^§ Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois 62794

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We have shown previously that nerve growth factor (NGF) down-regulates adenosine A_2A receptor (A_2AAR) mRNA in PC12 cells. To define cellular mechanisms that modulate A_2AAR expression, A_2AAR mRNA and protein levels were examined in three PC12 sublines: i) PC12nnr5 cells, which lack the high affinity NGF receptor TrkA, ii) srcDN2 cells, which overexpress kinase-defective Src, and iii) 17.26 cells, which overexpress a dominant-inhibitory Ras. In the absence of functional TrkA, Src, or Ras, NGF-induced down-regulation of A_2AAR mRNA and protein was significantly impaired. However, regulation of A_2AAR expression was reconstituted in PC12nnr5 cells stably transfected with TrkA. Whereas NGF stimulated the mitogen-activated protein kinases p38, extracellular regulated kinase 1 and 2 (ERK1/ERK2), and stress-activated protein kinase/c-Jun NH₂-terminal kinase (SAPK/JNK) in PC12 cells, these kinases were activated only partially or not at all in srcDN2 and 17.26 cells. Inhibiting ERK1/ERK2 with PD98059 or inhibiting SAPK/JNK by transfecting cells with a dominant-negative SAPK/JNK3 mutant partially blocked NGF-induced down-regulation of A_2AAR expression in PC12 cells. In contrast, inhibiting p38 with SB203580 had no effect on the regulation of A_2AAR mRNA and protein levels. Treating SAPK/JNK3 mutant-transfected PC12 cells with PD98059 completely abolished the NGF-induced decrease in A_2AAR mRNA and protein levels. These results reveal a role for ERK1/ERK2 and SAPK/JNK in regulating A_2AAR expression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Adenosine receptors are G-protein coupled receptors that mediate important physiological processes in both the central and peripheral nervous system, including vasodilation, respiratory depression, wakefulness, and spontaneous locomotor activity. There are four major adenosine receptor subtypes, A₁, A_2A, A_2B, and A₃; each is encoded by a distinct gene, and each has unique affinities for adenosine analogs and methylxanthine derivatives (1-3). In the developing rat brain, adenosine A_2A receptor (A_2AAR)¹ mRNA is expressed transiently in various regions (4). Moreover, a severalfold increase in A_2AAR protein levels occurs during early postnatal development in a number of brain regions, whereas a decrease in A_2AAR mRNA is observed in other regions (5).

PC12 cells, derived from a rat pheochromocytoma, have been used extensively as a model for neuronal differentiation and development (6). In response to NGF, these cells differentiate into sympathetic-like neurons and extend neurites (6). The signal transduction pathways activated by NGF originate at both high (TrkA) and low (p75) affinity receptors, and downstream targets of each receptor have been implicated in regulating expression of genes involved in differentiation, neurotransmission, and neuronal function (6-13). Stimulation of the receptor tyrosine kinase TrkA results in the activation of Ras, Src, phospholipase C-, SNT, and phosphoinositide 3-OH kinase (14-17). In PC12 cells, active Ras triggers a cascade of phosphorylation events leading to activation of ERK1/ERK2 via Raf-1 (18-20) or p38 MAP kinase and stress-activated protein kinase/c-Jun NH₂-terminal kinase (SAPK/JNK) via MAP kinase kinase kinase (21-23). p75 activation increases ceramide production (24) and activates NFB (25).

