title: Signaling through the neuropeptide GPCR PAC₁ 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.
authors.affiliation: Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland 20892-4090, USA.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Emery
authors.forename: Lee E
authors.initials: LE
authors.lastname: Eiden
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D051379:Mice; D016501:Neurites / Q000502:physiology*; D016716:PC12 Cells; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology
chemical_list: D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D000262:Adenylyl Cyclases