The spatial distribution of GPCR and Gβγ activity across a cell dictates PIP3 dynamics

Abstract

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 diseases, including cancer, 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. Though the mechanisms are unclear, PIP3 produced upon GPCR activation attenuates within minutes, indicating a tight temporal regulation. Our data show that subcellular redistributions of G proteins govern this PIP3 attenuation when GPCRs are activated globally, while localized GPCR activation induces sustained subcellular PIP3. Interestingly the observed PIP3 attenuation was Gγ subtype-dependent. Considering distinct cell-tissue-specific Gγ expression profiles, our findings not only demonstrate how the GPCR-induced PIP3 response is regulated depending on the GPCR activity gradient across a cell, but also show how diversely cells respond to spatial and temporal variability of external stimuli.

Engineered blue-shifted melanopsins for subcellular optogenetics

“…Therefore, to examine MeOp-induced localized PIP3 generation, we used RAW264.7 cells. RNAseq data shows that the endogenous Gαq level in RAW264.7 cells is significantly lower than other Gα subtypes 67 . Therefore, as described previously 67,68 , to increase Gβγ contribution from the Gq pathway, in addition to A333T or WT-MeOP and Akt-PH-mCh (PIP3 sensor), we also expressed Gαq-CFP in RAW264.7 cells.…”

“…RNAseq data shows that the endogenous Gq level in RAW264.7 cells is significantly lower than other G subtypes 67 . Therefore, as described previously 67,68 , to increase G contribution from the Gq pathway, in addition to A333T or WT-MeOP and Akt-PH-mCh (PIP3 sensor), we also expressed Gq-CFP in RAW264.7 cells. Next, we exposed one side of the cell to blue light (Fig.…”

“…Previously, we have shown localized blue opsin activation and the resultant Gγ-induced localized PIP3 and directional migration in live cells 30,67 . However, unlike RAW264.7 macrophages, due to the lack of appropriate G types, HeLa cells do not show PIP3 generation upon Gi/o GPCR activation 46 .…”

Spatiotemporal Optical Control of Gαq-PLCβ Interactions

“…In addition to Gγ9 translocation, we examined the Gβγ-activated phosphoinositide-3-kinases (PI3Ks) induced PIP3 generation in RAW264.7 cells. We employed RAW264.7 cells because, as shown previously, HeLa cells did not show an observable PIP3 generation due to the lack of high membrane affinity Gγs. , PIP3 generation was measured using the translocation of the fluorescently tagged PIP3 sensor (Akt-PH-Venus) from the cytosol to the plasma membrane. Since GRPR-induced PIP3 generation in cells only with endogenous Gαq is minor and is enhanced by Gαq expression (Figure B, left), for this experiment, we use RAW264.7 cells expressing GRPR, Opto-dHTH, iLID-CAAX, Akt-PH-Venus, and Gαq.…”

“…DNA constructs used were as follows: GRPR, mRFP-HTH, mRFP, Lyn-mRFP-HTH, Lyn-mRFP, CRY2-mCherry-HTH, Venus-iLID-HTH and variants, Lyn-SspB, mCherry-PH, Venus-PH, mCherry-DBD, mCherry-iLID-HTH, dHTH-mCherry-SspB, dHTH-51 residue linker-mCherry-SspB, Opto-dHTH, Lyn-mRFP-HTH L859E, Lyn-mRFP-p63 HTH, Lyn-mRFP-Kalirin HTH, Lyn-mRFP-Trio HTH, LARG-mCherry-SspB, Gαq-CFP, GRK2-YFP, Venus-miniGq, CIBN-CAAX, iLID-CAAX (Addgene plasmid # 85680), melanopsin, Akt-PH-mCherry, Akt-PH-Venus, Venus-rGBD, and mCherry-Gγ9. ,,, GRPR was a kind gift from the laboratory of Zhou-Feng Chen at Washington University, St. Louis, MO. mCherry-PH, Venus-PH, Gαq–CFP, mCherry-DBD, melanopsin, Akt-PH-mCherry, Akt-PH-Venus and fluorescently tagged Gγ9 were kindly provided by Dr. N. Gautam’s laboratory, Washington University in St Louis, MO.…”