Gene products regulated by NGF in PC12 cells include several G-protein coupled receptors, such as the M₄ muscarinic, secretin, and adenosine A_2A receptors (7, 26, 12). Using gene expression profiling (expressed sequence tags) coupled with Northern analysis, a decrease in A_2AAR mRNA could be demonstrated as early as 3 days (3 d) posttreatment with NGF and levels remained depressed for up to 12 d (7). In situ hybridization with an A_2AAR oligonucleotide probe detected a 50% decrease in the number of grains per cell in NGF-differentiated PC12 cells, confirming that NGF decreases A_2AAR mRNA levels (26). Corresponding to the changes in mRNA levels, immunoreactive A_2AAR protein declines by more than half after 7 d of NGF treatment, and the number of binding sites for the A_2AAR selective antagonist, [³H]SCH 58261, decreases by 3-fold (26). When PC12 cells are treated with A_2AAR agonists, a transient down-regulation of A_2AAR mRNA and protein occurs (27). Despite these observations, the specific cellular mechanisms regulating A_2AAR mRNA levels have not been thoroughly delineated. In this study, we provide the first insights into the downstream pathways employed by NGF to control A_2AAR expression in PC12 cells. Such pathways may likewise play an important role in the regulation of A_2AAR expression during brain development.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cell Culture-- Rat pheochromocytoma cells (PC12) were obtained from the American Type Tissue Culture collection (Manassas, VA). PC12nnr5, clone 106, and srcDN2 cells were a generous gift from Gordon Guroff at NICHD, National Institutes of Health (Bethesda, MD). The dominant-negative Ras cell line, 17.26, was obtained from Robert Maue at Dartmouth Medical Center (Hanover, NH). PC12 cell lines were maintained on rat tail collagen, Type IV (Upstate Biotechnology, Saranac Lake, NY) as described previously (7). SrcDN2, 17.26, and clone 106 were cultured in the presence of 300 µg/ml Geneticin (Life Technologies, Inc.). Cells were treated with PD98059 or SB203580 (Calbiochem, San Diego, CA) and mouse 2.5S NGF (Promega, Madison, WI) as described below.

Northern Blot Analysis-- Poly(A⁺) RNA was isolated, fractionated through a denaturing agarose gel, and transferred to Hybond N⁺ membranes (NEN Life Science Products) essentially as described previously (12). A ³²P-labeled 2.3-kilobase pair SstI/XhoI fragment from a rat A_2A cDNA clone and a 1.2-kilobase pair EcoRI/XhoI fragment from a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were used as probes (7). Expression levels of A_2AAR mRNA were normalized to GAPDH mRNA levels. Blots were analyzed on a Molecular Dynamics PhosphorImager.

Data are expressed as the mean ± S.E. of n independent experiments.

Ribonuclease Protection Assay-- At the times indicated, total RNA was isolated from PC12nnr5 cells. An RNase protection assay was performed essentially as described by Lee et al. (28). Construction of plasmid 118GAPDHpSP73 for generating an antisense riboprobe of the GAPDH mRNA was described previously (12). Plasmid 296A2ApSP72 for generating an antisense riboprobe template of the A_2AAR mRNA was constructed by subcloning an EcoRI/PvuII restriction fragment of 296 nucleotides into pSP72 (Promega). Riboprobes transcribed from EcoRI-linearized 296A2ApSP72 correspond to nucleotides 596-891 of the A_2AAR cDNA clone in pBluescript (Stratagene, La Jolla, CA) (7). Authenticity of the plasmid construct was verified by dideoxy sequencing.

Transfection of PC12 Cells with SAPK(K55A)-- PC12 cells were plated onto 100-mm dishes, grown to 70-80% confluency, and transiently transfected with 5 µg of SAPK(K55A) in expression vector pCMV5 (29) or an empty expression vector with LipofectAMINE 2000 (Life Technologies, Inc.). At 18-24 h after transfection, cells were treated with PD98059 and/or NGF, and they were harvested for total RNA at the indicated times.

