Cyclic AMP‐dependent Activation of ERK Via GLP‐1 Receptor Signaling Requires the Neuroendocrine Cell‐Selective Guanine Nucleotide Exchanger NCS‐RapGEF2

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.