YM‐254890 Promotes the Subcellular Redistribution of Gαq

Abstract

Uveal melanoma (UM), though a rare form of cancer, is the most common primary intraocular cancer in adults. Approximately 50% of patients succumb to metastatic UM for which there are no effective treatments resulting in a survival period of 2‐8 months. Interestingly, most patients with UM harbor a mutually exclusive mutation in Gαq or Gα11. However, no drugs are clinically approved to target these proteins to treat 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 and thereby inhibiting GDP to GTP exchange. However, their mechanism of action and potential to prevent uveal melanoma progression is not fully understood. While Gα subunits are known to signal at the plasma membrane (PM), some studies also show that these proteins traffic into the cytoplasm or to intracellular organelles particularly upon activation. The objective of this study is to understand the changes in localization of Gαq wild type (WT) and constitutively active GαqQ209L that occur by inhibition with YM. By immunofluorescence microscopy we show that Gαq WT and GαqQ209L display a subcellular redistribution from the PM to the cytoplasm upon YM treatment. We hypothesize that this relocalization is important for YM’s ability to inhibit GαqQ209L. By adding PM‐targeting motifs (myristylation, palmitoylation, or polybasic sites) to constitutively active Gαq we show that the PM‐restricted mutants do not redistribute into the cytoplasm upon YM treatment and, surprisingly, retain their ability to signal upon YM treatment. Interestingly, our signaling assays show that G protein‐coupled receptor (GPCR) activated PM‐restricted Gαq WT remains sensitive to inhibition by YM. Using pull down techniques to determine if YM promotes an inactive conformation of the PM‐restricted GαqQ209L mutants we show that both GαqQ209L and PM‐restricted GαqQ209L mutants display increased binding to Gβγ and decreased binding to RGS2 upon YM treatment, suggesting that YM promotes an inactive conformation in these PM‐restricted mutants in the same manner as with Gαq WT and GαqQ209L. Our studies suggest that an additional way in which YM inhibits GαqQ209L is by promoting the subcellular redistribution of constitutively active Gαq from the PM to the cytoplasm.