SDS-PAGE and Immunoblotting-- PC12 cells were plated onto 35-mm collagen-coated tissue culture dishes at 90% confluency and incubated 24 h. The medium was then replaced with Dulbecco's modified Eagle's medium lacking serum, and the cells were incubated for an additional 24 h. Serum starved monolayers were washed with cold phosphate-buffered saline, and cells were harvested/lysed in 95 °C SDS-PAGE sample buffer containing 50 mM Tris, pH 6.8, 10% glycerol, 0.1% bromphenol blue, 2% SDS, 0.7 M -mercaptoethanol, 50 mM sodium fluoride, 2 mM sodium orthovanadate, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 10.8 µg/ml aprotinin, and 10 µg/ml leupeptin. Lysates were placed on ice during sonication and reheated to 95 °C for 5 min. Proteins were separated on a 7.5% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech). Membranes were processed for Western analysis as described by the New England Biolabs (Beverly, MA) protocol supplied with the antibodies. Phospho-p44/42 MAP kinase (ERK1/ERK2) antibody, phospho-p38 MAP kinase antibody, p44/42 MAP kinase antibody, SAPK/JNK antibody, and p38 MAP kinase antibody were from New England Biolabs. Phospho-JNK (G-7) monoclonal antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-TrkA (Ab-1) monoclonal antibody was from Calbiochem. Goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (Upstate Biotechnology) allowed detection of proteins by the ECL+Plus detection system (Amersham Pharmacia Biotech).

Immunocomplex Kinase Assay-- Treated cells were washed in ice-cold phosphate-buffered saline, lysed in immune precipitation buffer (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 0.5% (v/v) Nonidet P-40, 1% Triton X-100, 2 mM sodium orthovanadate, 20 µg/ml aprotinin, 5 µg/ml leupeptin, 50 mM sodium fluoride) and disrupted by aspiration through a 21 gauge needle. Cell debris was removed by centrifugation. Supernatants were incubated with anti-p38 antibody (C20) (Santa Cruz Biotechnology) for 2 h at 4 °C. Immunocomplexes were precipitated with immune precipitation buffer equilibrated protein A-agarose (Sigma) for 2 h at 4 °C, washed three times with immune precipitation buffer and twice with kinase buffer (25 mM HEPES, pH 7.5, 10 mM MgCl₂, 25 mM sodium -glycerophosphate, 2 mM sodium orthovanadate, 0.5 mM dithiothreitol) and resuspended in kinase buffer containing 100 µM ATP, 5 µCi of [-³²P]ATP and 2 µg of glutathione S-transferase-ATF-2 substrate. Reactions were incubated 30 min at 30 °C and terminated by the addition of 2× SDS-PAGE loading buffer. Proteins were separated on a 7.5% SDS-PAGE gel and analyzed by autoradiography.

Radioligand Binding-- Crude membrane preparations were obtained as described recently (30). ¹²⁵I-ZM241358, an A_2AAR-specific antagonist was used to measure specific binding to A_2AARs in crude membrane preparations. Data are expressed as the mean ± S.E. of n independent determinations.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

To assess the contribution of individual components of the NGF signal transduction pathway leading to regulation of A_2AAR mRNA and protein, PC12 sublines lacking functional TrkA or overexpressing dominant-inhibitory forms of Ras or Src were used. The role of the three MAP kinase family members p38, ERK1/ERK2, and SAPK/JNK in NGF-mediated regulation of A_2AAR mRNA was also determined.

TrkA Is Required for Down-regulation of A_2AAR mRNA and Protein-- Earlier experiments have concentrated on the effects of long term (7-12 days) NGF treatment on A_2AAR expression in PC12 cells (7, 26). In the present study, shorter periods of treatment were examined to define initial pathways responsible for NGF regulation. Steady state A_2AAR mRNA declined to 62 and 43% of untreated control cells following 1 and 3 d of NGF treatment, respectively (Fig. 1A). Correspondingly, binding of the A_2AAR antagonist ¹²⁵I-ZM241358 to PC12 cells decreased by 50 and 45% following 1 and 3 d of NGF treatment, respectively (Fig. 1B). Thus, NGF-induced down-regulation of A_2AAR mRNA and protein is apparent as early as 24 h following NGF treatment, with a further decrease in A_2AAR mRNA occurring at 3 d.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   TrkA is required for NGF-induced down-regulation of A2AAR mRNA and protein. A, bottom, quantitation of steady state A2AAR mRNA in PC12, PC12nnr5 (nnr5), and clone 106 (106) cells treated for 0, 1, and 3 d with 50 ng/ml NGF. Data are expressed as the mean ± S.E. of four to eight independent determinations. Top, left and right panels, representative Northern blot; center panels, representative ribonuclease protection assay. B, quantitation of the percentage of change in antagonist binding to PC12, PC12nnr5, and clone 106 cells treated as in A. Data are expressed as the mean ± S.E. of three to six independent determinations. 125I-ZM241358 binding in untreated PC12, PC12nnr5, and clone 106 was 339 ± 18, 70 ± 4, and 207 ± 19 fmol/mg, respectively.

