Gαs protein binds ubiquitin to regulate endosomal sorting

[Speaker] Christine Lavoie:1
[Co-author] Xuezhi Li:1, Danny Letourneau:2, Brian Holleran:1, Richard Leduc:1, Pierre Lavigne:2
1:Pharmacology and Physiology, Universite de Sherbrooke, Canada, 2:Biochemitry, Universite de Sherbrooke, Canada

The Gαs subunit is classically involved in the signal transduction of G-protein-coupled receptors (GPCR) at the plasma membrane. However, recent evidence has revealed the presence of Gαs on endosomes and its non-canonical role in endosomal sorting of receptors to lysosomes. Yet, the mechanism of action of Gαs in this sorting step is still poorly characterized. Because ubiquitin is key in the endosomal sorting pathway, we investigated whether Gαs interacts with ubiquitin.

In cellulo and in vitro GST-pull-down assays were used to investigate the interaction between Gαs and ubiquitin. The specific residues implicated in this interaction have been mapped by mutagenesis and NMR titrations. Confocal microscopy and western blot analysis were used to investigate receptor sorting into intraluminal vesicles of endosomes for lysosomal degradation.

Gαs interacts directly with ubiquitin and colocalizes with ubiquitinated proteins in microdomains of early endosomes. A functional and conserved ubiquitin-interacting motif (UIM) was identified at the N-terminal extremity of Gαs. Most importantly, mutation of the UIM in Gαs prevented the transfer of EGFR (a classical example of ubiquitinated receptors) into intraluminal vesicles, leading to an accumulation of EGFR on the limiting membrane of endosomes, and thus, the delay of its degradation.

Although Gαs structure has been known for years, we found a novel motif in Gαs that allows its interaction with ubiquitin, a key signal for cargo sorting to the lysosomal pathway. These findings demonstrate a new role for Gαs as an integral component of the ubiquitin-dependent endosomal sorting machinery and highlight the dual role of Gαs in receptor trafficking and signaling for the fine-tuning of the cellular response.
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