GPCRs Cell-Based Second Messenger Assays
Creative Biolabs provides cell-based second messenger assays for GPCR research and drug development, especially for large and small drug screening projects.
Introduction of Second Messengers
Second messengers are one of the initiating components of intracellular signal transduction, which can trigger various physiological changes, such as proliferation, cell differentiation, migration, survival and apoptosis. The major classes of second messengers are cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositol triphosphate (IP3) and diacylglycerol (DAG), and calcium ions (Ca2+). The release of second messengers is response to the signal molecules exposed outside cells, usually hormones, growth factors or neurotransmitters.
Figure 1. Cellular distribution of the processes involved in the production of intracellular second messengers. (Zhou D R, et al., 2019)
Second Messenger Assays for GPCR
G protein-coupled receptors (GPCRs) are the most diverse group of membrane receptors in eukaryotes and play critical roles in an incredible array of biological functions. Almost one-third to one-half of marketed drugs act by binding to GPCRs. The binding of external signaling molecules to GPCRs cause a conformational change, which results in the interaction between GPCRs and G proteins, and in turn triggers the production of hundreds or even thousands of second messenger molecules.
Second messenger assays can be used to study ligand induced GPCR modulation. They also can be used to detect and characterize all classes of compounds, such as full antagonist, partial antagonist, full agonist, partial agonist, inverse agonist, inhibitors, and allosteric modulators. It is important to elucidate second messenger signaling cascades resulting from interactions between GPCRs and their ligands.
Figure 2. Schematic of the intracellular signaling pathways involved in the ethanol neurotoxicity, through GPCR. (Imam S Z, et al., 2018)
Our Services
We provide various assays for the detection and quantification of different second messengers.
cAMP is an important second messenger involved in many biological processes. cAMP assays are useful tools for elucidating novel functions of orphan GPCRs, understanding physiological implications, and screening for novel drugs targeting GPCRs. For small- and large-scale analysis, we provide various cAMP screening and detection assays based on reporter gene system, competitive chemiluminescent immunoassay, time-resolved fluorescence resonance energy transfer (TR-FRET) and colorimetric competitive ELISA method.
IP3, an inositol phosphate signaling molecule, is a second messenger involved in the activation of various calcium regulated intracellular signals. Detection of IP1, the downstream metabolite of IP3, can overcome the detection shortcomings of IP3 due to its short half-life. We provide IP3/IP1 assays based on fluorescence polarization (FP), luciferase fragment assisted complementation strategy and ELISA for pharmacologically characterization of GPCR ligands and high-throughput screening.
DAG, a key component of G-protein signaling, is involved in a complex network to initiate and regulate diverse downstream cellular processes. We provide DAG assays based on coupled enzymatic reaction system, ELISA and fluorescent sensors to detect and quantify DAG levels.
Calcium plays a central role in converting endogenous and exogenous signals to cellular responses when Gi and Gq-coupled receptors are stimulated. The evaluation of calcium flux can be used as a measurement for various cellular processes. We provide calcium mobilization assays based on atomic absorption spectrometry, microelectrode, photoprotein, synthetic fluorescent indicator and genetically encoded calcium indicator to measure the calcium flux in various types of cells.
Live Cell Imaging of Second Messenger Systems
Real-time measurement of multiple second messenger pathways in relevant cells plays an increasingly important role in GPCR research and drug discovery. We provide multiplex sensors to simultaneously measure different second messenger components in living cells on microplate reader or live-cell imaging system to facilitate biomedical research.
Features & Benefits
- Unmatched performance for GPCRs.
- Applicable to different types of luminometer or multi-format reader.
- Flexible protocols for directly measurement in adherent and suspension cells.
- High-quality results with fewer false positives.
- Simplified protocols to deliver fast results.
If you are interested in our cell-based second messenger assays or have any specific needs, please feel free to contact us for more details.
Reference
- Zhou D R, et al. Intracellular second messengers mediate stress inducible hormesis and Programmed Cell Death: A review. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2019.
- Imam S Z, et al. Ontogeny of Second Messenger Systems. Handbook of Developmental Neurotoxicology. Academic Press, 2018: 199-206.
- Siehler S. Cell-based assays in GPCR drug discovery. Biotechnology Journal: Healthcare Nutrition Technology, 2008, 3(4): 471-483.