AAV ACTOne Gi-GPCR Assay Kit-DRD4

The Dopamine Receptor D4 (DRD4) is a G protein-coupled receptor (GPCR) that belongs to the D2-like family of dopamine receptors, which also includes D2 and D3 receptors. DRD4 is primarily coupled with the Gi/Go class of G proteins, leading to inhibition of adenylate cyclase and a subsequent decrease in cyclic AMP (cAMP) levels. This receptor modulates several intracellular signaling pathways, influencing neuronal activity, neurotransmitter release, and cognitive functions. DRD4 is widely expressed in the central nervous system, particularly in areas of the prefrontal cortex that are associated with executive function, attention, and emotional regulation. It is also found in other brain regions involved in cognition, behavior, and motivation. The receptor is known for its genetic variability, particularly in its exon 3 region, where polymorphisms result in different numbers of a 48-base-pair repeat. These polymorphisms are associated with various behavioral traits and susceptibility to psychiatric conditions. DRD4 has been implicated in a range of psychological and behavioral disorders, including attention-deficit/hyperactivity disorder (ADHD), schizophrenia, bipolar disorder, and substance abuse. For instance, certain polymorphisms of DRD4 are linked to increased risk of ADHD and impulsive behaviors. Due to its role in these conditions, DRD4 is a target of interest for drug development aimed at modulating dopaminergic signaling in the brain. The receptor's involvement in the regulation of attention, behavior, and emotion makes it a key focus for understanding the neurobiological underpinnings of these complex traits and disorders.
This kit uses AAV vectors with a CMV promoter to co-express the DRD4 and cyclic nucleotide-gated (CNG) channel, allowing researchers to conduct high-throughput screening and functional analysis of potential DRD4-targeting compounds. The kit provides a sensitive and reliable method for evaluating the pharmacological properties of DRD4 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.