Signaling through the neuropeptide GPCR PAC 1 induces neuritogenesis via a single linear cAMP‐ and ERK‐dependent pathway using a novel cAMP sensor

ABSTRACT Both cAMP and ERK are necessary for neuroendocrine cell neuritogenesis, and pituitary adenylate cyclase‐activating polypeptide (PACAP) activates each. It is important to know whether cAMP and ERK are arranged in a novel, linear pathway or in two parallel pathways using known signaling mechanisms. Native cellular responses [cAMP elevation, ERK phosphorylation, cAMP responsive element binding (CREB) phosphorylation, and neuritogenesis] and promoter‐reporter gene activation after treatment with forskolin, cAMP analogs, and PACAP were measured in Neuroscreen‐1 (NS‐1) cells, a PC12 variant enabling simultaneous morphological, molecular biological, and biochemical analysis. Forskolin (25 μM) and cAMP analogs (8‐bromo‐cAMP, dibutyryl‐cAMP, and 8‐chlorophenylthio‐cAMP) stimulated ERK phosphorylation and neuritogenesis in NS‐1 cells. Both ERK phosphorylation and neuritogenesis were MEK dependent (blocked by 10 μM U0126) and PKA independent (insensitive to 30 μM H‐89 or 100 nM myristoylated protein kinase A inhibitor). CREB phosphorylation induced by PACAP was blocked by H‐89. The exchange protein activated by cAMP (Epac)‐selective 8‐(4‐chlorophenylthio)‐2′‐ O ‐Me‐cAMP (100–500 μM) activated Rap1 without affecting the other cAMP‐dependent processes. Thus, PACAP‐38 potently stimulated two distinct and independent cAMP pathways leading to CREB or ERK activation in NS‐1 cells. Drug concentrations for appropriate effect were derived from control data for all compounds. In summary, a novel PKA‐ and Epac‐independent signaling pathway: PACAP → adenylate cyclase → cAMP → ERK → neuritogenesis has been identified.—Emery, A. C., Eiden, L. E. Signaling through the neuropeptide GPCR PAC 1 induces neuritogenesis via a single linear cAMP‐ and ERK‐dependent pathway using a novel cAMP sensor. FASEB J. 26, 3199–3211 (2012). www.fasebj.org