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 (ATPgammaS) activation of the G protein-coupled receptor P2Y(2) in the presence of nerve growth factor leads to the colocalization and association of tyrosine receptor kinase A and P2Y(2) receptors and is required for enhanced neuronal differentiation. Consistent with these effects, ATPgammaS 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 P2Y(2) receptors abolished the ATPgammaS-mediated increase in neuronal differentiation. Moreover, in vivo injection of ATPgammaS into the sciatic nerve increased growth-associated protein-43 (GAP-43), a marker for axonal growth, in wild-type but not P2Y(2)(-/-) mice. The interactions of tyrosine kinase- and P2Y(2)-signaling pathways provide a paradigm for the regulation of neuronal differentiation and suggest a role for P2Y(2) as a morphogen receptor that potentiates neurotrophin signaling in neuronal development and regeneration.

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 D, respectively) and labeled with SMI31 (blue), and antibodies for P-Trk (red) and P2Y₂ receptor (green) (A). Triple-localization appears white. Shown are neurite length (E) and branch point (F) measurements. (G) Immunoblots of WT and P2Y₂^-/- DRG neurons treated as indicated for 10 min. (Scale bar: 30 μm.)

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 P2Y₂ receptor or a scrambled sequence. (B) Immunoblots of receptor and signaling proteins in PC12 cells treated with P2Y₂ siRNA (seq.1/seq.2) or scrambled sequence. (C) Fraction of PC12 cells expressing neurites treated with P2Y₂ siRNA (seq.1 solid, seq.2 checkered), K252a, U0126 for 72 h or a scrambled sequence and the indicated treatments. (D) PC12 cells treated with 100 μM ATPγS at the indicated NGF concentrations.

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 untreated (A), treated with 100 μM ATPγS(B), treated with 100 ng/ml NGF (C), or treated with the combination (D) for 1 h.(E) Transfected PC12 cells treated for 1 h with indicated treatments, lysated, and immunoprecipitated. (F) Native PC12 cells treated for 1 h with indicated treatments. Shown are K252a pretreated 15 min before agonist treatment and cell lysates immunoprecipitated. (G) Shown are P1 rat DRG neurons treated for 1 h with indicated treatments and cell lysates immunoprecipitated.

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. Shown are densitometries calculated as fold increase over un-injected contra-lateral sciatic nerve from within each treatment group. (Scale bar: 100 μm.)

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 NGF, leading to increased neurite formation and growth.