Gs regulation of endosome fusion suggests a role for signal transduction pathways in endocytosis.
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.
Colombo
M I
MI
Department of Cell Biology and Physiology, Washington University, School of Medicine, St. Louis, Missouri 63110.
Mayorga
L S
LS
Nishimoto
I
I
Ross
E M
EM
Stahl
P D
PD
D000595:Amino Acid Sequence; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D008561:Membrane Fusion / Q000187:drug effects; D008969:Molecular Sequence Data; D010766:Phosphorylation; D015398:Signal Transduction
D002772:Cholera Toxin; D019204:GTP-Binding Proteins