The Influence of Membrane Localization on Constitutively Active Gαq Signaling
Abstract
Uveal melanoma (UM), though a rare cancer, is the most common primary intraocular cancer in adults. Most patients with UM harbor a mutually exclusive mutation in either Gαq or Gα11; however, no drugs are clinically approved to target Gαq/11 in UM. YM-254890 (YM) and FR-900359 (FR) have gained much attention due to their ability to inhibit constitutively active Gαq by preventing the release of GDP. However, their mechanism of action remains to be fully understood. Additionally, while plasma membrane (PM) localization is essential for Gα signaling, little is known about the localization of Gαq in the context of UM, and no studies have reported the localization of Gαq upon inhibition by YM. The objectives of this thesis work were two-fold and included (1) determining if there are differences in localization of WT Gαq and GαqQ209L, and if mutant Gαq signaling can be disrupted by inhibiting palmitoylation and (2) determining the localization of WT and constitutively active GαqQ209L upon YM treatment. Importantly, the former provides insight into ways to inhibit constitutively active Gαq signaling in uveal melanoma, while the latter sheds light on the effects of YM treatment. To determine the localization of WT and constitutively active Gαq, live cell imaging and cellular fractionations were performed on HEK 293 cells and uveal melanoma cells. GαqQ209L mutants showed decreased localization at membranes compared to WT Gαq, suggesting increased palmitate turnover in constitutively active mutants. To test the hypothesis that palmitoylation is required for oncogenic GαqQ209L signaling, ERK phosphorylation and TEAD- and SRE-dependent luciferase activity were measured upon expression of palmitoylation-deficient GαqQ209L or after treatment with the palmitoylation inhibitor, 2-Bromopalmitate (2-BP). Palmitoylation-deficient GαqQ209L and 2-BP-treated GαqQ209L displayed a complete loss of PM localization and an inability to signal, demonstrating that disruption of membrane localization of GαqQ209L through inhibition of palmitoylation abrogates signaling. Immunofluorescence microscopy additionally showed that WT Gαq and GαqQ209L display a subcellular redistribution from the PM to the cytoplasm upon YM treatment, although WT Gαq displayed a more modest relocalization. To test the hypothesis that YM-induced relocalization is important for inhibition, PM-targeting motifs (myristylation, palmitoylation, or polybasic sites) were added to GαqQ209L to more tightly anchor GαqQ209L to the PM (termed PM-restricted GαqQ209L mutants). Immunofluorescence microscopy showed that PM-restricted GαqQ209L mutants do not redistribute into the cytoplasm upon YM treatment. Measurement of phosphorylated ERK and luciferase readouts determined that PM-restricted GαqQ209L mutants retain their ability to signal upon YM treatment, while PM-restricted WT Gαq remains sensitive to inhibition by YM. Pull-down techniques were used to indirectly determine if YM binds to PM-restricted GαqQ209L and promotes an inactive conformation. GαqQ209L and PM-restricted GαqQ209L mutants displayed increased binding to Gβγ and decreased binding to RGS2 and the DH/PHext domain of p63RhoGEF upon YM treatment, suggesting that the resistance of the PM-restricted mutants to YM is likely dependent on its sustained PM localization and potential differences in effector interaction in the cell, rather than an inability of the PM-restricted GαqQ209L mutants to bind to YM and adopt an inactive conformation. Collectively, these studies suggest that membrane localization is important for GαqQ209L signaling and that an additional way in which YM inhibits GαqQ209L is by promoting its subcellular redistribution from the PM to the cytoplasm.
Subject Area
Biochemistry|Molecular biology|Cellular biology
Recommended Citation
Randolph, Clinita, "The Influence of Membrane Localization on Constitutively Active Gαq Signaling" (2022). ProQuest ETD Collection - Thomas Jefferson University. AAI29396963.
https://jdc.jefferson.edu/dissertations/AAI29396963
