Biology subjects

Le Gouill, C.

Publications and source records attributed to Le Gouill, C..

6 recordsLinked to original sources

bioRxiv2024

Molecular determinants underlying differential recruitment of p115RhoGEF and PDZRhoGEF to activated Gα13

Heterotrimeric G proteins, particularly G12 and G13, are pivotal regulators of cellular signaling pathways. Their direct downstream effectors, which include p115RhoGEF and PDZRhoGEF, engage downstream signaling via Rho activation. Yet the molecular determinants that dictate their differential recruitment by G12/13 are not fully understood. Here, we combined quantitative computational residue-level analysis with site-directed mutagenesis and bioluminescence resonance energy transfer (BRET)-based assays to dissect G13 interactions with these RhoGEFs. We mapped the contributions of individual residues to binding and identified specific G13 residues in its helical domain, switch regions, and effector-binding site as key yet differential contributors to p115RhoGEF and PDZRhoGEF recruitment. Experimental validation with BRET confirmed that changes in many G13 residues impact p115RhoGEF more substantially than PDZRhoGEF, underscoring the specificity of G13 interactions with p115RhoGEF. Investigation of the p115RhoGEFs identified critical residues that contribute to interactions with G13 and G12. Our findings highlight residue-level differences in the molecular interactions of G13 with p115RhoGEF and PDZRhoGEF, providing insights into the specificity and regulation of G13-mediated signaling pathways. The resulting residue-level maps lay the groundwork for development of selective therapeutic strategies targeting G13-RhoGEF interactions.

biochemistry↗

bioRxiv2024

Multiple triggers converge to preferential effector coupling in the CB2R through a complex allosteric communication network

G protein-coupled receptors are important therapeutic drug targets for a wide range of diseases. Their ability to preferentially engage specific signaling pathways over others can be exploited to design drugs that target only disease-associated pathways leading to an improved safety profile. However, the underlying molecular mechanisms for preferential pathway engagement are complex and remain largely elusive. To elucidate the multifaceted actions at the receptor level that lead to preferential coupling, we employ a combination of techniques. Our approach integrates systematic mutagenesis of the CB2R and comprehensive profiling of Gi2 and {beta}-arrestin1 engagements with computer simulations to track mutant-induced impacts on receptor dynamics. Most importantly, our research discloses multiple triggers on a complex allosteric communication network (ACN) that converge to preferential CB2R coupling by modulating evolutionary conserved motifs (e.g., CWxP, NPxxY, sodium binding site). Potent triggers for a preferential Gi2 response exhibit high levels of connectivity and are located in proximity to connections with high information transmission. Our insights highlight the complexity of GPCR signaling and can guide the rational design of drug candidates tailored to evoke specific functional responses that can enhance the precision and efficacy of therapeutic interventions.

cell biology↗

bioRxiv2023

The G protein-coupled receptor TBXA2R activates ERMs to promote motility, invasion, and metastasis of triple-negative breast cancer cells.

Cell migration and invasion are critical processes for cancer cell metastasis, relying on the ability of cells to adapt their morphology. Proteins of the ezrin, radixin, and moesin (ERM) family are key regulators of cell morphogenesis and essential determinants of cancer cell metastasis. However, the mechanisms by which ERMs are activated in metastatic cells remain poorly understood. Here, we identify the thromboxane A2 receptor (TBXA2R), a G protein-coupled receptor overexpressed in multiple cancers, as a critical activator of ERMs, enhancing the motility and invasion of triple-negative breast cancer (TNBC) cells. We found that TBXA2R activates ERMs by engaging the Gq/11 and G12/13 subfamilies, the small GTPase RhoA, and its Ser/Thr kinase effectors SLK and LOK. Furthermore, we demonstrate that TBXA2R promotes TNBC cell motility and invasion in vitro and metastatic colonization in vivo, dependent on ERM function. These findings reveal a novel signaling axis by which a member of the largest class of receptors activates key metastatic determinants, thereby controlling various aspects of metastasis. This discovery opens new avenues for developing targeted therapies against cancer metastasis.

cancer biology↗

bioRxiv2021

Vasopressin V2 is a promiscuous G protein-coupled receptor that is biased by its peptide ligands

Activation of the G protein-coupled receptors by agonists may result in the activation of one or more G proteins, and in the recruitment of arrestins. The balance of activation of different pathways can be influenced by the ligand. Using BRET-based biosensors, we showed that the vasopressin V2 receptor activates or at least engages many different G proteins across all G protein subfamilies in response to its native agonist arginine vasopressin (AVP). This includes members of the Gi/o and G12/13 families that have not been previously reported. These signalling pathways are also activated by the synthetic peptide desmopressin and natural homologs of AVP, namely oxytocin and the non-mammalian hormone vasotocin. They demonstrated varying degrees of functional selectivity relative to AVP, as quantified using the operational model for quantifying ligand bias. Additionally, we modelled G protein activation as a Michaelis-Menten reaction. This approach provided a complementary way to quantify signalling bias, with an added benefit of clear separation of the effects of ligand affinity from the intrinsic activity of the receptor. These results showed that V2 receptor is not only promiscuous in its ability to engage several G proteins, but also that its signalling profile could be easily biased by small structural changes in the ligand.

pharmacology and toxicology↗

bioRxiv2020

Diverse chemotypes drive biased signaling by cannabinoid receptors

Cannabinoid CB1 and CB2 receptors are members of the G protein-coupled receptor family, which is the largest class of membrane proteins in the human genome. As part of the endocannabinoid system, they have many regulatory functions in the human body. Their malfunction therefore triggers a diverse set of undesired conditions, such as pain, neuropathy, nephropathy, pruritus, osteoporosis, cachexia and Alzheimers disease. Although drugs targeting the system exist, the molecular and functional mechanisms involved are still poorly understood, preventing the development of better therapeutics with fewer undesired effects. One path toward the development of better and safer medicines targeting cannabinoid receptors relies on the ability of some compounds to activate a subset of pathways engaged by the receptor while sparing or even inhibiting the others, a phenomenon known as biased signaling. To take advantage of this phenomenon for drug development, a better profiling of the pathways engaged by the receptors is required. Using a BRET-based signaling detection platform, we systematically analyzed the primary signaling cascades activated by CB1 and CB2 receptors, including 9 G protein and 2 {beta}-arrestin subtypes. Given that biased signaling is driven by ligand-specific distinct active conformations of the receptor, establishing a link between the signaling profiles elicited by different drugs and their chemotypes may help designing compounds that selectively activate beneficial pathways while avoiding those leading to undesired effects. We screened a selection of 35 structurally diverse ligands, including endocannabinoids, phytocannabinoids and synthetic compounds structurally similar or significantly different from natural cannabinoids. Our data show that biased signaling is a prominent feature of the cannabinoid receptor system and that, as predicted, ligands with different chemotypes have distinct signaling profiles. The study therefore allows for better understanding of cannabinoid receptors signaling and provides the information about tool compounds that can now be used to link signaling pathways to biological outcomes, aiding the design of improved therapeutics.

pharmacology and toxicology↗

bioRxiv2020

Selectivity Landscape of 100 Therapeutically Relevant GPCR Profiled by an Effector Translocation-Based BRET Platform

The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and {beta}arrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1,500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

pharmacology and toxicology↗