ABSTRACTElucidation of the underlying mechanism of dopamine signaling to ERK that underlies plasticity in dopamine D1 receptor expressingneurons leadingto acquired cocaine preference is incomplete. NCS-Rapgef2 is a novel cAMP effector, expressed in neuronal and endocrine cells in adult mammals, that is required for D1 dopamine receptor-dependent ERK phosphorylation in mouse brain. In this report, we studied the effects of abrogating NCS-Rapgef2 expression on cAMP-dependent ERK→Egr-1/zif268 signaling in cultured neuroendocrine cells; in D1 medium spiny neurons (MSNs) of nucleus accumbens slices; and in mouse brain in a region-specific manner. NCS-Rapgef2 gene deletion in the nucleus accumbens (NAc) in adult mice, using AAV-mediated expression of cre recombinase, eliminated cocaine-induced ERK phosphorylation and Egr-1/Zif268 upregulation in D1-MSNs and cocaine-induced behaviors including locomotor sensitization and conditioned place preference (CPP). Abrogation of NCS-Rapgef2 gene expression in medium prefrontal cortex and basolateral amygdala, by crossing mice bearing a floxed Rapgef2 allele with a cre mouse line driven by calcium/calmodulin-dependent kinase IIα promoter also eliminated cocaine-induced phospho-ERK activation and Egr-1/Zif268 induction, but without effect on the cocaine-induced behaviors. Our results indicate that NCS-Rapgef2 signaling to ERK in dopamine D1-receptor expressing neurons in the NAc, butnotin corticolimbic areas, contributes to cocaine-induced locomotor sensitization and CPP. Ablation of cocaine-dependent ERK activation by elimination of NCS-Rapgef2 occurred with no effect on phosphorylation of CREB in D1 dopaminoceptive neurons of NAc. This study reveals a new cAMP-dependent signaling pathway for cocaine-induced behavioral adaptations, mediated through NCS-Rapgef2/phospho-ERK activation, independently of PKA/CREB signaling.SIGNIFICANCE STATEMENTERK phosphorylation in dopamine D1 receptor expressing neurons exerts a pivotal role in psychostimulant-induced neuronal gene regulation and behavioraladaptation, including locomotor sensitization and drug preference in rodents. In this study, we examined the role of dopamine signaling through the D1 receptor via a novel pathway initiated through the cAMP-activated guanine nucleotide exchange factor NCS-Rapgef2 in mice. NCS-Rapgef2 in the nucleus accumbens is required for activation of ERK and Egr-1/Zif268 in D1 dopaminoceptive neurons after acute cocaine administration, and subsequentenhanced locomotor response anddrugseeking behavior after repeated cocaine administration. This novel component in dopamine signaling provides a potential new target for intervention in psychostimulant-shaped behaviors, and new understanding of how D1-MSNs encode the experience of psychomotor stimulant exposure.

Elucidation of the mechanism of dopamine signaling to ERK that underlies plasticity in dopamine D1 receptor-expressing neurons leading to acquired cocaine preference is incomplete. NCS-Rapgef2 is a novel cAMP effector, expressed in neuronal and endocrine cells in adult mammals, that is required for D1 dopamine receptor-dependent ERK phosphorylation in mouse brain. In this report, we studied the effects of abrogating NCS-Rapgef2 expression on cAMP-dependent ERKfiEgr-1/Zif268 signaling in cultured neuroendocrine cells; in D1 medium spiny neurons of NAc slices; and in either male or female mouse brain in a region-specific manner. NCS-Rapgef2 gene deletion in the NAc in adult mice, using adeno-associated virus-mediated expression of cre recombinase, eliminated cocaine-induced ERK phosphorylation and Egr-1/Zif268 upregulation in D1-medium spiny neurons and cocaine-induced behaviors, including locomotor sensitization and conditioned place preference. Abrogation of NCS-Rapgef2 gene expression in mPFC and BLA, by crossing mice bearing a floxed Rapgef2 allele with a cre mouse line driven by calcium/calmodulin-dependent kinase IIa promoter also eliminated cocaine-induced phospho-ERK activation and Egr-1/Zif268 induction, but without effect on the cocaine-induced behaviors. Our results indicate that NCS-Rapgef2 signaling to ERK in dopamine D1 receptor-expressing neurons in the NAc, but not in corticolimbic areas, contributes to cocaine-induced locomotor sensitization and conditioned place preference. Ablation of cocaine-dependent ERK activation by elimination of NCS-Rapgef2 occurred with no effect on phosphorylation of CREB in D1 dopaminoceptive neurons of NAc. This study reveals a new cAMP-dependent signaling pathway for cocaine-induced behavioral adaptations, mediated through NCS-Rapgef2/phospho-ERK activation, independently of PKA/CREB signaling.

Gs‐coupled GPCR‐stimulated neuritogenesis in PC12 and NS‐1 ‐ cells depends on activation of the MAP kinase ERK. Here, we examine changes in ERK activation (phosphorylation), and the time course of ERK‐dependent gene induction, to seek transcriptional determinants for this process. Quenching of ERK activation by inhibition of MEK with U0126 at any time point for at least 24 h following addition of PACAP resulted in arrest of neurite formation. Changes in the transcriptome profile throughout this time period revealed at least two phases of gene induction: an early phase dominated by induction of immediate‐early genes, and a later phase of gene induction after 4–6 h of exposure to PACAP with persistent elevation of phospho‐ERK levels. Genes induced by PACAP in both phases consisted in those whose induction was dependent on ERK (i.e., blocked by U0126), and some whose induction was blocked by the protein kinase A inhibitor H89. ERK‐dependent “late gene” transcripts included Gpr50, implicated earlier in facilitation of NGF‐induced neurite formation in NS‐1 cells. Gpr50 induction by PACAP, but not NGF, was dependent on the guanine nucleotide exchange factor RapGEF2, which has been shown to be required for PACAP‐induced neuritogenesis in NS‐1 cells. Expression of a Gpr50‐directed shRNA lowered basal levels of Gpr50 mRNA and attenuated Gpr50 mRNA and GPR50 protein induction by PACAP, with a corresponding attenuation of PACAP‐induced neuritogenesis. Gs‐GPCR‐stimulated neuritogenesis first requires immediate‐early gene induction, including that of Egr1 (Zif268/NGF1A/Krox24) as previously reported. This early phase of gene induction, however, is insufficient to maintain the neuritogenic process without ERK‐dependent induction of additional late genes, including Gpr50, upon continuous exposure to neurotrophic neuropeptide. Early (Egr1) and late (Gpr50) gene induction by NGF, like that for PACAP, was inhibited by U0126, but was independent of RapGEF2, confirming distinct modes of ERK activation by Gs‐coupled GPCRs and neurotrophic tyrosine receptor kinases, converging on a final common ERK‐dependent signaling pathway for neuritogenesis.

G‐protein coupled receptors activate various intracellular processes upon ligand engagement depending on coupling to adenylyl cyclase (Gs, Gi/o), phospholipase C (Gq), or RhoGEFs (G12/13). Cyclic AMP activation of the Rap‐Braf‐MEK‐ERK pathway after signaling initiated by the neuropeptide PACAP, via the Gs‐protein coupled receptor (GsPCR) PAC1, occurs uniquely through the neuritogenic cAMP sensor Rap guanine nucleotide exchange factor 2 (NCS‐RapGEF2) in NS‐1 neuroendocrine cells. We examined the expression of other Family B GsPCRs in this cell line and assessed cAMP elevation and neuritogenesis after treatment with their cognate peptide ligands. Exposure of NS‐1 cells to the VIPR1/2 agonist VIP, or the GLP1R agonist exendin‐4, did not induce neuritogenesis, or elevation of cAMP, presumably due to insufficient receptor protein expression. VIP and exendin‐4 did induce neuritogenesis after transduction of human VIPR1, VIPR2 and GLP1R into NS‐1 cells. Exendin‐4/GLP1R‐stimulated neuritogenesis was MEK‐ERK‐ dependent (blocked by U0126), indicating its use of the cAMP‐>RapGEF2‐>ERK neuritogenic signaling pathway previously identified for PACAP/PAC1 signaling in NS‐1 cells. NCS‐ RapGEF2 is expressed in the beta cell tumor‐derived cell lines MIN6 and INS‐1, and in human pancreatic islets. As in NS‐1 cells, exendin‐4 caused ERK phosphorylation in INS‐1 cells. Reduction in RapGEF2 expression after its specific shRNA treatment reduced exendin‐4‐induced ERK phosphorylation. Transcriptome analysis of INS‐1 cells after one hour exposure to exendin‐ 4 revealed an immediate early‐gene response that was composed of both ERK‐dependent and ERK‐independent signaling targets. ERK‐dependent targets, and presumably involving NCS‐RapGEF2 signaling, included Nr4a2, Btg1, Skil, Plkl2, Per1 and Dyrk3, while ERK‐independent targets and presumably involving PKA or Epac signaling, included Nr4a1, Nr4a3, Crem, Fosl2, Sik1 and Rgs2. We propose that cAMP signaling initiated by GLP‐1 in pancreatic beta cells causes parallel activation of multiple cAMP effectors, including NCS‐ RapGEF2, Epac, and protein kinase A, to separately control various facets of GLP‐1 action, including insulin secretion and transcriptional modulation. Identification of coupling of GsPCRs to ERK activation via NCS‐RapGEF2 provides a novel signaling mode for GLP‐1 in pancreatic beta cells and potentially brain and elsewhere. RapGEF2‐dependent ERK activation is likely to be a feature of additional nervous and endocrine targets of family B receptor ligands including PACAP, VIP, CRH, GIP, secretin, glucagon, PTH, calcitonin, and GHRH. Whether or not ligands for these receptors, including GLP‐1, might activate the NCS‐RapGEF2, PKA, or Epac cAMP‐dependent pathways in a biased fashion, with differential physiological effects, has yet to be investigated. This abstract is dedicated to the memory of Andrew C. Emery.

Cyclic AMP activation of the Rap-Braf-MEK-ERK pathway after signalling initiated by the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), via the G s -protein coupled receptor (G s PCR) PAC1, occurs uniquely through the neuritogenic cAMP sensor Rap guanine nucleotide exchange factor 2 (NCS-RapGEF2) in Neuroscreen-1 (NS-1) neuroendocrine cells. We examined the expression of other Family B G s PCRs in this cell line and assessed cAMP elevation and neuritogenesis after treatment with their cognate peptide ligands. Exposure of NS-1 cells to the VIPR1/2 agonist vasoactive intestinal polypeptide, or the GLP1R agonist exendin-4, did not induce neuritogenesis, or elevation of cAMP, presumably as a result of insufficient receptor protein expression. Vasoactive intestinal polypeptide and exendin-4 did induce neuritogenesis after transduction of human VIPR1, VIPR2 and GLP1R into NS-1 cells. Exendin-4/GLP1R-stimulated neuritogenesis was MEK-ERK-dependent (blocked by U0126), indicating its use of the cAMP→RapGEF2→ERK neuritogenic signalling pathway previously identified for PACAP/PAC1 signalling in NS-1 cells. NCS-RapGEF2 is expressed in the rodent insulinoma cell lines MIN6 and INS-1, as well as in human pancreatic islets. As in NS-1 cells, exendin-4 caused ERK phosphorylation in INS-1 cells. Reduction in RapGEF2 expression after RapGEF2-shRNA treatment reduced exendin-4-induced ERK phosphorylation. Transcriptome analysis of INS-1 cells after 1 hour of exposure to exendin-4 revealed an immediate earlygene response that was composed of both ERK-dependent and ERK-independent signalling targets. We propose that cAMP signalling initiated by glucagon-like peptide 1 (GLP-1) in pancreatic beta cells causes parallel activation of multiple cAMP effectors, including NCS-RapGEF2, Epac and protein kinase A, to separately control various facets of GLP-1 action, including insulin secretion and transcriptional modulation.