The GPR137 Knockout HEK293T Polyclonal Cells are a heterogeneous population of HEK293T cells engineered by CRISPR/Cas9-mediated disruption of the GPR137 gene, creating a loss-of-function model for this lysosomal orphan GPCR. The polyclonal nature encompasses multiple genetic edits, collectively ablating functional GPR137 expression without clonal isolation, thereby reducing clone-specific artefacts. Supplied as cryopreserved cells, this pool is suitable for direct recovery and expansion in autophagy and mTORC1 signaling studies. Each lot is quality controlled for viability and target-gene disruption.
HEK293T cells are human embryonic kidney epithelial cells stably expressing the SV40 large T antigen, facilitating high transfection efficiency and episomal plasmid replication. They are widely employed for transient protein expression, lentivirus production, and CRISPR genome engineering. Their intrinsic expression of mTORC1 pathway components, including the Rag GTPases and autophagy machinery, provides a relevant cellular context for dissecting lysosomal signaling. The rapid proliferation and experimental tractability of HEK293T cells enable robust phenotypic analysis of GPR137 knockout effects.
GPR137 localizes to lysosomal membranes and functions upstream of mTORC1, promoting kinase activation under nutrient-replete conditions. It achieves this by facilitating lysosomal positioning and cooperating with the Ragulator?CRag GTPase complex and RHEB, which in turn phosphorylates downstream effectors S6K and 4E-BP1, thereby suppressing autophagy. Loss of GPR137 impairs mTORC1 signaling, leading to dephosphorylation of S6K, increased LC3-II and p62 turnover, and enhanced autophagic flux. Additionally, GPR137 is transcriptionally regulated by TFEB, establishing a feedback loop between lysosomal biogenesis and nutrient sensing. Interacting partners include ARL8B and the Ragulator complex, positioning GPR137 as a critical node in lysosomal communication.
In HEK293T cells, GPR137 knockout unmasks its specific role in mTORC1?Cautophagy crosstalk. Comparative assays between wild-type and knockout cultures allow precise measurement of signaling outputs such as phospho-S6K levels and autophagic markers. The polyclonal nature diminishes clonal variation, offering a consistent model for mechanistic dissection. Complementation with wild-type GPR137 can validate functional domains, while pharmacological manipulation with mTOR inhibitors or lysosomal blockers further clarifies epistatic relationships. This model thus serves as a robust platform for studying lysosomal GPCR biology.
Key applications include western blotting for mTORC1 readouts (p-S6K, p-S6) and autophagy markers (LC3-II, p62), immunofluorescence with LAMP2 or LysoTracker to assess lysosomal positioning, and cell proliferation assays (MTT, colony formation). This model is particularly valuable for oncology research in glioblastoma and pancreatic ductal adenocarcinoma, where autophagy modulation impacts tumorigenesis. The polyclonal knockout cells are also amenable to drug screening and phosphoproteomic analysis. For additional technical information, please contact Ascent Research.
