title: Posttranslational processing of human and mouse urocortin 2: characterization and bioactivity of gene products.
abstract: Mouse (m) and human (h) urocortin 2 (Ucn 2) were identified by molecular cloning strategies and the primary sequence of their mature forms postulated by analogy to closely related members of the corticotropin-releasing factor (CRF) neuropeptide family. Because of the paucity of Ucn 2 proteins in native tissues, skin, muscle, and pancreatic cell lines were transduced with lentiviral constructs and secretion media were used to isolate and characterize Ucn 2 products and study processing. Primary structures were assigned using a combination of Edman degradation sequencing and mass spectrometry. For mUcn 2, transduced cells secreted a 39 amino acid peptide and the glycosylated prohormone lacking signal peptide; both forms were C-terminally amidated and highly potent to activate the type 2 CRF receptor. Chromatographic profiles of murine tissue extracts were consistent with cleavage of mUcn 2 prohormone to a peptidic form. By contrast to mUcn 2, mammalian cell lines transduced with hUcn 2 constructs secreted significant amounts of an 88 amino acid glycosylated hUcn 2 prohormone but were unable to further process this molecule. Similarly, WM-266-4 melanoma cells that express endogenous hUcn 2 secreted only the glycosylated prohormone lacking the signal peptide and unmodified at the C terminus. Although not amidated, hUcn 2 prohormone purified from overexpressing lines activated CRF receptor 2. Hypoxia and glycosylation, paradigms that might influence secretion or processing of gene products, did not significantly impact hUcn 2 prohormone cleavage. Our findings identify probable Ucn 2 processing products and should expedite the characterization of these proteins in mammalian tissues.
authors.affiliation: The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
authors.forename: Joan M
authors.initials: JM
authors.lastname: Vaughan
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Donaldson
authors.forename: Wolfgang H
authors.initials: WH
authors.lastname: Fischer
authors.forename: Marilyn H
authors.initials: MH
authors.lastname: Perrin
authors.forename: Jean E
authors.initials: JE
authors.lastname: Rivier
authors.forename: Paul E
authors.initials: PE
authors.lastname: Sawchenko
authors.forename: Wylie W
authors.initials: WW
authors.lastname: Vale
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015687:Cell Hypoxia; D002478:Cells, Cultured; D003346:Corticotropin-Releasing Hormone / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism; D014161:Transduction, Genetic; D054832:Urocortins / Q000737:chemistry / Q000378:metabolism
chemical_list: C101531:CRF receptor type 2; D018019:Receptors, Corticotropin-Releasing Hormone; C426705:UCN2 protein, human; D054832:Urocortins; C518358:urocortin 2, mouse; D003346:Corticotropin-Releasing Hormone