Non-canonical role of the heterotrimeric G protein Galphas (Gas) on endosomes

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Non canonical role of the heterotrimeric G protein Gas on endosomes******Heterotrimeric G proteins (a,ß,? subunits) are classically involved in signal transduction initiated at the plasma membrane (PM) by G protein-coupled receptors (GPCRs). However, accumulated evidence has highlighted the presence of heterotrimeric G proteins on intracellular organelles thereby suggesting that they have a potential role in GPCR signalling and trafficking. My research program is thus geared toward a better understanding of these new non-traditional aspects of the G-proteins.******Recent studies have confirmed that Gas is present on endosomes and has non-conventional roles in endosomal receptor signalling and trafficking. It is now accepted that canonical GPCR-Gas signalling occurs from endosomes as well as the PM. We have recently discovered a novel role of Gas on the endosomal sorting of GPCRs to lysosome for degradation. The present research program is divided into two aims that are a logical extension of these previous findings.******Hypothesis: At odds with the original concept of endocytosis, we believe that this process does not completely terminate the signalling of a receptor, but can instead divert it from one pathway to another. We propose that once the GPCR reaches the endosomes, it binds and activates Gas subunit in a non-canonical way, which regulates or recruits sorting components on these membranes to modulate receptor targeting to lysosomes. Our overall objective for the next 5 years is to outline the molecular composition and the mode of operation of this Gas-based traffic-detection-and-response.******AIM1. To determine whether and how Gas recognizes GPCR on endosomes***GPCRs are known for their capacity to bind and activate Ga proteins. We noticed that the GPCRs affected by Gas depletion (such as dOR, CXCR4) are not coupled to Gas in their signalling pathway at the plasma membrane. Various techniques including co-immunoprecipitation, GST-pull-down and BRET will be used to determine whether these GPCRs can interact directly or indirectly (via ß-arrestin or ubiquitin, two GPCR sorting signal for lysosome) with Gas on endosomes.******AIM2. To investigate whether Gai-coupled GPCRs trigger the activation of Gas on endosomes. ***To test the hypothesis of GPCR endosomal traffic-initiated Gas signalling, we will use traffic-synchronization protocols to examine whether the arrival of a GPCR traffic `pulse' at the endosomes elicits a Gas signalling response on this organelle. The presence of active Gas on intracellular membranes will be examined by confocal microscopy imaging using specific active Gas biosensor as well as by the quantification of cAMP.******Significance: The current dogma suggests that the signalling of GPCRs is tightly controlled by the endocytic pathways. Our work will highlight a novel concept in which the endosomal signalling of these receptors control their endosomal trafficking to lysosome.*****