The contribution of TrkA and p75 to the regulation of A_2AAR mRNA and protein were examined in PC12nnr5 cells, which are a PC12 subline that expresses p75, lacks functional TrkA receptors, and does not differentiate in response to NGF (31). As the basal steady-state level of A_2AAR mRNA is reduced in PC12nnr5 cells, a ribonuclease protection assay was performed to quantitate A_2AAR mRNA. As shown in Fig. 1, NGF failed to down-regulate both A_2AAR mRNA and protein levels. For comparison, the effects of NGF on A_2AAR expression were studied in clonal cell line 106. Clone 106, derived from PC12nnr5 cells stably transfected with TrkA, has levels of ¹²⁵I-NGF binding similar to those in native PC12 cells and differentiates in response to NGF (32). When cultures of clone 106 were treated with NGF for 1 or 3 d, both A_2AAR mRNA and protein levels were down-regulated to the same extent as native PC12 cells (Fig. 1). Because TrkA is implicated in A_2AAR mRNA and protein regulation, potential roles for TrkA-associated signaling components, Src and Ras, were examined.

NGF-mediated Down-regulation of A_2AAR mRNA and A_2AAR Protein Is Impaired by Dominant-negative Src-- As oncogenic Src mimics NGF-induced neurite outgrowth and phosphorylation of a similar set of cellular substrates, a role for Src in the signal transduction pathway initiated by NGF has been implicated (33). Therefore, the effects of NGF on A_2AAR mRNA and protein were examined in srcDN2 cells that overexpress a dominant-negative, kinase-defective Src mutant (34). Upon treatment of srcDN2 cells with NGF for 1 or 3 d, down-regulation of steady-state mRNA was not observed (92 and 100% of untreated cells, respectively) (Fig. 2). Likewise, A_2AAR protein levels remained near control levels following 1 and 3 d of NGF treatment (110 and 81%, respectively). Thus, Src appears to be critical for NGF-induced down-regulation of both A_2AAR mRNA and protein.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Dominant-inhibitory Src prevents down-regulation of A2AAR mRNA and protein by NGF. A, bottom, quantitation of steady state A2AAR mRNA in srcDN2 cells treated for 0, 1, and 3 d with 50 ng/ml NGF. Data are expressed as the mean ± S.E. of five independent determinations. Top, representative Northern blot. B, quantitation of percentage of change of antagonist binding to srcDN2 cells treated as in A. Data are expressed as the mean ± S.E. of three to five independent determinations. 125I-ZM241358 binding in untreated srcDN2 cells was 66 ± 21 fmol/mg.

Ras Mediates NGF-stimulated Down-regulation of A_2AAR mRNA and Protein-- PC12 cells undergo a Ras-dependent transient induction of several immediate-early genes within minutes of NGF treatment that precedes neurite outgrowth (35). The delayed response genes are transcriptionally active hours to days following NGF treatment (6, 7), and the induction of several genes, such as agrin, tau, transin, and SCG10, has been shown to be Ras-dependent (8, 10, 11). Furthermore, transcriptional down-regulation of the epidermal growth factor receptor requires Ras activity (36).

