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


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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    • "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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    ABSTRACT: Heterotrimeric G-proteins are signal transducers of heptahelical receptors. They consist of α and βγ subunits, both capable of interacting with several different effectors. Specific domains in their structures enable them to connect different intracellular signaling cascades, such as the adenylyl cyclase, phosphoinositol-bisphosphate or MAP kinase pathways. Their activity is synchronized by several components, one of them being a new protein family termed RGS (regulators of G-protein signaling). Members of this family inhibit the G-protein function. The intracellular localization of G-proteins indicates their role in plasma membrane- independent processes. Opioid receptors transmit their signals mainly via Gi/o proteins. Although the heterogeneity of opioid ligands (peptides and alkaloids) and their receptors (μ, δ, κ and suggested subtypes in these classes) reveals a complicated picture, their unique characteristic of a high dependence capacity can not be explained without the analysis of the G-protein function.
    Acta Biologica Szegediensis 01/2001; 45(1).
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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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    ABSTRACT: Antigen-evoked influx of extracellular Ca2+ into mast cells may occur via store-operated Ca2+ channels called calcium release–activated calcium (CRAC) channels. In mast cells of the rat basophilic leukemia cell line (RBL-2H3), cholera toxin (CT) potentiates antigen-driven uptake of 45Ca2+ through cAMP-independent means. Here, we have used perforated patch clamp recording at physiological temperature to test whether cholera toxin or its substrate, Gs, directly modulates the activity of CRAC channels. Cholera toxin dramatically amplified (two- to fourfold) the Ca2+ release–activated Ca2+ current (ICRAC) elicited by suboptimal concentrations of antigen, without itself inducing ICRAC, and this enhancement was not mimicked by cAMP elevation. In contrast, cholera toxin did not affect the induction of ICRAC by thapsigargin, an inhibitor of organelle Ca2+ pumps, or by intracellular dialysis with low Ca2+ pipette solutions. Thus, the activity of CRAC channels is not directly controlled by cholera toxin or Gsα. Nor was the potentiation of ICRAC due to enhancement of phosphoinositide hydrolysis or calcium release. Because Gs and the A subunit of cholera toxin bind to ADP ribosylation factor (ARF) and could modulate its activity, we tested the sensitivity of antigen-evoked ICRAC to brefeldin A, an inhibitor of ARF-dependent functions, including vesicle transport. Brefeldin A blocked the enhancement of antigen-evoked ICRAC without inhibiting ADP ribosylation of Gsα, but it did not affect ICRAC induced by suboptimal antigen or by thapsigargin. These data provide new evidence that CRAC channels are a major route for Fcε receptor I–triggered Ca2+ influx, and they suggest that ARF may modulate the induction of ICRAC by antigen.
    The Journal of Cell Biology 01/2000; 148(1):137-146. DOI:10.1083/jcb.148.1.137 · 9.83 Impact Factor
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    • "GTP stimulated the internalization of Hb±Hp complex on the EDTA-prepared hepatocytes in the presence of ATP, but not in the absence of ATP (Table 2). Colombo et al. [36] reported that the stimulation of early endosome fusion by GTP was observed in vitro. It looks like the early endosome fusion occurs on the hepatocytes in the our assay system . "
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    ABSTRACT: We have demonstrated the internalization of haemoglobin-haptoglobin (Hb-Hp) complex using rat hepatocytes prepared by EDTA perfusion, followed by Percoll. The isolated hepatocytes exhibited a saturation curve of the binding of fluorescein isothiocyanate-labelled haemoglobin-haptoglobin complex (FITC-Hb-Hp. Furthermore, competition between the binding of FITC-Hb-Hp and unlabelled Hb to the hepatocytes, was observed. The cells exhibited approximately 9 x 10(4) 'high affinity sites' (Kd approximately 1.2 microM) for the Hb-Hp complex. The data in toto suggest the presence of only one type of receptor i.e. the high affinity receptor (in both affinity and number of sites per cell). The results were similar to those obtained from rat hepatocytes prepared by collagenase digestion [1]. In order to verify whether EDTA-prepared hepatocytes could be used for the study of receptor-mediated endocytosis, the internalization of pre-bound Hb-Hp in the isolated hepatocytes was assessed by two methods. First, acid-insensitive FITC-Hb-Hp time-dependently increased following incubation at 37 degrees C. Secondly, Hb-Hp became inaccessible to the exogenous FITC-anti-haemoglobin antibody. These processes were dependent on ATP, but independent of Ca2+ and stimulated by GTP. The results demonstrate that the receptor-mediated endocytosis of Hb-Hp occurred in the EDTA-prepared hepatocytes.
    The International Journal of Biochemistry & Cell Biology 09/1998; 30(8):923-31. DOI:10.1016/S1357-2725(98)00035-1 · 4.05 Impact Factor
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