A heterotrimeric G protein, Gαi-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells

Renal Unit, Massachusetts General Hospital, Boston.
The Journal of Cell Biology (Impact Factor: 9.83). 10/1991; 114(6):1113-24.
Source: PubMed


A heterotrimeric G alpha i subunit, alpha i-3, is localized on Golgi membranes in LLC-PK1 and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence. The alpha i-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The alpha i-3 subunit is found on isolated rat liver Golgi membranes by Western blotting and G alpha i-3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLC-PK1 cells were stably transfected with G alpha i-3 on an MT-1, inducible promoter in order to overexpress alpha i-3 on Golgi membranes. The intracellular processing and constitutive secretion of the basement membrane heparan sulfate proteoglycan (HSPG) was measured in LLC-PK1 cells. Overexpression of alpha i-3 on Golgi membranes in transfected cells retarded the secretion of HSPG and accumulated precursors in the medial-trans-Golgi. This effect was reversed by treatment of cells with pertussis toxin which results in ADP-ribosylation and functional uncoupling of G alpha i-3 on Golgi membranes. These results provide evidence for a novel role for the pertussis toxin sensitive G alpha i-3 protein in Golgi trafficking of a constitutively secreted protein in epithelial cells.

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    • "the control of membrane trafficking and regulate exocytosis from intracellular organelles (Stow et al., 1991; Aridor et al., 1993). In addition, G proteins of the Gi family have been reported to control the trafficking of aquaporin 2 channels in kidney epithelial cells (Valenti et al., 1998), and also the trafficking and distribution of connexin 43 hemichannels in Novikoff hepatoma cells (Lampe et al., 2001). "

    The Journal of General Physiology 09/2015; 146(3). DOI:10.1085/jgp.201511462 · 4.79 Impact Factor
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    • "Molecular Biology of the Cell homeostasis. In pharmacological studies, Golgi-localized Gβγ was shown to be required for post-Golgi anterograde transport (Stow et al., 1991; Irannejad and Wedegaertner, 2010). However, the GPCRs associated with Golgi-localized Gβγ are not known. "
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    ABSTRACT: The importance of endosome-to-trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51-VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore, GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for the efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. Also, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. © 2015 by The American Society for Cell Biology.
    Molecular biology of the cell 07/2015; 26(17). DOI:10.1091/mbc.E14-11-1568 · 4.47 Impact Factor
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    • "Heterotrimeric G proteins were detected in the Golgi over two decades ago (Barr et al., 1992; Stow et al., 1991), and numerous studies have provided clues that they may regulate membrane traffic and maintain the structural integrity of the Golgi (Cancino and Luini, 2013). However, the concept of G protein activation at the Golgi and the potential impact of such activation are met with skepticism, primarily due to the lack of direct proof of G protein activation. "
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    ABSTRACT: A long-held tenet of heterotrimeric G protein signal transduction is that it is triggered by G protein-coupled receptors (GPCRs) at the PM. Here, we demonstrate that Gi is activated in the Golgi by GIV/Girdin, a non-receptor guanine-nucleotide exchange factor (GEF). GIV-dependent activation of Gi at the Golgi maintains the finiteness of the cyclical activation of ADP-ribosylation factor 1 (Arf1), a fundamental step in vesicle traffic in all eukaryotes. Several interactions with other major components of Golgi trafficking-e.g., active Arf1, its regulator, ArfGAP2/3, and the adaptor protein β-COP-enable GIV to coordinately regulate Arf1 signaling. When the GIV-Gαi pathway is selectively inhibited, levels of GTP-bound Arf1 are elevated and protein transport along the secretory pathway is delayed. These findings define a paradigm in non-canonical G protein signaling at the Golgi, which places GIV-GEF at the crossroads between signals gated by the trimeric G proteins and the Arf family of monomeric GTPases. Copyright © 2015 Elsevier Inc. All rights reserved.
    Developmental Cell 04/2015; 33(2). DOI:10.1016/j.devcel.2015.02.009 · 9.71 Impact Factor
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