The data shown in Fig. 3 demonstrate that NGF can also mediate down-regulation of A_2AAR mRNA in a Ras-dependent manner. In 17.26 cells expressing a dominant-negative Ras mutant (35), steady state mRNA levels following 1 and 3 d of NGF treatment were 88 and 81% of untreated control cells, respectively, which is a less dramatic decrease than that seen in native PC12 (compare Figs. 1A and 3A). A similar impairment of NGF-induced down-regulation of protein was observed in 17.26 cells (compare Figs. 1B and 3B). The loss of NGF-mediated down-regulation of A_2AAR mRNA and protein in 17.26 and srcDN2 cells was not due to a loss of TrkA receptor levels. As determined by Western analysis using an anti-TrkA antibody, expression of TrkA protein in srcDN2 and 17.26 cells was comparable to that found in native PC12 cells (data not shown). In agreement with our findings, srcDN2, 17.26, and native PC12 cells exhibited similar levels of ¹²⁵I-NGF binding to TrkA (32). Taken together, these results indicate that both Src and Ras are necessary TrkA-signaling components that regulate A_2AAR mRNA and protein levels. Ras has multiple downstream effectors that activate divergent signaling pathways (reviewed in Ref. 37), such as Raf-1 and MAP kinase kinase kinase (22). The most thoroughly described Ras activated pathway is the Raf-dependent activation of ERK1/ERK2 (38, 39). More recently, Raf-independent Ras-activated MAP kinase pathways have been identified. For example, the Ras effector MAP kinase kinase kinase 1 activates SEK, which in turn activates SAPK/JNK and p38 (40-43). As such, the activity of p38, ERK1/ERK2, and SAPK/JNK was examined in PC12, srcDN2, and 17.26 cells.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Down-regulation of A2AAR mRNA and protein is blocked by dominant-negative Ras. A, bottom, quantitation of steady state A2AAR mRNA in 17.26 PC12 cells treated for 0, 1, and 3 d with 50 ng/ml NGF. Data are expressed as the mean ± S.E. of four to seven independent determinations. Top, representative Northern blot. B, quantitation of the percentage of change in antagonist binding to 17.26 PC12 cells treated as in A. Data are expressed as the mean ± S.E. of four or five independent determinations. 125I-ZM241358 binding in untreated 17.26 cells was 85 ± 20 fmol/mg.

MAP Kinase Family Members Are Activated in PC12 Cells but Not in Sublines Expressing Dominant-inhibitory Src or Ras Mutants-- NGF activation of p38, ERK1/ERK2, and SAPK/JNK was assessed with phosphorylation state-specific antibodies (Fig. 4). In native PC12 cells, NGF activated p38 and ERK1/ERK2 at early time points (15 and 30 min), whereas SAPK/JNK was not activated until 3 d. In agreement with previous reports, expression of dominant-negative Ras in 17.26 cells inhibited NGF activation of ERK1/ERK2 (18, 20) and SAPK/JNK (22), confirming that growth factor activation of these two MAP kinases is Ras-dependent. Likewise, activation of p38 above basal levels in NGF-treated 17.26 cells was not observed. As demonstrated previously (34), ERK1/ERK2 was activated in srcDN2 cells albeit to a slightly lesser extent than native PC12 cells. Moreover, NGF did not effectively activate p38 or SAPK/JNK in srcDN2 cells (Fig. 4). These findings demonstrate that Src is required for the full activation of the MAP kinases. As these kinases are distal components of the NGF signal transduction pathway, and their activation was impaired or inhibited by dominant-negative signaling components more proximal to TrkA, it is possible that one or more of these MAP kinase family members plays a role in mediating the effects of NGF on A_2AAR mRNA. Notably, a recent report demonstrated that two MAP kinase family members, JNK and ERK1/ERK2, had opposing effects on tau promoter activity and affected promoter activity over different time frames (10).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   NGF activation of MAP kinases in native PC12 cells and srcDN2 or 17.26 sublines. To assess activation of p38 MAP kinase and ERK1/ERK2, 24 h serum-starved PC12 (WT), srcDN2, and 17.26 cells were left untreated or incubated for 15 or 30 min with 100 ng/ml NGF. To assess SAPK/JNK activation, cells were untreated or treated with 100 ng/ml NGF for 1 or 3 d in Dulbecco's modified Eagle's medium containing 1% serum. Representative Western blot are shown from experiments that were repeated at least twice. Blots were probed with phosphorylation state-specific antibodies, stripped, and reprobed with an antibody that recognized the respective MAP kinase subfamily member, independent of phosphorylation state. Left, p38 MAP kinase; center, ERK1/ERK2; right, SAPK/JNK.

