title: Cyclic Adenosine 3',5'-Monophosphate Elevation and Biological Signaling through a Secretin Family Gs-Coupled G Protein-Coupled Receptor Are Restricted to a Single Adenylate Cyclase Isoform.
abstract: PC12 cells express five adenylate cyclase (AC) isoforms, most abundantly AC6 and AC7. These two ACs were individually silenced using lentiviral short hairpin RNAs, which lead to a decrease (≥80%) of the protein product of each transcript. These stable PC12 sublines were then used to examine potential AC isoform preference for signaling through a family B G protein-coupled receptor (GPCR). Cells were challenged with the endogenous agonist of the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1), pituitary adenylate cyclase-activating polypeptide (PACAP)-38, or the diterpene forskolin as an AC-proximal control. Intracellular cAMP levels were elevated by forskolin about equally in wild-type, AC6, and AC7 knockdown cells. The ability of PACAP-38 and forskolin to activate three cAMP sensors downstream of AC [protein kinase A (PKA), exchange protein activated by cAMP (Epac) 2/Rapgef4, and neuritogenic cAMP sensor (NCS)/Rapgef2] was examined by monitoring the phosphorylation status of their respective targets, cAMP response element-binding protein, p38, and extracellular signal-regulated kinase. Forskolin stimulation of each downstream target of cAMP was unaffected by knockdown of either AC6 or AC7. PACAP-38 activation of all downstream targets of cAMP was unaffected by AC7 knockdown, but abolished following AC6 knockdown. Membrane cholesterol depletion with methyl-β-cyclodextrin mimicked the effects of AC6 silencing on PACAP signaling, without attenuating forskolin signaling. These data suggest that vicinal constraint of the GPCR PAC1 and AC6 determines the exclusive requirement for this AC in PACAP signaling, but that the coupling of the cAMP sensors PKA, Epac2/Rapgef4, and NCS/Rapgef2, to their respective downstream signaling targets, determines how cAMP signaling is parcellated to physiologic responses, such as neuritogenesis, upon GPCR-Gs activation in neuroendocrine cells.
authors.affiliation: Sections on Molecular Neuroscience (A.C.E., X.-H.L., L.E.E.) and Directed Gene Transfer (W.X., M.V.E.), Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland.
authors.forename: Andrew C
authors.identifier: http://orcid.org/0000-0002-6760-2570
authors.initials: AC
authors.lastname: Emery
authors.affiliation: Sections on Molecular Neuroscience (A.C.E., X.-H.L., L.E.E.) and Directed Gene Transfer (W.X., M.V.E.), Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland.
authors.forename: Xiu-Huai
authors.initials: XH
authors.lastname: Liu
authors.affiliation: Sections on Molecular Neuroscience (A.C.E., X.-H.L., L.E.E.) and Directed Gene Transfer (W.X., M.V.E.), Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland.
authors.forename: Wenqin
authors.initials: W
authors.lastname: Xu
authors.affiliation: Sections on Molecular Neuroscience (A.C.E., X.-H.L., L.E.E.) and Directed Gene Transfer (W.X., M.V.E.), Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland eidenl@mail.nih.gov eidenm@mail.nih.gov.
authors.forename: Maribeth V
authors.initials: MV
authors.lastname: Eiden
authors.affiliation: Sections on Molecular Neuroscience (A.C.E., X.-H.L., L.E.E.) and Directed Gene Transfer (W.X., M.V.E.), Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland eidenl@mail.nih.gov eidenm@mail.nih.gov.
authors.forename: Lee E
authors.initials: LE
authors.lastname: Eiden
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation; D002784:Cholesterol / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology; D051381:Rats; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000378:metabolism*; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D020662:Guanine Nucleotide Exchange Factors; D007527:Isoenzymes; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; C000599193:Rapgef2 protein, rat; C531571:Rapgef4 protein, rat; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D005576:Colforsin; D002784:Cholesterol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; C121100:adenylyl cyclase 6