title: Characterization of the intracellular localization, processing, and secretion of two glial cell line-derived neurotrophic factor splice isoforms.
abstract: Endocrine and neuronal cells have highly developed secretion mechanisms, and the secretion can be either constitutive or regulated by physiological stimuli. In the constitutive pathway, intracellular transport vesicles undergo immediate fusion reactions after arrival at the target. In regulated secretion, vesicles accumulate near the target membrane until triggered to fuse, typically by a local rise in free Ca(2+). In the present study, we characterize the processing and secretion mechanisms of the glial cell line-derived neurotrophic factor (GDNF). Although the function of GDNF has been extensively studied, very little is known about the basic cell biology of GDNF and its precursor forms (alpha)pro-GDNF and (beta)pro-GDNF that have different pro-regions. Our results show that both (alpha)pro-GDNF and (beta)pro-GDNF are secreted. We demonstrate that KCl-induced depolarization increases the secretion of (beta)pro-GDNF and corresponding mature GDNF, but not (alpha)pro-GDNF and corresponding mature GDNF, to the cell medium in a Ca(2+)-dependent manner. In parallel with this, immunofluorescence analysis of cells show that (alpha)pro-GDNF/GDNF is localized mostly in the Golgi complex, whereas (beta)pro-GDNF/GDNF is localized primarily in secretogranin II and Rab3A-positive vesicles of the regulated secretory pathway. In addition, we find that matrix metalloproteinases and plasmin that cleave pro-BDNF and pro-NGF are not responsible for the cleavage of pro-GDNF, whereas furin endoproteinase, PACE4, and proprotein convertases PC5A, PC5B, and PC7 can cleave pro-GDNF into mature GDNF. Thus, the processing and secretion mechanisms of GDNF are different from those of BDNF and NGF.
authors.affiliation: Institute of Biotechnology, University of Helsinki, Viikki Biocenter, FIN-00014 University of Helsinki, Finland.
authors.forename: Liina
authors.initials: L
authors.lastname: Lonka-Nevalaita
authors.forename: Maria
authors.initials: M
authors.lastname: Lume
authors.forename: Satu
authors.initials: S
authors.lastname: Leppänen
authors.forename: Eija
authors.initials: E
authors.lastname: Jokitalo
authors.forename: Johan
authors.initials: J
authors.lastname: Peränen
authors.forename: Mart
authors.initials: M
authors.lastname: Saarma
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016501:Neurites / Q000378:metabolism; D016716:PC12 Cells; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D051381:Rats; D022142:Secretory Vesicles / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C495039:GDNF protein, human; C495040:Gdnf protein, mouse; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D020033:Protein Isoforms; D011498:Protein Precursors