Role of ERK1/ERK2, p38, and SAPK/JNK in Regulating A_2AAR mRNA and Protein-- To examine potential roles of the individual MAP kinases in affecting down-regulation of A_2AAR mRNA by NGF, the synthetic compound SB203580 was used to inhibit p38 (44). Although p38 activation was impaired in srcDN2 and 17.26 cells, this kinase does not appear to be involved in A_2AAR mRNA regulation as SB203580 failed to inhibit NGF-induced down-regulation of A_2AAR mRNA in native PC12 cells (Fig. 5A). Similarly, another p38 inhibitor, SB202190, also failed to inhibit A_2AAR mRNA down-regulation (data not shown). Inhibition of p38 activity in PC12 cells was verified by an in vitro immunocomplex kinase assay. Whereas p38 from lysates of NGF-stimulated cells phosphorylated glutathione S-transferase-ATF-2 (4-fold above basal levels), p38 from NGF-stimulated cells pretreated with SB203580 did not appreciably phosphorylate its substrate (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   ERK1/ERK2 MAP kinase and SAPK/JNK are required for maximal down-regulation of A2AAR mRNA. PC12 cells were treated with 20 µM SB203580 (A) or 50 µM PD98059 (B) for 1 h prior to the addition of 50 ng/ml NGF for 1 or 3 d. C, PC12 cells were transiently transfected with SAPK(K55A) and incubated with 50 ng/ml NGF for 1 or 3 d. D, SAPK(K55A) transfectants were co-treated with 50 µM PD98059 and 50 ng/ml NGF for 3 d, whereas vector-only transfectants were treated with just NGF. A2AAR mRNA was quantitated by Northern analysis as described under "Experimental Procedures." Top panels (A-D), representative Northern blots. Data are expressed as the mean ± S.E. of at least three independent determinations. Vector-only transfections were repeated twice.

ERK1/ERK2 activity was inhibited by employing the MAP kinase kinase inhibitor PD98059 (45). Cells co-treated with NGF and PD98059 demonstrated significantly less down-regulation of A_2AAR mRNA than cells treated with NGF alone, suggesting that ERK1/ERK2 plays at least a partial role in controlling A_2AAR mRNA steady state levels (compare Figs. 5B and 1A). As reported previously (45), PC12 cells pretreated with PD98059 did not extend neurites following 3 d NGF as did cells treated with NGF alone (data not shown). Western analysis also confirmed that PD98059 inhibited ERK1/ERK2, but not p38 and SAPK/JNK, activity in PC12 cells treated with NGF (data not shown).

