AAV ACTOne Gi-GPCR Assay Kit-CXCR4

C-X-C Chemokine Receptor Type 4 (CXCR4) is a G protein-coupled receptor (GPCR) that primarily binds to the chemokine CXCL12, also known as stromal cell-derived factor-1 (SDF-1). CXCR4 plays a crucial role in various physiological processes, including immune cell trafficking, hematopoiesis, and organ development. It is widely expressed in many tissues, including immune cells, endothelial cells, and various types of stem cells. CXCR4 is particularly important in the migration and homing of hematopoietic stem cells to the bone marrow, as well as in the immune system's response to injury and infection. Additionally, CXCR4 and its ligand CXCL12 are involved in the development of the central nervous, cardiovascular, and immune systems. Beyond its normal physiological roles, CXCR4 is implicated in various pathological conditions. It is notably overexpressed in many types of cancer, where it promotes tumor growth, metastasis, and angiogenesis. The CXCR4/CXCL12 axis also plays a role in HIV infection, as CXCR4 serves as a co-receptor for the virus, allowing it to enter and infect host cells. Given its involvement in cancer progression, HIV infection, and other diseases, CXCR4 is a significant target for therapeutic intervention. Drugs targeting CXCR4, such as antagonists and inhibitors, are being developed to treat cancer, HIV, and other conditions where CXCR4 plays a key role.
This kit uses AAV vectors with a CMV promoter to co-express the CXCR4 and cyclic nucleotide-gated (CNG) channel, allowing researchers to conduct high-throughput screening and functional analysis of potential CXCR4-targeting compounds. The kit provides a sensitive and reliable method for evaluating the pharmacological properties of CXCR4 drugs, such as agonists and antagonists, in a live-cell environment.

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Background

ACTOne™ is the only high-throughput GPCR screening technology that can directly measure the intracellular changes of the secondary messenger cyclic AMP (cAMP) in living cells, in real-time. It uses a proprietary modified cyclic nucleotide-gated (CNG) channel, which is co-localized with adenylate cyclase at the plasma membrane, as a biosensor of cAMP activity. The CNG channel opens when the cAMP level near the plasma membrane increases, resulting in ion flux and cell membrane depolarization. The influx of cations through the CNG channel can be quantified using fluorescent ion indicators or membrane potential (MP) dyes. It provides information on real time intracellular cAMP changes and is highly sensitive. By combining kinetic and endpoint readouts, we are able to capture and analyze transient responses from endogenous GPCRs and weak responses caused by weak Gs or Gi coupled GPCR activities. Using ACTOne, we are able to detect the subcellular cAMP concentration changes directly caused by GPCR activation. Real-time kinetic readouts minimize artifacts, and provide greater content and more statistically relevant data. The intensity of signal increase caused by GPCR activation is directly related to the receptor number on cell surface. Using ACTOne assay, we were able to detect activities of some endogenous Gs coupled receptors in HEK293 cells that have not been reported in literature. In addition, we have also detected weak Gs coupled activity of a GPCR that was widely considered to be only linked to Gq coupled pathway. The ACTOne assay also provides a useful tool for GPCR de-orphanization.