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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    • "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 · 9.69 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 · 4.24 Impact Factor
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    • "In vitro endosome fusion was found here to be remarkably inhibited by addition of cAMP. Further research will be required to determine whether this inhibition is a primary interaction of cAMP with the fusion machinery or a secondary effect on other regulators of endosomal fusion such as Gs (Colombo et al., 1994). In any case, the cAMP effect provides evidence that cytosolic second messengers modulate endosome fusion in vitro and may provide an explanation for the inhibition of endocytosis in intact cells by cAMP agonists. "
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    ABSTRACT: A quantitative real-time assay of cell-free endosomal vesicle fusion was developed and applied to study fusion mechanisms in endosomes from baby hamster kidney (BHK-21) cells. The assay is based on an irreversible approximately 10-fold increase in BODIPY-avidin fluorescence on binding of biotinylated conjugates. BODIPY-avidin and biotin-dextran were internalized for 10 min at 37 degrees C into separate populations of BHK-21 cells, and endosome fractions were prepared. Postnuclear supernatant fractions underwent ATP- and temperature-dependent fusion, as measured in a sensitive custom-built microfluorimeter by the continuous increase in BODIPY-avidin fluorescence. Fusion processes of efficiency > 2.5% could be detected with 200-ms time resolution in sample volumes of 50 microL containing endosomes derived from approximately 4 x 10(4) cells. The fusion time course consisted of a distinct lag phase (up to 10 min) in which little fusion occurred, followed by an approximately exponential rise (t 1/2 10-30 min; fusion efficiency approximately 15%). The lag phase was reduced by preincubation of separate endosome fractions with ATP at 37 degrees C and by coincubation of endosomes at 22 degrees C before the assay, suggesting a rate-limiting step involving binding of a soluble protein to the endosome membrane. Endosome fusion was strongly inhibited by GTP gamma S, N-ethylmaleimide, and AIF4-. Endosome fusion was not affected by phorbol myristate acetate but was significantly inhibited by cAMP and bovine brain calmodulin. The results establish a sensitive real-time fluorescence assay to quantify the kinetics and extent of endosome fusion in a cell-free system and demonstrate regulation of early endosome fusion by cytosolic second messengers.
    Biophysical Journal 08/1996; 71(1):487-94. DOI:10.1016/S0006-3495(96)79250-0 · 3.97 Impact Factor
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