To inhibit SAPK/JNK, PC12 cells were transiently transfected with SAPK(K55A), a kinase-defective construct of SAPK/JNK3. Following 1 and 3 d of NGF treatment, PC12 cells expressing SAPK(K55A) did not extend neurites, whereas empty vector-transfected PC12 cells extended neurites to the same extent as nontransfected cells (data not shown). These findings are in agreement with studies demonstrating that differentiation of PC12 cells requires the SAPK/JNK signal transduction pathway (46). As shown in Fig. 5C, PC12 cells expressing SAPK(K55A) did not undergo NGF-induced down-regulation of A_2AAR mRNA following 1 d of treatment (102% of untreated cells). After 3 d of NGF incubation, SAPK(K55A) transfectants had a slight decrease in A_2AAR mRNA (79% of untreated cells) that was not as great as that of empty vector-transfected cells. When kinase-defective SAPK transfectants were co-incubated with PD98059 and NGF for 3 d, down-regulation of A_2AAR mRNA was completely blocked (Fig. 5D). In contrast, when cells transfected with an empty vector were treated for 3 d with NGF alone, A_2AAR mRNA was down-regulated to the same extent (42%) as wild-type PC12 cells (Fig. 1A). As inhibition of either ERK1/ERK2 or SAPK/JNK individually results in partial inhibition of A_2AAR mRNA regulation, these results indicate that ERK1/ERK2 and SAPK/JNK are both required for complete down-regulation of A_2AAR mRNA.

The capacity of NGF to down-regulate A_2AAR mRNA following inhibition of the different MAP kinases was mimicked at the protein level (Fig. 6). It will be of interest in the future to determine whether NGF utilizes other mechanisms (besides mRNA regulation) to down-regulate A_2AAR protein (e.g. ubiquitin-mediated protein degradation).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   ERK1/ERK2 MAP kinase and SAPK/JNK are required for maximal down-regulation of A2AAR protein. PC12 cells were treated with 20 µM SB203580 (SB) or 50 µM PD98059 (PD) for 1 h prior to the addition of 50 ng/ml NGF or saline vehicle for 3 d. PC12 cells were transiently transfected with the dominant-negative SAPK(K55A) mutant (K55A) and incubated with 50 ng/ml NGF or saline for 3 d. Alternatively, SAPK(K55A) transfectants were pretreated with 50 µM PD98059 for 1 h prior to addition of 50 ng/ml NGF or saline for 3 d. A2AAR protein was quantitated by radioligand binding assay as described under "Experimental Procedures." Data are expressed as the mean ± S.E. of three independent determinations. 125I-ZM241358 binding in untreated PC12 (control) was 285 ± 46 fmol/mg.

Concluding Remarks-- To summarize, the data presented here demonstrate that NGF-induced down-regulation of A_2AAR mRNA and protein levels is TrkA-, Src-, and Ras-dependent. Furthermore, the MAP kinase family members ERK1/ERK2 and SAPK/JNK are distal signal transduction components activated by NGF and are implicated here as having important roles in mediating regulation of A_2AAR mRNA. Recent reports have demonstrated a role for mitogen-activated protein kinase family members in regulating mRNA stability. NGF-induced stabilization of the M₄ muscarinic receptor mRNA requires ERK1/ERK2 (12) and p38 plays a role in stabilizing cyclooxygenase-2 mRNA (47, 48). As NGF destabilizes A_2AAR mRNA transcripts,² we are currently examining the role of ERK1/ERK2 and SAPK/JNK in NGF-mediated A_2AAR mRNA destabilization.

	ACKNOWLEDGEMENTS

We thank Dr. G. Guroff for the PC12nnr5, clone 106, and srcDN2 cells and Dr. R. Maue for the 17.26 cells. The SAPK(K55A) construct in pCMV5 was kindly provided by Dr. M. Cobb.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants NS352321 (to N. H. L.) and HL56316 (to V. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, The Institute for Genomic Research, 9712 Medical Center Dr., Rockville, MD 20850. Tel.: 301-838-3529; Fax: 301-838-0208; E-mail: nhlee@tigr.org.

² R. L. Malek and N. H. Lee, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: A_2AAR, adenosine A_2A receptor; NGF, nerve growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, mitogen-activated protein; SAPK/JNK, stress-activated protein kinase/c-Jun NH₂-terminal kinase; ZM241358, 4-(2-(7-amino-2-(2-fury)(1,2,4)triazolo(2,3-)(1,3,5)triazin-5-amino)ethyl)phenol; ERK, extracellular regulated kinase; d, day(s); PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES