TrkA Reporter Assay System (Human)

This Human Tropomyosin Receptor Kinase A (TrkA, NTRK1) assay kit is an all-inclusive luciferase reporter assay system that includes, in addition to TrkA Reporter Cells, two optimized media for use in recovering the cryopreserved cells and in preparing test samples, reference activator NGF-beta, Luciferase Detection Reagent, a cell culture-ready assay plate, and a detailed protocol. Thaw, Feed, Dose, Read Fast, reproducible, easy-to-analyze results are only four steps away Many luciferase reporter assays require the user to grow their own cells and take time to optimize the results. INDIGO's reporter cells contain the receptor of interest and the luciferase reporter gene. Reporter cells have been optimized to provide extreme sensitivity to quantify even small changes in receptor activity. With INDIGO's cell-based reporter assays, the process is as easy as Thaw, Feed, Dose, and Read. TrkA Reporter Cells TrkA Reporter Cells are prepared using INDIGO's proprietary CryoMite(TM) process. This proprietary cryopreservation process, enables long-term preservation of our unique reporter cells, so we can ship our cryopreserved reporter cells and assay reagents to you via overnight delivery, for your immediate use. Or, you can store the assay kits at -80°C. Once thawed, reporter cells are ready for immediate use so there is no need to take time on intermediate spin-and-wash steps, viability determinations, or cell titer adjustments. TrkA Luciferase Reporter INDIGO's TrkA assay kits feature a luciferase detection reagent specially formulated to provide stable light emission between 5 and 90+ minutes after initiating the luciferase reaction. Incorporating a 5-minute reaction-rest period ensures that light emission profiles attain maximal stability, thereby allowing assay plates to be processed in batch. By doing so, the signal output from all sample wells, from one plate to the next, may be directly compared within an experimental set. Protein function: Receptor tyrosine kinase involved in the development and the maturation of the central and peripheral nervous systems through regulation of proliferation, differentiation and survival of sympathetic and nervous neurons. High affinity receptor for NGF which is its primary ligand (PubMed:1850821, PubMed:1849459, PubMed:1281417, PubMed:8325889, PubMed:15488758, PubMed:22649032, PubMed:17196528, PubMed:27445338). Can also bind and be activated by NTF3/neurotrophin- 3. However, NTF3 only supports axonal extension through NTRK1 but has no effect on neuron survival. Upon dimeric NGF ligand- binding, undergoes homodimerization, autophosphorylation and activation (PubMed:1281417). Recruits, phosphorylates and/or activates several downstream effectors including SHC1, FRS2, SH2B1, SH2B2 and PLCG1 that regulate distinct overlapping signaling cascades driving cell survival and differentiation. Through SHC1 and FRS2 activates a GRB2-Ras-MAPK cascade that regulates cell differentiation and survival. Through PLCG1 controls NF-Kappa-B activation and the transcription of genes involved in cell survival. Through SHC1 and SH2B1 controls a Ras-PI3 kinase-AKT1 signaling cascade that is also regulating survival. In absence of ligand and activation, may promote cell death, making the survival of neurons dependent on trophic factors. [The UniProt Consortium] Tropomyosin receptor kinases A, B and C constitute a family of receptors denoted as TrkA, TrkB, and TrkC. Trk's are single-pass transmembrane receptors that contain an extracellular ligand-binding domain, transmembrane domain, and an intracellular tyrosine kinase domain. Neurotrophins, the physiological activators of Trk receptors, comprise a group of proteins that include Nerve Growth Factor beta (NGF-beta), Neurotrophin 3 (NT-3), Neurotrophin 4 (NT-4) and Brain Derived-Neurotrophic Factor (BDNF). The active forms of these neurotrophins are disulfide-linked homodimer polypeptides. The binding of neurotrophins to Trk's triggers receptor homodimerization and autophosphorylation. The intrinsic tyrosine kinase activities of activated, dimeric Trk receptor initiate intracellular signaling cascades that include RAS-MAPK, PI3-AKT, and PLCgamma pathways. Trk's are expressed in multiple tissue types, and are primarily involved in neuronal development, neuronal proliferation, and avoidance of programmed cell death. Chromosomal rearrangements of NTRK1-3 may result in gene fusions have been clinically validated as oncogenic drivers in a wide array of human cancers. Not surprising, the Trk receptors command much interest as targets for drug development and drug safety screening. Topical recombinant human nerve growth factor beta (rhNGF-beta) has been FDA-approved for the treatment for patients with neurotropic keratitis, as well as Trk inhibitors for the treatment of patients with solid tumors harboring NTRK gene fusions. It is also widely accepted that neurotrophins contribute to the pain suffered in osteoarthritis, and anti-NGF antibodies are efficacious in reducing these pain symptoms. INDIGO's TrkA Reporter Cells include the luciferase reporter gene functionally linked to tandem consensus sequences of NFAT genetic response elements upstream of a minimal promoter. Activated NFAT binds to these response elements to seed the formation of a complete transcription complex that drives Luc gene expression. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in human TrkA activity. Therefore, the principal application of this reporter assay is in the screening of test compounds to quantify any functional activity that they may exert against TrkA, or the coupled Ca+2calcineurin / NFAT signal transduction pathway.