AAV ACTOne Gi-GPCR Assay Kit-S1PR1

Sphingolipid G-Protein-Coupled Receptor 1 (S1PR1), also known as Sphingosine-1-Phosphate Receptor 1, is a G protein-coupled receptor (GPCR) that specifically binds to sphingosine-1-phosphate (S1P), a bioactive lipid involved in a wide range of physiological processes. S1PR1 is one of five S1P receptors (S1PR1-5) and is highly important in regulating immune function, vascular development, and cell migration. When S1PR1 is activated by its ligand, S1P, it primarily couples with the Gi class of G proteins, leading to inhibition of adenylate cyclase and a reduction in cyclic AMP (cAMP) levels. This receptor also activates several other downstream signaling pathways, including those involving phospholipase C (PLC), Ras, and PI3K/Akt, which contribute to its diverse biological effects. S1PR1 is widely expressed in various tissues, including the immune system, cardiovascular system, and central nervous system. In the immune system, S1PR1 plays a critical role in the regulation of lymphocyte trafficking. It controls the egress of lymphocytes from lymphoid organs into the bloodstream, which is essential for normal immune surveillance. In the vascular system, S1PR1 is involved in maintaining vascular integrity and promoting endothelial cell survival and migration. Given its pivotal role in immune regulation, S1PR1 is a key target for therapeutic interventions in autoimmune diseases. The drug fingolimod (Gilenya), an S1PR modulator, was the first oral treatment approved for multiple sclerosis (MS) and works by preventing lymphocytes from leaving the lymph nodes, thereby reducing autoimmune attacks on the central nervous system. Research into S1PR1 continues to explore its broader roles in cancer, cardiovascular diseases, and neurodegenerative disorders, with the goal of developing new therapies that leverage its regulatory functions in cell migration, survival, and immune response.
This kit uses AAV vectors with a CMV promoter to co-express the S1PR1 and cyclic nucleotide-gated (CNG) channel, allowing researchers to conduct high-throughput screening and functional analysis of potential S1PR1-targeting compounds. The kit provides a sensitive and reliable method for evaluating the pharmacological properties of S1PR1 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.