Disruption of the interaction between mutationally activated Gαq and Gβγ attenuates aberrant signaling

Heterotrimeric G protein stimulation via G protein–coupled receptors promotes downstream proliferative signaling. Mutations can occur in Gα proteins which prevent GTP hydrolysis; this allows the G proteins to signal independently of G protein–coupled receptors and can result in various cancers, such as uveal melanoma (UM). Most UM cases harbor Q209L, Q209P, or R183C mutations in Gαq/11 proteins, rendering the proteins constitutively active (CA). Although it is generally thought that active, GTP-bound Gα subunits are dissociated from and signal independently of Gβγ, accumulating evidence indicates that some CA Gα mutants, such as Gαq/11, retain binding to Gβγ, and this interaction is necessary for signaling. Here, we demonstrate that disrupting the interaction between Gβγ and Gαq is sufficient to inhibit aberrant signaling driven by CA Gαq. Introduction of the I25A point mutation in the N-terminal α helical domain of CA Gαq to inhibit Gβγ binding, overexpression of the G protein Gαo to sequester Gβγ, and siRNA depletion of Gβ subunits inhibited or abolished CA Gαq signaling to the MAPK and YAP pathways. Moreover, in HEK 293 cells and in UM cell lines, we show that Gαq-Q209P and Gαq-R183C are more sensitive to the loss of Gβγ interaction than Gαq-Q209L. Our study challenges the idea that CA Gαq/11 signals independently of Gβγ and demonstrates differential sensitivity between the Gαq-Q209L, Gαq-Q209P, and Gαq-R183C mutants. Heterotrimeric G protein stimulation via G protein–coupled receptors promotes downstream proliferative signaling. Mutations can occur in Gα proteins which prevent GTP hydrolysis; this allows the G proteins to signal independently of G protein–coupled receptors and can result in various cancers, such as uveal melanoma (UM). Most UM cases harbor Q209L, Q209P, or R183C mutations in Gαq/11 proteins, rendering the proteins constitutively active (CA). Although it is generally thought that active, GTP-bound Gα subunits are dissociated from and signal independently of Gβγ, accumulating evidence indicates that some CA Gα mutants, such as Gαq/11, retain binding to Gβγ, and this interaction is necessary for signaling. Here, we demonstrate that disrupting the interaction between Gβγ and Gαq is sufficient to inhibit aberrant signaling driven by CA Gαq. Introduction of the I25A point mutation in the N-terminal α helical domain of CA Gαq to inhibit Gβγ binding, overexpression of the G protein Gαo to sequester Gβγ, and siRNA depletion of Gβ subunits inhibited or abolished CA Gαq signaling to the MAPK and YAP pathways. Moreover, in HEK 293 cells and in UM cell lines, we show that Gαq-Q209P and Gαq-R183C are more sensitive to the loss of Gβγ interaction than Gαq-Q209L. Our study challenges the idea that CA Gαq/11 signals independently of Gβγ and demonstrates differential sensitivity between the Gαq-Q209L, Gαq-Q209P, and Gαq-R183C mutants. Heterotrimeric G proteins (Gαβγ) are canonically activated through G protein–coupled receptors (GPCRs), which allows for GDP release from the Gα subunit and further GTP binding. It is generally accepted that GTP binding on the Gα subunit promotes an active conformation and allows for the dissociation from Gβγ. Active GTP-bound Gα proteins can further bind and stimulate downstream effectors (1Oldham W.M. Hamm H.E. Heterotrimeric G protein activation by G-protein-coupled receptors.Nat. Rev. Mol. Cell Biol. 2008; 9: 60-71Crossref PubMed Scopus (842) Google Scholar, 2Wu D.Q. Lee C.H. Rhee S.G. Simon M.I. Activation of phospholipase C by the alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells.J. Biol. Chem. 1992; 267: 1811-1817Abstract Full Text PDF PubMed Google Scholar). Hydrolysis of GTP on the Gα subunit promotes reassociation of the inactive Gαβγ heterotrimer and completes the cycle. Several conserved amino acids, found in all Gα subunits, are critical for the intrinsic GTP hydrolysis activity, and mutations of these residues prevent GTP hydrolysis and “lock” the Gα protein in a constitutively active (CA), GTP-bound state (3Lapadula D. Benovic J.L. Targeting oncogenic Galphaq/11 in uveal melanoma.Cancers (Basel). 