title: Meltrin beta (ADAM19) mediates ectodomain shedding of Neuregulin beta1 in the Golgi apparatus: fluorescence correlation spectroscopic observation of the dynamics of ectodomain shedding in living cells.
abstract: Membrane-anchored Neuregulin beta1 sheds its ectodomain as soluble factors. Two proteases that belong to a disintegrin and metalloprotease (ADAM) family are known to cleave Neuregulin beta1. One is tumor necrosis factor-alpha converting enzyme (TACE/ADAM17). The other is Meltrin beta (ADAM19). Against our expectation that shedding by ADAM proteases occurs at the cell surface, here we found that Meltrin beta mediates the ectodomain shedding of Neuregulin beta1 in the Golgi apparatus. Meltrin beta was localized in and around the Golgi apparatus in developing sensory neurons. Subcellular fractionation revealed that Meltrin beta generated soluble Neuregulin beta1 in Golgi-enriched fractions while TACE-cleaved Neuregulin beta1 was recovered in lighter fractions. To examine whether Meltrin beta-mediated ectodomain shedding occurs in the Golgi apparatus in living cells, we took advantage of different diffusion properties of cleavage products from those of membrane-anchored precursor proteins. Fluorescence correlation spectroscopy (FCS) is the most sensitive method to determine milli approximately submillisecond diffusion in vivo. Protease-active Meltrin beta caused a shift in autocorrelation function in FCS of green fluorescent protein (GFP)-tagged Neuregulin beta1 in the Golgi apparatus, suggesting a conversion of Neuregulin beta1 molecules from membrane-anchored to soluble forms in that organelle. The Golgi apparatus is a site of processing Neuregulin beta1 by Meltrin beta.
authors.affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Tomoichi
authors.initials: T
authors.lastname: Yokozeki
authors.forename: Shuji
authors.initials: S
authors.lastname: Wakatsuki
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Hatsuzawa
authors.forename: Roy A
authors.initials: RA
authors.lastname: Black
authors.forename: Ikuo
authors.initials: I
authors.lastname: Wada
authors.forename: Atsuko
authors.initials: A
authors.lastname: Sehara-Fujisawa
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005727:Ganglia, Spinal / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D020890:Neuregulin-1; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
chemical_list: D009419:Nerve Tissue Proteins; D020890:Neuregulin-1; C490581:Nrg1 protein, mouse; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D051722:ADAM Proteins; C096116:Adam19 protein, mouse