Kankanamge, D. | Search bioRxiv

bioRxiv2023

Spatiotemporal optical control of Gαq-PLCβ interactions

Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins, including G protein-coupled receptors (GPCRs). The Gq pathway activation by GPCRs is a major signaling axis with a broad physiological and pathological significance. Compared to other G members, GqGTP activates many crucial effectors, including PLC{beta} (Phospholipase C{beta}) and Rho GEFs (Rho guanine nucleotide exchange factors). PLC{beta} regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal - debilitating diseases, including cancer, epilepsy, Huntingtons Disease, and Alzheimers Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLC{beta} signaling controls cellular physiology has been difficult. Since activated PLC{beta} induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GqGTP-PLC{beta} interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLC{beta} signaling control, our data indicate that the molecular competition between RhoGEFs and PLC{beta} for GqGTP determines the potency of Gq-GPCR-governed directional cell migration.

molecular biology↗

bioRxiv2022

Molecular regulation of GPCR-G-protein-governed PIP3 generation and its adaptation

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a plasma membrane-bound signaling phospholipid involved in many cellular signaling pathways that control crucial cellular processes and behaviors, including cytoskeleton remodeling, metabolism, chemotaxis, and apoptosis. Therefore, defective PIP3 signaling is implicated in various disease driving processes, including cancer metastasis, diabetes, obesity, and cardiovascular diseases. Upon activation by G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), phosphoinositide-3-kinases (PI3Ks) phosphorylate phosphatidylinositol (4,5) bisphosphate (PIP2), generating PIP3. Interestingly, though the mechanisms are unclear, PIP3 produced upon GPCR activation attenuates within minutes, indicating a tight temporal regulation. Our data show the subcellular redistributions of G proteins govern this PIP3 attenuation in the presence of sustained receptor stimulation, and thus meet the definition of signaling adaptation. Interestingly the observed adaptation of PIP3 was G{gamma} subtype-dependent. Considering distinct cell-tissue-specific G{gamma} expression profiles, our findings not only demonstrate how the GPCR-induced PIP3 response is adapted but also show how diversely this adaptation process is regulated by the dominant G{gamma}s of a cell.

cell biology↗