G(S) REGULATION OF ENDOSOME FUSION SUGGESTS A ROLE FOR SIGNAL-TRANSDUCTION PATHWAYS IN ENDOCYTOSIS

Work from several laboratories indicates that guanine nucleotide-bindi ng proteins (GTP-binding proteins) are required for intracellular vesi cular transport. In a previous report we presented evidence indicating that one or more heterotrimeric G proteins regulate fusion between en dosomes (Colombo, M. I., Mayorga, L. S., Casey, P.J., and Stahl, P D. (1992) Science 255, 1695-1697). We now report on experiments showing t hat G(s) plays a role in endosome fusion. We have used several reagent s known to modulate G(s) function including (i) peptides corresponding to the cytoplasmic domains of G protein-coupled receptors and peptide s that mimic interaction of receptors with G proteins, (ii) anti-G pro tein antibodies, and (iii) cholera toxin. Synthetic peptides correspon ding to the third cytoplasmic loop of the beta 2-adrenergic receptor w hich putatively interact with G(alpha s) inhibited endosomal fusion. T he inhibitory effect of these peptides was prevented by a short preinc ubation of endosomes with guanosine-5'-3-O-(thio)triphosphate or by ph osphorylating the peptide with cAMP-dependent protein kinase. The invo lvement of G(s) 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 abrogat ed fusion in the presence of NAD, whereas no effect was observed in th e absence of the cofactor. Taken together these findings indicate a ro le for G(s) in either the mechanism or the regulation of fusion among endosomes. These results raise the possibility that signal transductio n through cytoplasmic domains of receptors may participate in the regu lation of endocytic trafficking.