title: C-terminal amidation of PACAP-38 and PACAP-27 is dispensable for biological activity at the PAC1 receptor.
abstract: PACAP-27 and PACAP-38 are the exclusive physiological ligands for the mammalian PAC1 receptor. The role of C-terminal amidation of these ligands at that receptor was examined in neuroendocrine cells expressing the PAC1 receptor endogenously and in non-neuroendocrine cells in which the human and rat PAC1 receptors were expressed from stable single-copy genes driven by the CMV promoter, providing stoichiometrically appropriate levels of this Gs-coupled GPCR in order to examine the potency and intrinsic activity of PACAP ligands and their des-amidated congeners. We found that replacement of the C-terminal glycine residues of PACAP-27 and -38 with a free acid; or extension of either peptide with the two to three amino acids normally found at these positions in PACAP processing intermediates in vivo following endoproteolytic cleavage and after exoproteolytic trimming and glycine-directed amidated, were equivalent in potency to the fully processed peptides in a variety of cell-based assays. These included real-time monitoring of cyclic AMP generation in both NS-1 neuroendocrine cells and non-neuroendocrine HEK293 cells; PKA-dependent gene activation in HEK293 cells; and neuritogenesis and cell growth arrest in NS-1 cells. The specific implications for the role of amidation in arming of secretin-related neuropeptides for biological function, and the general implications for neuropeptide-based delivery in the context of gene therapy, are discussed.
authors.affiliation: Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, MD 20892, USA.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Emery
authors.affiliation: Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, MD 20892, USA.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Alvarez
authors.affiliation: Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, MD 20892, USA.
authors.forename: Philip
authors.initials: P
authors.lastname: Abboud
authors.affiliation: Section on Directed Gene Transfer, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, MD 20892, USA.
authors.forename: Wenqin
authors.initials: W
authors.lastname: Xu
authors.affiliation: Section on Directed Gene Transfer, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, MD 20892, USA.
authors.forename: Craig D
authors.initials: CD
authors.lastname: Westover
authors.affiliation: Section on Directed Gene Transfer, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, MD 20892, USA.
authors.forename: Maribeth V
authors.initials: MV
authors.lastname: Eiden
authors.affiliation: Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, MD 20892, USA. Electronic address: eidenl@mail.nih.gov.
authors.forename: Lee E
authors.initials: LE
authors.lastname: Eiden
mesh_terms: D000577:Amides / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology / Q000502:physiology*; D051381:Rats; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000502:physiology*; D015290:Second Messenger Systems
chemical_list: D000577:Amides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D000242:Cyclic AMP
keywords: Amidation; Cyclic AMP; Nervous system; Neuropeptide; PAC1 receptor; PACAP