Abstract 4384: Differential epitope tagging of oncogenic GNAQ mutants produces distinct effects on downstream signaling of GNAQ

Abstract GNAQ Q209P and Q209L are common driver mutations in uveal melanoma, leading to constitutive activation of downstream signaling of GNAQ. It is not fully understood whether the two GNAQ mutants have similar effects on downstream signaling pathways. Due to lack of available specific GNAQ antibody, epitope tagging is a well-established tool for protein interaction studies using mass spectrometry. Several studies used an epitope tag inserted into an internal region or the c-terminus of GNAQ to study its function. However, the functional consequences of these modalities on effector engagement are not fully elucidated. Here, we engineered both GNAQ wild-type and GNAQ mutants (Q209L and Q209P) without epitope tag as well as three different commonly used tagging modalities. A 3xflag tag of 22 amino acids was inserted after residue 122 of GNAQ. A MYC/DDK tag consisting of 18 amino acids was incorporated before the stop codon of GNAQ. An internal Glu-Glu epitope tag (EE tag) of 6 amino acids was introduced internally by mutating residues AYLPTQ(171-176) of GNAQ to EYMPTE. To assess the impact of the different modalities on the function of GNAQ mutants, we introduced them into 293FT cells and examined the known oncogenic signaling outputs downstream of GNAQ: MAPK, PKC and FAK signaling. We found that all three tags of both GNAQQ209L and GNAQQ209P activated MAPK signaling at similar levels as the corresponding untagged GNAQ mutants as evidenced by increases of pERK and pp90RSK level compared to GNAQ wild-type controls. By contrast, both GNAQQ209L and GNAQQ209P tagged with MYC/DDK induced lower p-PKD (a readout of PKC) and p-FAK (a readout of FAK activation) compared to EE-tagged or untagged, suggesting that MYC/DDK tag fused to the c-terminus of GNAQ affects specific effectors downstream of GNAQ. Furthermore, GNAQQ209P with the 3xflag tag induced lower levels of p-PKD and p-FAK than 3xflag tagged GNAQQ209L did, indicating that GNAQQ209P may have a different three-dimensional conformation compared to GNAQQ209L with different downstream effectors engagement. Moreover, the GNAQ/11 inhibitor YM-254890 inhibited phosphorylation of ERK, PKD, FAK in a concentration dependent manner in 293FT cells transfected with GNAQ mutants tagged with MYC/DDK but had no effect on MAPK, PKC and FAK signaling in cells expressing EE-tagged GNAQ mutants. These data suggest that EE tag has no effect on the function of GNAQ , but interferes with YM-254890 binding. Our findings highlight unappreciated consequences of epitope-tagging of the oncogene GNAQ that are relevant for studies on the interactome of mutant GNAQ and drug response. Citation Format: Chinchu Jagadan Ushakumari, Aashish Manglik, Boris C Bastian, Xu Chen. Differential epitope tagging of oncogenic GNAQ mutants produces distinct effects on downstream signaling of GNAQ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4384.