AAV ACTOne GPCR Assay Kits
GPCRs
G protein-coupled receptors (GPCRs) are a large, diverse group of membrane proteins that play a key role in cellular communication and signal transduction, regulating a wide range of physiological processes. As one of the most extensively studied drug targets, over 30% of FDA-approved drugs are designed to target GPCRs.
- Gi-GPCRs: Inhibit adenylyl cyclase activity, reducing cAMP production and causing inhibitory cellular effects.
- Gs-GPCRs: Stimulate adenylyl cyclase, increasing cAMP production and promoting stimulatory effects in the cell.
- Gq-GPCRs: Activate phospholipase C (PLC), which triggers calcium release and activates protein kinase C (PKC).
- G12/13-GPCRs: Regulate the cytoskeleton and cell motility via Rho GTPases, influencing cell movement and shape.
This classification highlights how different GPCR subtypes control various signaling pathways, impacting cell behavior and physiological responses.
ACTOne GPCR Assay
ACTOne™ is the only high-throughput GPCR screening technology that directly measures intracellular changes in cyclic AMP (cAMP) in living cells, in real time. It utilizes a proprietary cyclic nucleotide-gated (CNG) channel, which serves as a biosensor for cAMP activity and is co-localized with adenylate cyclase at the plasma membrane. When cAMP levels rise, the CNG channel opens, allowing ion flux that causes cell membrane depolarization. This ion influx can be quantified using fluorescent ion indicators or membrane potential (MP) dyes, offering highly sensitive, real-time insights into intracellular cAMP dynamics.
By combining both kinetic and endpoint readouts, ACTOne™ captures and analyzes transient responses from endogenous GPCRs, including weak signals from low-activity Gs or Gi coupled GPCRs. This method allows direct detection of subcellular cAMP concentration changes triggered by GPCR activation, minimizing artifacts and providing more statistically robust data. Importantly, the signal intensity correlates with the number of receptors on the cell surface, offering a quantitative view of receptor activity.
Through ACTOne™, previously unreported Gs-coupled receptor activities in HEK293 cells have been detected, as well as weak Gs activity in a GPCR typically thought to be linked only to Gq pathways. This powerful assay is also an effective tool for GPCR de-orphanization, aiding in the identification of unknown GPCR functions.
AAV ACTOne GPCR Assay Kits
GPCR stable cell lines are extensively used for drug screening due to their reliability and consistent expression of specific GPCRs. These engineered cell lines are ideal for high-throughput screening of potential drug candidates, allowing researchers to efficiently evaluate pharmacological properties such as potency, efficacy, and selectivity. However, these stable cell lines have limited receptor diversity, and developing and validating them can be time-consuming and costly, especially for receptors that are difficult to express or require specific post-translational modifications. Additionally, stable cell lines typically express only one type of GPCR, whereas native tissues often co-express multiple GPCRs and other receptors, which can interact and influence each other.
In contrast, the AAV ACTOne GPCR Assay Kits utilize AAV vectors to co-express both cyclic nucleotide-gated (CNG) channels and GPCRs. This approach offers significant advantages over stable cell lines, including faster setup, greater physiological relevance, reduced risk of genetic drift, simplified workflows, flexibility in co-expression, dynamic control of expression, and improved cost and time efficiency. These benefits position the AAV ACTOne GPCR Assay Kit as a powerful alternative for GPCR drug screening and research.
Components in AAV ACTOne GPCR Assay Kits
The AAV ACTOne GPCR Assay Kits are live-cell-based cAMP biosensor systems designed for rapid and high-throughput functional screening of G-protein coupled receptors (GPCRs). Kits are available for both Gi-GPCR and Gs-GPCR assays.
Each kit includes two main components:
AAV-CNG/GPCRs enable transient expression of both CNG and GPCRs. AAV-CNG/GPCR is a mixture of AAV-CNG and AAV-GPCR. Both viruses use CMV as promoter, which is known for driving strong and rapid expression of transgenes in a wide variety of cell types. The ratio of two viruses are optimized in each kit.
ACTOne MP Dye allows for real-time, sensitive detection of GPCR functionality.
Advantages of AAV ACTOne GPCR Assay Kits
- Combined ACTOne and AAV Technologies
- Transient Expression of CNG and GPCR
- Real Time Assay on Living Cell
- High Sensitivity and Big assay window
- Both Kinetic and Endpoint Assay
- Easy to use by mixing the AAVs with cells
- Faster setup and time efficiency
- Reduced risk of genetic drift,
- Simplified workflows,
- flexibility in dynamic control of expression
- Low Cost: $498 for one plate