Article

Gs Regulation of Endosome Fusion Suggests a Role for Signal Transduction Pathways in Endocytosis

Department of Cell Biology and Physiology, Washington University, School of Medicine, St. Louis, Missouri 63110.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/1994; 269(21):14919-23.
Source: PubMed

ABSTRACT

Work from several laboratories indicates that guanine nucleotide-binding proteins (GTP-binding proteins) are required for intracellular vesicular transport. In a previous report we presented evidence indicating that one or more heterotrimeric G proteins regulate fusion between endosomes (Colombo, M. I., Mayorga, L. S., Casey, P. J., and Stahl, P. D. (1992) Science 255, 1695-1697). We now report on experiments showing that Gs plays a role in endosome fusion. We have used several reagents known to modulate Gs function including (i) peptides corresponding to the cytoplasmic domains of G protein-coupled receptors and peptides that mimic interaction of receptors with G proteins, (ii) anti-G protein antibodies, and (iii) cholera toxin. Synthetic peptides corresponding to the third cytoplasmic loop of the beta 2-adrenergic receptor which putatively interact with G alpha s inhibited endosomal fusion. The inhibitory effect of these peptides was prevented by a short preincubation of endosomes with guanosine-5'-3-O-(thio)triphosphate or by phosphorylating the peptide with cAMP-dependent protein kinase. The involvement of Gs in endosome recognition and/or the fusion process was assessed by testing an antibody against the COOH terminus of G alpha s. Anti-G alpha s IgG completely abolished fusion between endosomes. Lastly, preincubation of endosomal vesicles with cholera toxin abrogated fusion in the presence of NAD, whereas no effect was observed in the absence of the cofactor. Taken together these findings indicate a role for Gs in either the mechanism or the regulation of fusion among endosomes. These results raise the possibility that signal transduction through cytoplasmic domains of receptors may participate in the regulation of endocytic trafficking.

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    • "Gs has been detected in endocytic vesicles obtained from liver (Van Dyke, 2004), it associates with tubulin and the microtubule cytoskeleton in neuronal cells (Roychowdhury et al., 1999; Sarma et al., 2003), and Gs is enriched in the trans-Golgi network of rat pancreatic cells (Denker et al., 1996). Several studies have indicated other functional roles for Gs apart from activation of adenylyl cyclase, including regulation of apical transport in liver epithelia (Pimplikar and Simons, 1993), regulation of endosome fusions (Colombo et al., 1994), and controlling the trafficking and degradation of epidermal growth factor receptors (Zheng et al., 2004). How Gs is trafficked to these cellular locations and the mechanism governing its association with these subcellular compartments remain unclear. "
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    • "Coat assembly and the sorting of newly synthesized proteins secreted constitutively in polarized cells appear to be controlled by heterotrimer Gproteins (Ktistakis et al. 1992; Robinson and Kreis 1992; Pimplikar and Simons 1993). The processes of exocytotic and endocytotic membrane fusion are also under the stimulatory control of G i and the inhibitory control of G o (Bomsel and Mostov 1992; Ahnert-Hilger et al. 1994; Colombo et al. 1994; Helms 1995). A role of G-proteins in the maintenance of the highly specialized structure of the blood-brain barrier has also been suggested (Brett et al. 1989; Hoyer et al. 1991; Raub 1996; Fábián et al. 1998). "
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    • "MDCK epithelial cells , CT acts via Gs to stimulate transcytosis of occupied poly - Ig re - ceptors and increase apical transport of vesicles bearing influenza hemagglutinin ( Bomsel and Mostov , 1993 ; Pim - plikar and Simons , 1993 ) . Activation of Gs with CT also inhibits endosome fusion in J774 macrophages , a process thought to involve ARF ( Colombo et al . , 1994 ) . The ARF - directed reagent BFA inhibits so - called constitutive secre - tion as well as insulin - triggered exocytosis of vesicles bear - ing the GLUT4 glucose transporter in rat adipocytes ( Lachaal et al . , 1994 ) , Ca 2ϩ - induced exocytosis of secre - tory granules in melanotrophs ( Rupnik et al . , 1995 ) , cAMP - induced del"
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