FGFR/beta-Klotho Reporter Assay System [Human]

The family of Fibroblast Growth Factors (FGFs) comprise approximately 23 members that are related by core sequence and structure conservation, with the majority of FGFs being secreted signaling proteins. Secreted FGFs are predominantly autocrine and paracrine factors, with only three members evolved to function as endocrine factors. FGFs bind and activate FGF Receptors (FGFRs) which, themselves, are members of the family of high-affinity tyrosine kinase receptors. Paracrine FGFs show high affinity towards the extracellular matrix (ECM) component heparin sulfate (HS) and are thus retained in the ECM and function locally. In contrast, the atypical endocrine subfamily of FGFs, that comprise FGF-19, FGF-21, and FGF-23, have reduced affinity for HS and can therefore escape from the ECM into the circulation to reach target distant organs. FGF-21 a liver secreted endocrine FGF, has been identified as a factor that has multiple beneficial effects on obesity-related disorders. For example, it is a potent activator of glucose uptake in primary human adipocytes. In addition, FGF-21 has been shown to protect diet-induced obesity and diabetic illness in animals. Consequently, the associated metabolic benefits of the endocrine FGFs have promoted considerable interest in therapeutic development and drug safety screening. INDIGO's Human Fibroblast Growth Factor Receptor Receptor 1c and beta-Klotho (FGFR/beta-Klotho) assay utilizes proprietary human cells that have been engineered to provide constitutive expression of the Human Fibroblast Growth Factor Receptor 1c and beta-Klotho. FGFR1c and beta-Klotho are both single-pass transmembrane proteins. FGFR1c has an extracellular ligand-binding domain, transmembrane domain, and intracellular tyrosine kinase domain. It has been established that FGFR association with the co-receptor beta-Klotho generates a scaffold that is essential for endocrine growth factor binding interactions, such as those with FGF-21 and FGF-19. Following growth factor binding, the activated tyrosine kinase activities of the FGFR initiate intracellular signaling cascades that may include RAS-MAPK, PI3-AKT, PLCgamma and/or STAT pathways. It is FGFR1c/beta-Klotho signal transduction via the RAS-MAPK-ERK1/2 cascade that is exploited by the reporter cells in this assay. INDIGO's FGFR/beta-Klotho Reporter Cells contain the luciferase reporter gene functionally linked to an Elk-1 responsive promoter. Activated Elk-1 binds to the response elements to initiate the formation of a complete transcription complex that drives Luc gene expression. Quantifying relative changes in luciferase activity in the treated reporter cells relative to the untreated cells provides a sensitive, dose-dependent surrogate measure of drug-induced changes in FGFR1c/beta-Klotho activity. The principal application of this reporter assay is in the screening of test materials to quantify any functional interactions, either activating or inhibitory, that they may exert against FGFR1c/beta-Klotho or the coupled RAS-MAPK pathway. beta-Klotho Protein function: Contributes to the transcriptional repression of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis. Probably inactive as a glycosidase. Increases the ability of FGFR1 and FGFR4 to bind FGF21. [The UniProt Consortium] This Human Fibroblast Growth Factor Receptor 1c and beta-Klotho (FGFR/beta-Klotho) assay kit is an all-inclusive assay system that includes addition to FGFR/beta-Klotho Reporter Cells, an optimized culture medium for use in reviving the cryopreserved cells, a culture medium for use in preparing test sample treatments, the physiological endocrine activator FGF-21, Luciferase Detection Reagents, and a cell culture-ready assay plate. FGFR/beta-Klotho Reporter Cells are transiently transfected and prepared as frozen stocks using INDIGO's proprietary CryoMite(TM) process. This cryo-preservation method allows for the immediate dispensing of healthy, division-competent reporter cells into assay plates. There is no need for cumbersome intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup. INDIGO's FGFR/beta-Klotho 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. FGFR/beta-Klotho assay kits are offered in different assay formats to accommodate researchers' needs: 3x 32, 1x 96, and 1x 384 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications.