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Proc Natl Acad Sci U S A. Dec 27, 2005; 102(52): 19138–19143.
Published online Dec 19, 2005. doi:  10.1073/pnas.0505913102
PMCID: PMC1323158
Neuroscience

P2Y2 receptor activates nerve growth factor/TrkA signaling to enhance neuronal differentiation

Abstract

Neurotrophins are essential for neuronal differentiation, but the onset and the intensity of neurotrophin signaling within the neuronal microenvironment are poorly understood. We tested the hypothesis that extracellular nucleotides and their cognate receptors regulate neurotrophin-mediated differentiation. We found that 5′-O-(3-thio)triphosphate (ATPγS) activation of the G protein-coupled receptor P2Y2 in the presence of nerve growth factor leads to the colocalization and association of tyrosine receptor kinase A and P2Y2 receptors and is required for enhanced neuronal differentiation. Consistent with these effects, ATPγS promotes phosphorylation of tyrosine receptor kinase A, early response kinase 1/2, and p38, thereby enhancing sensitivity to nerve growth factor and accelerating neurite formation in both PC12 cells and dorsal root ganglion neurons. Genetic or small interfering RNA depletion of P2Y2 receptors abolished the ATPγS-mediated increase in neuronal differentiation. Moreover, in vivo injection of ATPγS into the sciatic nerve increased growth-associated protein-43 (GAP-43), a marker for axonal growth, in wild-type but not P2Y2-/- mice. The interactions of tyrosine kinase- and P2Y2-signaling pathways provide a paradigm for the regulation of neuronal differentiation and suggest a role for P2Y2 as a morphogen receptor that potentiates neurotrophin signaling in neuronal development and regeneration.

Keywords: neurotrophin signaling, P2Y2, tyrosine receptor kinase A

Neurite formation, a process extending from the cell soma and led by a growth cone, is a primary morphological event in neuronal differentiation, ultimately facilitating synaptic connections by neurons (1). Neuronal regeneration depends on the formation of neurites to repair injured or lost connections. These neuronal growth events require appropriate spatial and temporal expression and action of both initiation signals and promoter molecules (2). Neurotrophins, such as nerve growth factor (NGF), are key regulators of neuronal differentiation. NGF is released by target tissues, initiates neurite generation, maintains neuronal survival, prevents apoptosis, and promotes synapse formation (3-5). NGF signaling via the tyrosine receptor kinase A (TrkA) leads to stimulation of early response kinase 1/2 (ERK1/2) via Ras/Raf pathways, which is required for differentiation, as well as the activation of the survival kinase, Akt (6). The regulation of NGF/TrkA signaling is determined by availability of NGF, as well as TrkA activation, characterized by receptor autophosphorylation, internalization, and retrograde transport from axons to cell bodies (7). Recent evidence has shown that receptor crosstalk is an important mechanism for regulation of neurotrophin signaling (6). The NGF/TrkA-signaling pathway converges with pain-related ion channels to regulate NGF-mediated heat sensitivity of sensory neurons (5) and with adenosine A2A receptors to regulate neuronal survival (8). The contributions of other signal transduction pathways integrated with NGF/TrkA signaling to modulate neuronal differentiation remain largely unknown.

It has been suggested that extracellular nucleotides may influence neuronal differentiation, migration, and survival (9), such as enhancement of NGF-dependent neurite outgrowth from PC12 cells (10). Nucleotides in the nervous system are stored in, and released from, synaptic vesicles and glial cells at mM concentrations, thereby affecting neurotransmitter release and glial calcium wave propagation, respectively (11, 12). ATP has been proposed as an activity-dependent signaling molecule that regulates glial-glial and glial-neuron communication (12, 13).

Extracellular nucleotide signaling occurs via purinergic receptor 2 (P2) ionotropic (P2X) and metabotropic (P2Y) G protein-coupled receptors (GPCRs). Seven P2X receptors (P2X1 to -7) and eight P2Y (P2Y1, -2, -4, -6, and -11 to -14) receptors, each with unique agonist response profiles, have been identified (14). Both P2Y and P2X receptors are expressed in the nervous system. P2X receptors regulate synaptic transmission (15), pain (16), and respiration (17). The role of P2Y receptors in the nervous system is less well characterized, but they can couple to neuronal ion channels (18) and modulate pain responses (19). The contribution of individual P2 receptor subtypes to neuronal functions and their interaction with signal transduction pathways in the nervous system remain largely unknown. In this study, we used several complementary approaches to assess the role of P2Y receptors in neuronal differentiation. The results, which indicate an important contribution for P2Y2 receptors in this differentiation via interaction between the TrkA and P2Y2 signaling pathways, define mechanisms for merging the interaction of neurotrophins and extracellular nucleotides.

Materials and Methods

Reagents. Reagents were acquired as follows: ATPγS, AMPCP, and ARL67156 (Sigma); NGF (Invitrogen); K252a (Calbiochem); and U0126 (Cell Signaling Technology, Beverly, MA).

Morphology. PC12 cells were plated at 60% confluency on collagen/poly-d-lysine-coated plates and incubated with 100 ng/ml NGF with other treatments for 72 h. ATPγS, AMPCP, and ARL67156 were added once for a 3-day interval at 100 μM, 250 μM, and 50 μM, respectively. K252a (10 nM) and U0126 (10 μM) were applied once. Images were taken every 24 h for 3 days by using an Axiocam charge-coupled device (CCD) camera on an Axiophot Zeiss microscope. The fractions of PC12 cells expressing a neurite that was at least one cell body in length were counted. All conditions were assessed in triplicate.

Immunoblot Analysis. Protein samples, loaded at equal concentrations, were separated on 10% or 12% precast SDS polyacrylamide gels (Invitrogen) and then transferred to poly(vinylidene difluoride) membranes. Membranes were blocked in 20 mM PBS, 1% Tween 20 with 1.5% nonfat dry milk and then incubated with primary antibody at 4°C overnight. Antibodies used were as follows: phosphorylated (P)-TrkA, P-ERK1/2, ERK1/2, P-p38, protein 38 (p38), Myc, and HA (Cell Signaling Technology); P2Y2 and P2Y4 (Alomone Labs, Jerusalem, Israel); actin and TrkA (Santa Cruz Biotechnology); GAPDH (Novus Biologicals, Littleton, CO); and rowth-associated protein-43 (GAP-43) (Chemicon, Temecula, CA). Secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) were visualized with ECL reagent (Amersham Pharmacia).

Immunoprecipitation. PC12 and dorsal root ganglion (DRG) cells were treated with the indicated pharmacological/neurotrophic agents for 1 h. Cell lysate was precleared with protein G agarose beads (Roche, Indianapolis) for 1 h and incubated with the primary immunoprecipitating antibody for 3 h, and protein G agarose bead was added for another 3 h. The beads were isolated and washed, and the immunoprecipitated protein was used in immunoblot analysis.

Immunofluorescence Analysis. Cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton, and incubated with P2Y2 (Alomone Labs), P-TrkA 496 (Santa Cruz Biotechnology), and SMI31 (Sternberger Monoclonals, Lutherville, MD) (1:100) antibodies overnight at 4°C. Cells were incubated with FITC or Cy3-conjugated antibody (1:250; Jackson Immunoresearch) for 2 h. Confocal images of cells were taken at ×40 on a Nikon E600FN microscope with a Radiance 2000 (Bio-Rad) confocal. Cells were imaged in a Z-series stack in 0.5 μM slices [colocalization measurements were made by using imagej software (NIH)].

Immunostaining. Sciatic nerves were immunostained as described (20).

PC12 Cell Transfection. PC12 cells were transfected with pcDNA hemagglutinin (HA)-tagged TrkA (gift of M. Chao), pLXSN Myc-tagged P2Y2 (gift of S. Wolff and T. K. Harden) constructs, and with predesigned small interfering RNA (siRNA) (Ambion, Austin TX; ID no. 50110, 143692) for P2Y2 with Lipofectamine 2000 (Invitrogen) per the manufacturer's instructions.

Cell Isolation and Culture. DRG neurons were dissected and trypsin dissociated from P1 WT or P2Y2-/- mice (21) (gift of B. Koller). Cultures were grown on laminin/poly-d-lysine/collagen-coated eight-chambered slides for 24 h in MEM with 10% heat-inactivated FBS, l-glutamine, and glucose in the presence of 50 ng/ml NGF. PC12 cells were grown as described by Taupenot et al. (22).

Sciatic Nerve Injection. Sciatic nerves were injected as described (23, 24). The left sciatic nerves of P1 mice were injected with 6 μg of ATPγS in 6 μl of PBS, 2 μg NGF in PBS, or the combination. The right sciatic nerve of each mouse served as a control and was injected with 6 μl of PBS. Results of three independent experiments of three animals per condition are shown.

Statistical Analysis. Data were analyzed by a paired t test, one-way analysis of variance followed by Tukey's multiple comparison test or linear regression. Significance was assigned to P < 0.05. For quantitative RT-PCR, an 8-fold change was used as a significant response between treatments. Neurite length and branch point measurements were performed in a blinded fashion by two independent observers.

Results

Genetic or siRNA Depletion of P2Y2 Receptors Eliminates ATPγS-Enhanced Neurite Formation and Signaling in DRG Neurons and PC12 Cells. Neurite formation is a hallmark event in neuronal differentiation, neurodevelopment and regeneration after injury (25, 26). ATPγS enhances neuronal differentiation in PC12 cells (see ref. 27 and Fig. 6 A-D, along with Figs. 7-9, which are published as supporting information on the PNAS web site). A general P2 inhibitor (suramin) but not a P2X inhibitor (PPADS) suppressed ATPγS-enhanced NGF-mediated neuronal differentiation in PC12 cells (Fig. 7A). Because selective antagonists do not exist for P2Y2 receptors, we used both a mouse knockout and siRNA to deplete P2Y2 and examine its contribution in ATPγS-mediated enhancement of neuronal differentiation.

DRG neurons express both TrkA (28) and P2 receptors (29), and neurite formation in DRG neurons is regulated by TrkA (30). Because neonatal DRG neurons undergo apoptosis in the absence of NGF, neurite growth experiments were conducted in the presence of the neurotrophin (31). To assess the signaling effects of ATPγS independent of NGF, we used DRG neurons derived from 4-week old mice, which do not require NGF for survival (32).

DRG neurons from WT and P2Y2-/- mice were immunostained for activated TrkA (P-Trk) and P2Y2 receptors to assess their colocalization along with the neurofilament marker SMI31 after 24 h growth with either NGF alone or NGF with ATPγS. P-Trk (red) and P2Y2 (green) colocalized at the SMI31-positive cell bodies and neurites (blue) in WT DRG neurons in both treatment groups (Fig. 1 A and B, white). Neurites expressed P-Trk along the neurite processes and in the cell body, whereas P2Y2 expression was greater in the soma, colocalizing with P-Trk (Fig. 1 A and B). P2Y2-/- DRG neurons stained positive for SMI31 (blue) and P-Trk (red) but not P2Y2 receptor (Fig. 1 C and D, purple). There was no measurable difference between numbers of TrkA-expressing DRG neurons in WT versus P2Y2-/- mice (data not shown), suggesting the changes in P-Trk expression do not result from a decrease in TrkA-positive neurons. ATPγS in the presence of NGF enhanced neurite growth in control DRG neurons (Fig. 1B), when compared with DRG neurons treated with NGF alone (Fig. 1A).

Fig. 1.
Neurite growth, P2Y2-TrkA colocalization, and signaling in DRG neurons. Shown are dissociated DRG neurons from WT and P2Y2-/- P1 mice grown for 24 h with 50 ng/ml NGF alone (A and C, respectively), or 100 μM ATPγS and 50 ng/ml NGF (B and ...

DRG neurons from P2Y2-/- mice did not show changes in neuronal differentiation when treated with ATPγS in the presence of NGF compared with NGF treatment alone (Fig. 1 C and D). Quantification demonstrated a 3-fold increase (P < 0.001) in neurite length (Fig. 1E) and 5-fold increase in neurite branching (P < 0.001) (Fig. 1F) in WT DRG but no change (P > 0.1) in P2Y2-/- DRG neurite length (Fig. 1E) and branching (Fig. 1F). We obtained similar results in 4-week-old DRG cells from WT and P2Y2-/- mice (data not shown), thus demonstrating that enhancement by ATPγS of NGF-induced neuronal differentiation requires P2Y2 receptors.

Immunoblot analysis of murine DRG neurons also demonstrated the requirement of P2Y2 receptors for enhanced NGF signaling. ATPγS-mediated P-ERK1/2 formation and increase in NGF-promoted P-Trk occurred in DRG neurons from WT but not P2Y2-/- mice (Fig. 1G). Phosphorylation of p38 was similar in WT and P2Y2-/- DRG neurons (Fig. 1G). Abolition of ATPγS-mediated signaling changes in the P2Y2-/- DRG neurons is in accord with results from siRNA P2Y2 receptor depletion in PC12 cells (cf. Fig. 2 B-D vs. 1 A-G). Use of two distinct P2Y2 receptor siRNAs in PC12 cells decreased P2Y2 receptor mRNA (Fig. 2A) and significantly (P < 0.001) reduced P2Y2 receptor protein compared with cells treated with empty vector (Fig. 2B). No change in P2Y4 occurred in either treatment group. Reduced expression of P2Y2 abolished the enhanced P-TrkA formation in response to ATPγS in the presence of NGF and the increase in P-ERK1/2 in response to ATPγS alone (Fig. 2B) and inhibited ATPγS-promoted enhancement of NGF-mediated neurite formation (Fig. 2C). Thus, use of P2Y2-knockout animals and P2Y2 receptor siRNA with PC12 cells both demonstrate that P2Y2 is the nucleotide receptor that activates TrkA phosphorylation and signaling to enhance neuronal differentiation.

Fig. 2.
siRNA reduction of P2Y2 eliminates ATPγS-enhancement of NGF-induced neurite formation and signaling. (A) Quantitative RT-PCR of PC12 cells independently treated with two nonoverlapping siRNAs (seq.1 orange, seq.2 orange checkered) against the ...

Inhibitors of TrkA (K252a) and ERK1/2 (U0126) can abolish their downstream signaling effects (33, 34). To assess involvement of TrkA and ERK1/2 in the enhancement by ATPγS of NGF-promoted neurite formation, we treated PC12 cells with either K252a or U0126; K252a abolished neurite formation by NGF alone or together with ATPγS whereas U0126 blocked enhancement of NGF-promoted neurite formation by ATPγS (Fig. 2C). K252a did not inhibit the enhanced P-ERK1/2 formation by ATPγS independent of NGF (data not shown). The results showing enhanced neurite formation by ATPγS are consistent with findings demonstrating mitogen-activated protein kinase-dependent enhancement of neurite outgrowth from PC12 cells (35) and further support the notion that ATPγS mediates enhanced NGF-promoted neurite formation through increased activation of both TrkA and ERK1/2.

P2Y-Specific Receptor Agonists Enhance Neurite Formation in PC12 Cells Through Increased NGF Sensitivity. Extracellular ATP is degraded to ADP, AMP, and ultimately adenosine by endogenous ecto-nucleotidases and 5′ nucleotidases expressed on the surface of many cell types, including PC12 cells (36, 37). The ability of ATPγS to enhance neurite formation in the presence of NGF led us to test whether nucleotides sensitize PC12 cells to NGF. As shown in Fig. 2D,ATPγS (100 μM) significantly (P < 0.01) increased the fraction of neurite-bearing PC12 cells and left-shifted the NGF concentration-response curve, indicating sensitization to NGF by ATPγS. PC12 cells express multiple ecto-nucleotidases that hydrolyze ATP and UTP, and 5′nucleotidases that generate adenosine from AMP and uridine from UMP (36). Inhibition of ecto-nucleotidase activity by ARL67156 (ecto-nucleotidase inhibitor) and AMPCP (5′nucleotidase inhibitor) (the combination did not by themselves cause neurite formation or mimic ATP action on NGF response, data not shown), to prevent the hydrolysis of ATP and UTP (38, 39) significantly (P < 0.05) left-shifted the NGF concentration-response curve (Fig. 2D).

P2Y2 and TrkA Receptors Coimmunoprecipitate in PC12 and DRG Cells. Because the data suggested P2Y2-TrkA signaling pathway interaction in the regulation of neuronal differentiation, we examined the cellular localization of P2Y2 and TrkA as well as their physical interaction by coimmunoprecipitation. In accord with the data from immunoblot studies (Fig. 6E), we detected low levels of P-TrkA in the absence of NGF (Fig. 3 A and B). Addition of NGF caused colocalization of the P-TrkA (red) with P2Y2 receptors (green) (Fig. 3 C and D, yellow). NGF-treated cells (Fig. 3 C and D) had 4-fold greater fluorescence intensity of individually labeled receptors compared with untreated or ATPγS-treated cells, consistent with enhanced P-TrkA and P2Y2 expression. Cells treated with NGF together with ATPγS had 76% colocalization whereas untreated, ATPγS-treated, and NGF-treated cells demonstrated 3.2%, 14%, and 52% colocalization, respectively. By contrast, P2Y12 receptors and P-TrkA did not colocalize (data not shown).

Fig. 3.
P-TrkA and P2Y2 receptors coimmunoprecipitate upon NGF induction. Shown are confocal immunofluorescence images of PC12 cells incubated with P-TrkA (red) and P2Y2 (green) antibodies. The merged images show colocalization (yellow). Shown are PC12 cells ...

To further assess the interaction of the P2Y2 and TrkA receptors, we transfected PC12 cells with Myc-tagged P2Y2 and HA-tagged TrkA expression vectors. Immunoblot analysis of coimmunoprecipitated proteins demonstrated that the TrkA and P2Y2 receptors physically interact when activated by ATPγS, NGF, or the combination (Fig. 3E). Moreover, immunoprecipitation of untransfected PC12 cell lysates using a phospho-specific TrkA antibody (to assess colocalization of the endogenous proteins) revealed that NGF-activated TrkA (P-TrkA) interacts with P2Y2, an interaction that was increased by ATPγS and abolished by treatment with K252a (Fig. 3F). Coimmunoprecipitation of P2Y2 and TrkA receptors from P1 rat DRG neurons treated with NGF alone or together with ATPγS (1h) also showed physical interaction of these receptors (Fig. 3G).

ATPγS Increases GAP-43 Expression in Sciatic Nerves. To test whether P2Y2 activation increases a marker of neuronal growth in vivo, we treated neonatal P2Y2-/- and WT mouse sciatic nerves [which express TrkA, (40)], with NGF, ATPγS, or the combination. After 48 h, we assessed expression of GAP-43, a marker of neuronal growth, extension, and plasticity (41, 42). Untreated sciatic nerve from WT (Fig. 4A) or P2Y2-/- mice (Fig. 4C) showed similar basal levels of GAP-43 (green) although P2Y2-/- mice lacked expression of P2Y2 receptor (Fig. 4E). Injection of ATPγS induced an increase in GAP-43 in sciatic nerves of WT (Fig. 4B) but not P2Y2-/- mice (Fig. 4D). Sciatic nerves from WT treated with ATPγS had a 1.8-fold increase of GAP-43 compared with controls; treatment with ATPγS in combination with NGF further increased GAP-43 expression (Fig. 4E). No changes were observed with treatment of sciatic nerves from P2Y2-/- mice (Fig. 4E). GAP-43 immunostaining of ATPγS-injected WT sciatic nerves showed an increase in GAP-43 expression as early as 12 h postinjection, which was sustained for 24 h (Fig. 8A). Bielschowski silver axon staining did not reveal morphological differences of control versus treated sciatic nerve axons (Fig. 8B). Western blots demonstrated ERK1/2-dependent up-regulation of GAP-43 expression by ATPγS in DRG neurons, an effect blocked by the ERK kinase inhibitor U0126 (Fig. 8C). Overall, these results suggest that P2Y2 regulates expression of GAP-43 in the sciatic nerve.

Fig. 4.
ATPγS increases GAP-43 and NGF increases P2Y2 levels in vivo. Shown are untreated (A and C) or ATPγS-injected (B and D) sciatic nerves stained for SMI31 (blue) and GAP-43 (green). (E) Sciatic nerves probed for indicated protein markers. ...

Discussion

The current data provide evidence from a variety of complementary approaches for interaction of NGF and extracellular nucleotides acting at P2Y2 receptors in the enhancement of neuronal differentiation in PC12 cells, DRG neurons, and sciatic nerve. The TrkA and P2Y2 receptors coimmunoprecipitated in cells stimulated with NGF or ATPγS, indicating a physical association regulated by receptor activation. In vivo treatment of P2Y2-expressing (but not P2Y2-null) sciatic nerves with ATPγS increased levels of GAP-43, a marker for neuronal growth and differentiation (43).

A model for the interaction between NGF/TrkA and P2Y2 signaling (Fig. 5) begins with TrkA activation, which enhances its physical association with, and protein levels of, P2Y2 receptors. The convergence of NGF/TrkA with ATP/P2Y2 signaling results in increased ERK1/2 activation by P-TrkA, leading to increased neurite formation. Activation of P2Y2 receptors increases P-ERK1/2 formation independent of NGF, although the increase is not sufficient to induce neurite formation in the absence of neurotrophin signaling (Figs. (Figs.2C2C and 6F). Overall, our results suggest that enhancement of P-TrkA formation by P2Y2 activation is a crucial interaction between NGF/TrkA and P2Y2 receptor signaling. It is intriguing that, during embryonic development, both TrkA (44) and P2Y2 (45) are expressed by sensory spinal ganglia. Such findings, together with the current data, implicate P2Y2-NGF/TrkA interaction in neuronal development in vivo.

Fig. 5.
Model of interaction of P2Y2 with TrkA receptors. Activation of TrkA by NGF (P-TrkA) leads to ERK1/2 (P-ERK1/2) activation and up-regulation of the P2Y2 receptor. UTP or ATP activates the P2Y2 receptor, which increases P-TrkA levels in the presence of ...

Release of nucleotides occurs in both neurons and glia and can modulate neurotransmission (46, 47), but little is known regarding the role of released nucleotides in the developing nervous system. Our data suggest that the enhanced NGF-mediated neuronal differentiation may represent a physiological role for extracellular nucleotide signaling, a proposal consistent with results we obtained from inhibition of nucleotidases (Fig. 9). Autocrine/paracrine action of released nucleotides thus may modulate neurotrophin-promoted neuronal differentiation during development. Expression of mRNA for multiple P2Y receptors, including P2Y2, has been detected in DRG neurons (29); in situ hybridization demonstrates P2Y2 mRNA in 77% of such neurons in vivo (19). Furthermore, P2Y receptor transcripts are ubiquitously found in human central and peripheral nervous tissue samples (48); P2Y and certain P2X receptors are likely to be important physiologically as well as in neurological injury and disease (49).

We hypothesize that released nucleotides function as neuronal morphogens, akin to the action of another extracellular acting nucleotide, cAMP, in Dictyostelium (50). If one were to equate nucleotide release as the molecular “gas pedal,” then the enzymes that hydrolyze these molecules would be the “brake.” Multiple ecto-nucleotidases have been identified on neurons and glia during development (51, 52). The temporal and spatial abundance of nucleotidases have the potential to influence extracellular nucleotide signaling and, as suggested here, neurite formation and neuronal differentiation.

Multiple types of neurons and cells in the nervous system express P2Y receptors. Although we assessed functions of the P2Y2 receptor in vitro and in vivo, the roles of other P2 receptors in vivo cannot be discounted. PC12 cells express six of the eight known P2Y receptors (Fig. 7 B-E), results that confirm and extend previous findings (27). Only P2Y (and not P2X) receptors respond to UTP, e.g., P2Y2 (ATP = UTP) and P2Y4 (UTP) (53). The enhancement in NGF-promoted neurite expression (Fig. 9 B-D) by ATP and UTP, together with the siRNA and knockout data (Figs. (Figs.11 and and2),2), implicates P2Y2 receptors as a primary mediator of this effect. P2Y2 receptors mediate excitation of DRG neurons, perhaps in the activity-dependent regulation of pain (19). Interestingly, TrkA knockout mice show extensive loss of DRG neurons and fail to respond to painful stimuli (28). Our data showing colocalization and coimmunoprecipitation of P2Y2 and TrkA in DRG neurons provide evidence of physical interaction between these receptors, suggesting involvement of P2Y2 and TrkA in neurite extension, neuropathic pain, and nociception.

Nucleotide release occurs after injury, which, via P2Y receptor activation, may modulate axonal regeneration (54). Our study shows P2Y2/TrkA receptor interaction in nociceptive (TrkA) neurons (28). Additional tyrosine kinase receptors, TrkB and TrkC, are expressed by motor and proprioceptive neurons (55), which may also crosstalk with P2Y2 receptors. Future work will address these interactions and their roles in nerve injury and regeneration.

Pharmacological inhibition of P-TrkA (by K252a) abolished neurite formation and colocalization of P-TrkA with P2Y2, whereas partial inhibition of P-ERK1/2 (by U0126) blunted the enhancement of neurite formation by ATPγS without inhibiting ATPγS-enhanced increased in P-TrkA in the presence of NGF. These results identify two independent activities of the P2Y2 receptor for enhancing neurotrophin-mediated differentiation: (i) increase in level of activated neurotrophin receptor and (ii) increased activation of ERK1/2. In undifferentiated PC12 cells, P2Y2 receptors increase intracellular calcium levels (56), which may activate PKC, Ras, and Raf, thereby activating ERK1/2 (57). Activation of Raf by P2Y2 receptors and TrkA could represent a convergence point for the two signaling pathways.

Our study provides in vivo evidence for involvement of GPCRs in neuronal differentiation. In previous work, PACAP (via its GPCR) and adenosine (via A2A, also a GPCR) enhanced neuronal survival independent of NGF (58). Here we show that ATPγS (via P2Y2 receptors) enhances NGF-dependent neuronal differentiation and neurite extension. The interaction of P2Y2 signaling on neurotrophin-mediated action identifies a mechanism for enhanced neuronal differentiation by extracellular nucleotides. Although our work emphasizes neuronal differentiation, selective P2Y2 receptor agonists might have potential as pharmacological agents to aid in neuronal regeneration after injury or disease.

Supplementary Material

Supporting Figures:

Acknowledgments

We thank Rik Bundey [University of California at San Diego (UCSD)] for assistance with the QRT-PCR, Laurent Taupenot (UCSD) for PC12 cells, Sam Wolff and Ken Harden [University of North Carolina (UNC), Chapel Hill] and Moses Chao (New York University) for P2Y2 and TrkA receptor plasmids, and Beverly Koller and Wendy Zinzow (UNC) for the P2Y2-/- mouse. We thank Gary Laevsky (UCSD) for confocal imaging at the National Center for Microscopy and Imaging Research, San Diego, supported by National Institutes of Health Grant RR04050 (to Dr. Mark Ellisman). This work was supported by National Institute of General Medical Sciences Training Grant GM007240; by National Institute on Drug Abuse Training Grant DA07315-03; by a grant from the American Foundation for Aging Research (to D.B.A); by the Wadsworth Foundation; by UCSD Academic Senate Grant RE521H; by National Multiple Sclerosis Society Research Grant RG3370; by National Institutes of Health (NIH) Grants NS051470 and NS052189 (to K.A.); by NIH Grants GM66232 and HL58120; and by a UCSD Academic Senate Grant (to P.A.I.).

Notes

Author contributions: D.B.A., K.A., and P.A.I. designed research; D.B.A. and K.A. performed research; K.A. and P.A.I. contributed new reagents/analytic tools; D.B.A., K.A., and P.A.I. analyzed data; and D.B.A., K.A., and P.A.I. wrote the paper.

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: ATPγS, adenosine 5′-O-(3-thio)triphosphate; DRG, dorsal root ganglion; ERK1/2, early response kinase 1/2; GPCR, G protein coupled receptor; GAP-43, growth-associated protein-43; P, phosphorylated; p38, protein 38; P2, purinergic receptor 2; TrkA, tyrosine receptor kinase A; NGF, nerve growth factor; siRNA, small interfering RNA.

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