AAV ACTOne Gs-GPCR Assay Kit-MC3R

The Melanocortin 3 Receptor (MC3R) is a G protein-coupled receptor (GPCR) that is primarily expressed in the brain, particularly in the hypothalamus. MC3R is activated by melanocortin peptides, including α-MSH and ACTH. It plays a significant role in energy homeostasis, regulation of appetite, and body weight. MC3R is also involved in the regulation of circadian rhythms and immune function. Dysfunction or mutations in MC3R are associated with obesity and other metabolic disorders.
This kit uses AAV vectors with a CMV promoter to co-express the MC3R and cyclic nucleotide-gated (CNG) channel, allowing researchers to conduct high-throughput screening and functional analysis of potential MC3R-targeting compounds. The kit provides a sensitive and reliable method for evaluating the pharmacological properties of MC3R 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.