2021; 13Crossref PubMed Scopus (5) Google Scholar, 4Offermanns S. G-proteins as transducers in transmembrane signalling.Prog. Biophys. Mol. Biol. 2003; 83: 101-130Crossref PubMed Scopus (221) Google Scholar). Aberrant CA mutations in Gα proteins have been identified in various human tumors and diseases. For example, Gαs CA mutations have been identified in a subset of pancreatic tumors (5Landis C.A. Masters S.B. Spada A. Pace A.M. Bourne H.R. Vallar L. GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours.Nature. 1989; 340: 692-696Crossref PubMed Scopus (1246) Google Scholar, 6O'Hayre M. Vazquez-Prado J. Kufareva I. Stawiski E.W. Handel T.M. Seshagiri S. et al.The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer.Nat. Rev. Cancer. 2013; 13: 412-424Crossref PubMed Scopus (398) Google Scholar). Mutations in Gαq and Gα11 have also been described at residues Q209 and R183 in the majority of patients with uveal melanoma (UM). Q209L or Q209P mutations in Gαq or Gα11 occur in 90% of UM patients, and mutations at R183 occur in about 5% of patients (7Chua V. Lapadula D. Randolph C. Benovic J.L. Wedegaertner P.B. Aplin A.E. Dysregulated GPCR signaling and therapeutic options in uveal melanoma.Mol. Cancer Res. 2017; 15: 501-506Crossref PubMed Scopus (59) Google Scholar, 8Robertson A.G. Shih J. Yau C. Gibb E.A. Oba J. 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Uveal melanoma: epidemiologic aspects.Ophthalmol. Clin. North Am. 2005; 18 (viii): 75-84Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 12Shields C.L. Kaliki S. Livesey M. Walker B. Garoon R. Bucci M. et al.Association of ocular and oculodermal melanocytosis with the rate of uveal melanoma metastasis: analysis of 7872 consecutive eyes.JAMA Ophthalmol. 2013; 131: 993-1003Crossref PubMed Scopus (106) Google Scholar). The constitutive activity of Gαq/11 in UM, driven by the activating Q209L/P and R183 mutations, promotes the stimulation of two major signaling pathways: 1) the mitogen-activated protein kinase (MAPK) pathway and 2) the Yes-associated protein and Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) pathway. The MAPK cascade is stimulated by the direct binding of CA Gαq/11 to phospholipase C–β (PLC-β), which hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol further activates PKC, which phosphorylates and activates RasGRP3. The further activation of Ras promotes the stimulation of the MAPK cascade and results in the phosphorylation and activation of ERK. Phospho-ERK (pERK) dimerizes and translocates into the nucleus to bind transcription factors and promote the transcription of proliferative genes (13Chen X. Wu Q. Depeille P. Chen P. Thornton S. Kalirai H. et al.RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma.Cancer Cell. 2017; 31: 685-696.e6Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Mutationally active Gαq/11 also stimulates the YAP/TAZ pathway through direct binding and activation of the RhoGEF Trio. In an inactive state, YAP is phosphorylated and remains cytoplasmic through mediators of the Hippo pathway. Activation of Trio leads to the stimulation of focal adhesion kinase, which inhibits mediators of the Hippo pathway and ultimately leads to the dephosphorylation and nuclear translocation of YAP. YAP binds to the TEA domain (TEAD) transcription factor in the nucleus to promote the transcription of genes involved in proliferation and cell survival (14Feng X. Arang N. Rigiracciolo D.C. Lee J.S. Yeerna H. Wang Z. et al.A platform of synthetic lethal gene interaction networks reveals that the GNAQ uveal melanoma oncogene controls the Hippo pathway through FAK.Cancer Cell. 2019; 35: 457-472.e5Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 15Feng X. Degese M.S. Iglesias-Bartolome R. Vaque J.P. Molinolo A.A. Rodrigues M. et al.Hippo-independent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated rho GTPase signaling circuitry.Cancer Cell. 2014; 25: 831-845Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). To date, inhibitors for downstream targets of CA Gαq/11 have been proven to be generally unsuccessful in disrupting the progression of metastatic UM (16Carvajal R.D. Sosman J.A. Quevedo J.F. Milhem M.M. Joshua A.M. Kudchadkar R.R. et al.Effect of selumetinib vs chemotherapy on progression-free survival in uveal melanoma: a randomized clinical trial.JAMA. 2014; 311: 2397-2405Crossref PubMed Scopus (328) Google Scholar, 17Steeb T. Wessely A. Ruzicka T. Heppt M.V. Berking C. How to MEK the best of uveal melanoma: a systematic review on the efficacy and safety of MEK inhibitors in metastatic or unresectable uveal melanoma.Eur. J. Cancer. 