AAV ACTOne Gi-GPCR Assay Kit-CCR5

Chemokine (C-C motif) Receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that binds to specific chemokines, including CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES). CCR5 is primarily expressed on the surface of immune cells such as T cells, macrophages, dendritic cells, and some types of B cells. The receptor plays a critical role in directing the migration of these cells to sites of infection or inflammation, thereby contributing to the immune response. CCR5 is well-known for its role as a co-receptor for the human immunodeficiency virus (HIV). The virus uses CCR5, along with CD4, to enter and infect host cells. This makes CCR5 a key target in HIV research and treatment, with CCR5 antagonists (such as Maraviroc) being developed to block this interaction and prevent the virus from infecting cells. In addition to its involvement in HIV infection, CCR5 is implicated in various other pathological conditions, including inflammatory diseases, cancer, and autoimmune disorders. Its role in immune cell trafficking and inflammatory responses makes CCR5 a potential target for therapies aimed at treating these conditions. Notably, individuals with a naturally occurring mutation in the CCR5 gene (CCR5-Δ32) are resistant to certain strains of HIV, highlighting the receptor's importance in viral pathogenesis.
This kit uses AAV vectors with a CMV promoter to co-express the CCR5 and cyclic nucleotide-gated (CNG) channel, allowing researchers to conduct high-throughput screening and functional analysis of potential CCR5-targeting compounds. The kit provides a sensitive and reliable method for evaluating the pharmacological properties of CCR5 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.