Gs-coupled GPCR-mediated Activation of Rapgef2- and ERK-dependent Gene Regulation and Neuritogenesis in NS-1 Cells.

Gs-coupled GPCRs signal through the second messenger cAMP. In the neuroendocrine NS-1 cell line, cAMP acts through three parcellated signal pathways using distinct cAMP sensors as third messenger: protein kinase A (PKA) is linked to neuroendocrine-specific gene expression, the exchange protein (EPAC) to cell proliferation arrest, and the neuritogenic cAMP sensor NCS-Rapgef2 to neuritogenesis (Xu et al., J. Neuroendocrinol. 33: e12974). NCS-RapGEF2-mediated neuritogenesis occurs via the Rap-Braf-MEK-ERK pathway, which is activated by a variety of Gs-coupled GPCRs, including the endogenously expressed PAC1 receptor, and receptors for GLP-1 (GLP1R) and VIP (VIPR1, VIPR2) when exogenously expressed in NS-1 cells, followed by exposure to exendin-4 or VIP (Xu et al., ibid) or by ADRB2, after exposure to isoproterenol. We have noted previously that other Gs-coupled GPCRs expressed in NS-1 cells mediate measurable cAMP elevation, which however is apparently below the threshold required for triggering and maintaining neuritogenesis (Xu et al., ibid). Here, we identify changes in the NS-1 cell transcriptome associated with PACAP activation of PAC1 receptor and the role of ERK phosphorylation in sustaining gene expression potentially associated with cAMP-dependent neuritogenesis. Continuous ERK phosphorylation is required for neuritogenic signaling: inhibition of ERK phosphorylation with U0126 for up to 24 hours after PACAP treatment arrests neuritogenesis, while inhibition of PKA with H89 has no effect on neuritogenesis. NS-1 cells were treated for 24 hours with PACAP in the presence and absence of either the MEK inhibitor U0126 or the PKA inhibitor H89, and subjected to transcriptomic analysis. We identified 25 genes significantly upregulated by PACAP but downregulated (P<0.001, >2 fold, n=3) by co-treatment with U0126. DCLK1 is a serine/threonine protein kinase important for neuronal development. Dclk1 mRNA is upregulated ~six-fold at 8 hr and four-fold at 24 hr after PACAP treatment, and this was reduced to 2 fold compared to no treat) and its upregulation by PACAP is also blocked by U0126. The gene encoding the orphan GPCR, GPR50, is upregulated more than 100-fold by 8 hours after PACAP, and induction of GPR50 mRNA is blocked more than 97% by U0126, with partial blockade (80%) by H89. We are currently investigating the impact of GPR50 on neuritogenesis using GPR50 knockdown with shRNA in NS-1 cells. Forty-five genes upregulated by PACAP and blocked by H89, but not U0126, include Slit2 and Gas1. Regulation of these genes by Gs-coupled GPCR signaling through PKA may mediate NS-1 cell responses separate from activation of neuritogenesis. Cyclic AMP elevation associated with Gs-coupled GPCR signaling in NS-1 cells results in upregulation of both RapGEF2/ERK-dependent and PKA-dependent gene induction. A threshold for cAMP elevation, either in amount or duration of elevation, or both, appears to be a requirement for Gs-coupled GPCR-mediated neuritogenesis. NS-1 cells provide a model for investigating the functional importance of RapGEF2- and PKA-dependent gene regulation initiated by cAMP in neuroendocrine cells.