Biology subjects

Lukasheva, V.

Publications and source records attributed to Lukasheva, V..

3 recordsLinked to original sources

bioRxiv2026

Unconventional Interplay Between GPCRs and RTKs Signaling Pathways Through SH2 Domain-Containing Proteins

Crosstalk across two major receptor families involved in signal transduction, namely receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), have been observed at different levels of their signaling cascades. Using newly developed enhanced bystander bioluminescence resonance energy transfer (ebBRET)-based biosensors that monitor the recruitment of SH2 domains to activated RTKs, we assessed the ability of GPCRs to modulate cellular localization of SH2 domains. Receptor-mediated activation of either Gq/11 or G12/13 but not Gs or Gi/o (e.g., thromboxane A2 receptor, TP, and type-2 protease activated receptor, PAR2) resulted in the plasma membrane (PM) dissociation of SH2 domains derived from RTKs effectors such as GRB2, STAT5 and PLC{gamma}1. The role of Gq/11, G12/13, Rho and downstream kinases in the subcellular SH2 domain redistribution was further confirmed using both pharmacological and genetic approaches. BRET imaging and spectrometric analyses showed that the dissociation of SH2 domains from the PM was accompanied by their accumulation in the nucleus and a reduction in RTK signaling activity, as determined using a STAT5 transcriptional assay. The effect of Gq/11 and G12/13 activation on STAT5 transcriptional activity was observed both in engineered systems and in HeLa cells endogenously expressing all the components of the regulatory mechanism. The Gq/11 / G12/13-mediated redistribution of SH2 domain-containing proteins represents an undescribed mechanism through which GPCRs regulate RTKs activity. Significance StatementThis study reveals a novel crosstalk mechanism between G protein coupled receptors and receptor tyrosine kinases showing that Gq/11 and G12/13 activation triggers Rho-dependent translocation of SH2-containing effector proteins, such as GRB2, PLC{gamma}1 and STAT5. This process causes compartmentalization inside the nucleus and thus reduces their availability at the plasma membrane, leading to attenuated RTK responses.

cell biology↗

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↗

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↗