2018; 103: 41-51Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 18Brouwer N.J. Konstantinou E.K. Gragoudas E.S. Marinkovic M. Luyten G.P.M. Kim I.K. et al.Targeting the YAP/TAZ pathway in uveal and conjunctival melanoma with verteporfin.Invest Ophthalmol. Vis. Sci. 2021; 62: 3Crossref PubMed Google Scholar). Because Gαq/11 stimulates multiple pathways, directly targeting CA Gαq/11 may be a more promising therapeutic strategy for metastatic UM patients. There are currently no FDA-approved drugs that target Gαq/11; however, the compounds YM-254890 (YM) and FR900359 (FR) have been shown to inhibit both WT and CA Gαq/11 and have shown promising results in cancer cell models (19Lapadula D. Farias E. Randolph C.E. Purwin T.J. McGrath D. Charpentier T.H. et al.Effects of oncogenic Galphaq and Galpha11 inhibition by FR900359 in uveal melanoma.Mol. Cancer Res. 2019; 17: 963-973Crossref PubMed Scopus (60) Google Scholar, 20Annala S. Feng X. Shridhar N. Eryilmaz F. Patt J. Yang J. et al.Direct targeting of Galphaq and Galpha11 oncoproteins in cancer cells.Sci. Signal. 2019; 12eaau5948Crossref PubMed Scopus (72) Google Scholar, 21Onken M.D. Makepeace C.M. Kaltenbronn K.M. Kanai S.M. Todd T.D. 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Sci. 2021; 4: 888-897Crossref PubMed Scopus (12) Google Scholar). However, both YM and FR to date have had varying effects on UM tumor arrest and regression in preclinical UM mouse models (24Onken M.D. Makepeace C.M. Kaltenbronn K.M. Choi J. Hernandez-Aya L. Weilbaecher K.N. et al.Targeting primary and metastatic uveal melanoma with a G protein inhibitor.J. Biol. Chem. 2021; 296: 100403Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 25Hitchman T.D. Bayshtok G. Ceraudo E. Moore A.R. Lee C. Jia R. et al.Combined inhibition of Galphaq and MEK enhances therapeutic efficacy in uveal melanoma.Clin. Cancer Res. 2021; 27: 1476-1490Crossref PubMed Scopus (24) Google Scholar). Therefore, understanding how CA Gαq/11 is regulated in cells and further exploring other methods of inhibiting oncogenic Gαq/11 may provide therapeutic benefits for metastatic UM patients. In an inactive, GDP-bound state, Gα proteins form heterotrimers with Gβγ. This interaction with Gβγ aids in plasma membrane localization to neighboring GPCRs and helps stabilize the complex for GTP exchange (26Evanko D.S. Thiyagarajan M.M. Siderovski D.P. Wedegaertner P.B. Gbeta gamma isoforms selectively rescue plasma membrane localization and palmitoylation of mutant Galphas and Galphaq.J. Biol. Chem. 2001; 276: 23945-23953Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 27Evanko D.S. Thiyagarajan M.M. Wedegaertner P.B. Interaction with Gbetagamma is required for membrane targeting and palmitoylation of Galpha(s) and Galpha(q).J. Biol. Chem. 2000; 275: 1327-1336Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 28Fishburn C.S. Pollitt S.K. Bourne H.R. Localization of a peripheral membrane protein: gbetagamma targets Galpha(Z).Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1085-1090Crossref PubMed Scopus (51) Google Scholar, 29Duc N.M. Kim H.R. Chung K.Y. 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However, work in our lab and others suggest that the CA Gαq-Q209L mutant retains a functionally important interaction with Gβγ via the N-terminal α helical domain (32Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. The 2.0 A crystal structure of a heterotrimeric G protein.Nature. 1996; 379: 311-319Crossref PubMed Scopus (1056) Google Scholar, 33Nishimura A. Kitano K. Takasaki J. Taniguchi M. Mizuno N. Tago K. et al.Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 13666-13671Crossref PubMed Scopus (182) Google Scholar, 34Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. et al.The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2.Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1021) Google Scholar). Previous work in our lab indicates that a single point mutation (I25A) in the N-terminal α helix of WT Gαq is sufficient to disrupt binding to Gβγ, and this disruption of binding further prevents GPCR-dependent signaling of WT Gαq (35Evanko D.S. Thiyagarajan M.M. Takida S. Wedegaertner P.B. Loss of association between activated Galpha q and Gbetagamma disrupts receptor-dependent and receptor-independent signaling.Cell Signal. 2005; 17: 1218-1228Crossref PubMed Scopus (30) Google Scholar). The I25A N-terminal Gβγ-binding mutation was also sufficient to disrupt overactive inositol phosphate (IP) production for the CA Gαq-R183C mutant. Understanding the role of Gβγ in the oncogenic signaling activity of CA Gαq could address the gaps in knowledge of the cellular regulation of CA Gαq and further offer Gβγ as a potential target for disrupting aberrant signaling in UM. Here, we tested the hypothesis that disrupting the interaction between Gβγ and the CA Gαq mutants commonly found in UM patients—Q209L (QL), Q209P (QP), and R183C (RC)—is sufficient to inhibit oncogenic signaling of these mutationally active Gαq. We provide evidence that disrupting the interaction between CA Gαq and Gβγ significantly inhibits Gαq-mediated activation of the MAPK and YAP/TAZ pathways. Surprisingly, we show differential sensitivity between Gαq-QL and the Gαq-QP/RC mutants in that oncogenic signaling by Gαq-QP and Gαq-RC is more sensitive to the disruption of Gβγ binding than Gαq-QL. Previous work has shown that the activation of downstream effectors of Gαq, such as PLC-β, requires the binding of Gαq to Gβγ (35Evanko D.S. Thiyagarajan M.M. Takida S. Wedegaertner P.B. Loss of association between activated Galpha q and Gbetagamma disrupts receptor-dependent and receptor-independent signaling.Cell Signal. 2005; 17: 1218-1228Crossref PubMed Scopus (30) Google Scholar). Furthermore, recent studies have indicated that CA Gαq-Q209L proteins retain binding to Gβγ at the N-terminal α-helical region (36Cervantes-Villagrana R.D. Adame-Garcia S.R. Garcia-Jimenez I. Color-Aparicio V.M. Beltran-Navarro Y.M. Konig G.M. et al.Gbetagamma signaling to the chemotactic effector P-REX1 and mammalian cell migration is directly regulated by Galphaq and Galpha13 proteins.J. Biol. Chem. 2019; 294: 531-546Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). It has been established that the I25A point mutation within the N-terminal α-helical domain of Gαq is sufficient to disrupt the interaction between Gαq and Gβγ and consequently inhibit the GPCR-dependent signaling of WT Gαq (35Evanko D.S. Thiyagarajan M.M. Takida S. Wedegaertner P.B. Loss of association between activated Galpha q and Gbetagamma disrupts receptor-dependent and receptor-independent signaling.Cell Signal. 2005; 17: 1218-1228Crossref PubMed Scopus (30) Google Scholar). Thus, we hypothesized that introducing the I25A mutation to disrupt binding to Gβγ would inhibit oncogenic signaling through the CA Gαq-Q209L (QL), Q209P (QP), and R183C (RC) mutants. To monitor the activity of the YAP/TAZ pathway, we transfected the CA mutants, with and without the I25A mutation, into HEK 293 Gαq/11 CRISPR-Cas9 KO cells and monitored YAP activity using the TEAD luciferase reporter assay, which is stimulated in response to YAP activation and nuclear translocation. Gαq-QL, Gαq-QP, and Gαq-RC all showed strong activation in the assay. Cells expressing the pcDNA3 vector and WT Gαq were used as negative controls and indicated no significant change in TEAD luciferase reporter activity (Fig. 1A). The Gαq-QL-C9,10S palmitoylation-deficient mutant was used as a negative control for Gαq-QL. The C9,10S mutation in WT Gαq has been previously established to prevent plasma membrane localization and consequently prevent GPCR-dependent activation (37Wedegaertner P.B. Chu D.H. Wilson P.T. Levis M.J. Bourne H.R. Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha.J. Biol. Chem. 1993; 268: 25001-25008Abstract Full Text PDF PubMed Google Scholar); likewise, we now show that Gαq-QL-C9,10S is unable to stimulate TEAD-dependent luciferase activity. Interestingly, Gαq-I25A-QL displayed a decreased ability compared to Gαq-QL to activate TEAD luciferase activity, yet retained significantly higher activity than the Gαq-QL-C9,10S control. In contrast, the introduction of the I25A mutation abolished the ability of Gαq-QP and Gαq-RC to stimulate TEAD luciferase. These data demonstrate that the introduction of the I25A mutation inhibits oncogenic signaling to the YAP pathway by the CA Gαq-QL/P and Gαq-RC mutants. These results also suggest potential differential sensitivity between the CA Gαq-QL and Gαq-QP/RC mutants. To further monitor YAP activity and the differential sensitivity between the CA Gαq-I25A mutants, we used immunofluorescence microscopy to examine the cellular localization of YAP (Fig. 1B). It has been previously established that activation of the Rho-dependent YAP pathway results in stabilization and dephosphorylation of YAP and its subsequent translocation into the nucleus (38Liu-Chittenden Y. Huang B. Shim J.S. Chen Q. Lee S.J. Anders R.A. et al.Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP.Genes Dev. 2012; 26: 1300-1305Crossref PubMed Scopus (1012) Google Scholar, 39Ramos A. Camargo F.D. The Hippo signaling pathway and stem cell biology.Trends Cell Biol. 2012; 22: 339-346Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). The YAP pathway was not stimulated in cells expressing the pcDNA3 vector and WT Gαq, as shown by the low expression and cytoplasmic localization of YAP. Expression of Gαq-QL indicated strong nuclear localization of YAP, and treatment with the Gαq/11 inhibitor YM-254890 (YM) as a control resulted in the loss of nuclear YAP and its accumulation in the cytoplasm. Gαq-I25A-QL also promoted nuclear localization of YAP, consistent with its retention of signaling in the TEAD-luciferase assay (Fig. 1A). Cells expressing CA Gαq-QP and Gαq-RC had robust nuclear YAP localization, indicating stimulation of this pathway. However, Gαq-I25A-QP and Gαq-I25A-RC failed to stimulate the nuclear localization of YAP, as indicated by the cytoplasmic localization of YAP. Similar to the TEAD-luciferase reporter assay results, this data suggests that Gαq-I25A-QL is more resistant to disruption of signaling than the Gαq-I25A-QP and Gαq-I25A-RC mutants. In immunofluorescence microscopy studies, we also examined the subcellular localization of the CA Gαq mutants with and without the I25A mutation (Fig. S1). Previous work has demonstrated that the interaction with Gβγ is crucial for palmitoylation and further membrane localization of Gαq (27Evanko D.S. Thiyagarajan M.M. Wedegaertner P.B. Interaction with Gbetagamma is required for membrane targeting and palmitoylation of Galpha(s) and Galpha(q).J. Biol. Chem. 2000; 275: 1327-1336Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Gαq-QL and Gαq-RC were localized to the plasma membrane in transfected cells. The introduction of the I25A mutation does not significantly impact the plasma membrane localization of Gαq-QL but results in partial loss of plasma membrane localization of Gαq-RC (Fig. S1). Interestingly, Gαq-QP had only partial plasma membrane association, and introduction of the I25A mutation resulted in an almost complete loss of plasma membrane localization (Fig. S1). Thus, decreased plasma membrane localization of Gαq-RC and Gαq-QP when interaction with Gβγ is disrupted through introduction of the I25A mutation provides at least a partial mechanistic explanation for their loss of signaling. These differences in cellular localization of the Gαq-I25A-QL, Gαq-I25A-QP, and Gαq-I25A-RC (Fig. S1) likely contribute to the differential sensitivity in aberrant cell signaling (Fig. 1). Along with stimulation of the YAP pathway, CA Gαq/11 also canonically stimulates PLC-β and further activates the MAPK cascade. To monitor the effects of the I25A mutants on the MAPK pathway, we quantified pERK levels in HEK 293 Gαq/11 KO cells transfected with the CA Gαq constructs and the I25A mutants (Fig. 1, C and D). Expression of CA Gαq-QL resulted in pERK stimulation >4-fold compared to cells transfected with pcDNA3 or WT Gαq. The Gαq-I25A-QL mutant also activated pERK at a slightly decreased level compared to Gαq-QL, although the difference was not statistically significant. Interestingly, the Gαq-QP and Gαq-RC mutants stimulated pERK, but pERK activity was significantly decreased in cells expressing the Gαq-I25A-QP and Gαq-I25A-RC mutants (Fig. 1, C and D). The effects of the I25A mutants on MAPK activity were further validated with the serum response element (SRE) luciferase reporter assay, which monitors both Rho and MAPK-dependent activity (Fig. 1E). As indicated, the CA Gαq-QL mutant robustly stimulated SRE-dependent luciferase activity. Introduction of the I25A mutation into Gαq-QL (Gαq-I25A-QL) strongly inhibited the ability to stimulate SRE luciferase activity, similar to the lack of signaling by the Gαq-QL-C9,10S palmitoylation-deficient mutant and the loss of signaling upon treatment of cells expressing Gαq-QL with YM. As expected, the CA Gαq-QP and Gαq-RC mutants also robustly stimulated SRE luciferase activity, while the Gαq-I25A-QP and Gαq-I25A-RC mutants completely failed to stimulate luciferase activity, similar to cells treated with YM (Fig. 1E). The decreased signaling by Gαq-I25A-QL in comparison to Gαq-QL is much more drastic in the SRE luciferase assay than the pERK immunoblot assay (Fig. 1, C and D). Although this is somewhat surprising since the SRE luciferase readout is downstream of the MAPK pathway, we note that the SRE luciferase assay provides a very high signal-to-noise readout for Gαq-QL signaling (>100-fold above basal vector alone or WT Gαq) and is consistently very sensitive to disruption of Gαq-QL signaling (e.g., Gαq-I25A-QL only signals 5-6-fold above basal). Taken together, these results indicate that the introduction of the I25A mutation to disrupt binding to Gβγ inhibited oncogenic signaling of the CA Gαq mutants through the YAP and MAPK pathways, and the Gαq-QP and Gαq-RC mutants are more sensitive to the I25A mutation than Gαq-QL. Previous studies in our lab have elucidated that CA Gαq-QL has an increased association with Gβγ compared to Gαq-RC (35Evanko D.S. Thiyagarajan M.M. Takida S. Wedegaertner P.B. Loss of association between activated Galpha q and Gbetagamma disrupts receptor-dependent and receptor-independent signaling.Cell Signal. 2005; 17: 1218-1228Crossref PubMed Scopus (30) Google Scholar). Furthermore, we have also shown that Gαq-I25A-QL maintains a substantial association with Gβγ compared to an almost complete loss of Gβγ association when the I25A mutation is introduced into WT Gαq or Gαq-RC (35Evanko D.S. Thiyagarajan M.M. Takida S. Wedegaertner P.B. Loss of association between activated Galpha q and Gbetagamma disrupts receptor-dependent and receptor-independent signaling.Cell Signal. 2005; 17: 1218-1228Crossref PubMed Scopus (30) Google Scholar). It has also been previously established that Gαq-QP has reduced binding to effector proteins, such as p63RhoGEF, Trio, and GRK2 (40Maziarz M. Leyme A. Marivin A. Luebbers A. Patel P.P. Chen Z. et al.Atypical activation of the G protein Galphaq by the oncogenic mutation Q209P.J. Biol. Chem. 2018; 293: 19586-19599Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Thus, we wanted to further characterize the interactions between Gβγ and CA Gαq-QL/P and Gαq-RC and the corresponding I25A mutants. To study this, we transiently transfected the WT or CA Gαq and I25A mutants into HEK293 cells which stably expressed 6x-His-Gβ1y2 and pulled down Gβ1 using Ni-NTA beads. The relative association of Gαq bound to Gβ1γ2 was revealed through immunoblotting (Fig. 2A) and was further quantified (Fig. 2B). As expected, WT Gαq displayed a strong pull down with Gβ1γ2, but the introduction of the I25A mutation into WT Gαq strongly decreased this interaction, indicating that the I25A mutation disrupts binding between Gαq and Gβγ. Interestingly, Gαq-QL had a similar binding association to Gβ1γ2 with that of WT Gαq, and Gαq-QL bound significantly stronger to Gβ1γ2 than Gαq-QP and Gαq-RC (Fig. 2, A and B). Furthermore, Gαq-I25A-QL was more strongly bound to Gβ1γ2 than Gαq-I25A-QP and Gαq-I25A-RC (Fig. 2, A and B). We did not see a statistically significant difference in association with Gβγ between Gαq-QP and Gαq-RC and their corresponding I25A mutants. This is likely due to the low initial binding to Gβγ with both Gαq-QP and Gαq-RC. Although we previously demonstrated the surprising ability of Gαq-QL to interact strongly with Gβγ and the poor interaction of Gαq-RC with Gβγ, we now show a dramatically decreased association of Gαq-QP with Gβγ compared to Gαq-QL, even though both have a mutation of Q209. This differential binding to Gβγ between the CA Gαq mutants likely contributes to the varying sensitivity in oncogenic signal disruption between the I25A mutants (Fig. 1). Considering that inhibiting the association between the CA Gαq mutants and Gβγ with the N