AAV ACTOne Gi-GPCR Assay Kit-CCR4

Chemokine (C-C motif) Receptor 4 (CCR4) is a type of G protein-coupled receptor (GPCR) that primarily binds to specific chemokines, including CCL17 (TARC) and CCL22 (MDC). CCR4 is expressed predominantly on the surface of certain immune cells, such as T helper 2 (Th2) cells, regulatory T cells (Tregs), and certain types of dendritic cells. It plays a crucial role in the immune system by directing the migration of these cells to sites of inflammation or to lymphoid tissues. CCR4 is involved in various physiological processes, including immune surveillance, inflammation, and the regulation of immune responses. Its role is particularly significant in the context of allergic reactions, autoimmune diseases, and cancer, where it can influence the recruitment of immune cells to tumors or inflamed tissues. Given its role in immune cell trafficking, CCR4 is a target of interest in therapies for conditions such as asthma, atopic dermatitis, and certain types of cancer, including T-cell lymphomas. Inhibitors or antagonists of CCR4 are being explored as potential treatments to modulate immune responses or limit the spread of cancer.
This kit uses AAV vectors with a CMV promoter to co-express the CCR4 and cyclic nucleotide-gated (CNG) channel, allowing researchers to conduct high-throughput screening and functional analysis of potential CCR4-targeting compounds. The kit provides a sensitive and reliable method for evaluating the pharmacological properties of CCR4 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.