Cayman Platelet-Derived Growth Factor Receptors α and β Reporter Assay System

This assay utilizes proprietary human cells that have been engineered to provide constitutive expression of the Human Platelet-Derived Growth Factor Receptors α and β, referred to herein as PDGFRα/β. PDGFRα and PDGFRβ are both single-pass transmembrane receptors that contain respective extracellular ligand-binding domains, transmembrane domains, and intracellular tyrosine kinase domains.

Platelet-derived growth factor (PDGF), the physiological activator of PDGFRs, consists of four polypeptide members: A, B, C and D1, 2. The biologically active forms of PDGF proteins are both homo-dimers and hetero-dimers of disulfide-linked polypeptides. These function to promote cell migration, proliferation, and survival. Binding of dimeric PDGF triggers conformational changes that drive the assembly of homo-dimeric (Rα:Rα, Rβ:Rβ) and/or hetero-dimeric (Rα:Rβ) receptors, and the activation of their respective cytosolic tyrosine kinase domains. Because these reporter cells constitutively express both PDGFRα and PDGFRβ, it is anticipated that all three forms of the activated receptor dimers are present. If desired, reporter cells expressing either homo-dimeric PDGFR can be made available (please inquire).

The tyrosine kinase activities of activated, dimeric PDGFR’s initiate intracellular signaling cascades that include RAS-MAPK, PI3-AKT, PLCγ and STAT pathways. For example, activation of the PLCγ pathway leads to an increase of intracellular calcium. One prominent outcome of the PDGF/PDGFR > PLCγ pathway is that calcineurin, a calcium-dependent phosphatase, dephosphorylates and activates the transcription factor NFAT. It is PDGFR signal transduction via the Ca⁺²∙calcineurin / NFAT cascade that is exploited by the reporter cells provided in this kit.

INDIGOs PDGFRα/β Reporter Cells contain the luciferase reporter gene functionally linked to tandem consensus sequences of NFAT response elements upstream of a minimal promoter. Activated NFAT binds to these response elements to initiate the formation of a complete transcription complex that drives Luc gene expression.

PDGF activates PDGFRα/β in a dose-dependent manner, thereby triggering the Ca⁺²∙calcineurin/NFAT signal transduction pathway. 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 PDGFRα/β activity. Accordingly, 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 PDGFRα/β, or the coupled Ca⁺²∙calcineurin/NFAT signal transduction pathway.

The clinical use of recombinant PDGF-BB has led to the successful treatment of chronic or diabetes-related non-healing lower extremity wounds. In addition, PDGF-BB has also been used in clinics for reducing Parkinsonian symptoms. However, dysfunctional PDGFR signaling can lead to a range of physiological disorders. For example, enhanced signaling of PDGFRs has been implicated in the pathogenesis of atherosclerosis, pulmonary fibrosis, angiogenesis, and tumorigenesis. Consequently, the PDGF receptors continue to command much interest as targets for drug development and drug safety screening.

INDIGO’s Reporter Cells are transiently transfected and prepared as frozen stocks using a proprietary CryoMite™ process. This cryo-preservation method allows for the immediate dispensing of healthy, division-competent reporter cells into assay plates. There is no need for intermediate treatment steps such as spin-and-rinse of cells, viability determinations or cell titer adjustments prior to assay setup. This assay kit provides the convenience of an allinclusive cell-based reporter assay system for PDGFRα/β signaling. In addition to Reporter Cells, included are an optimized culture medium for use in reviving the cryopreserved cells, a culture medium for use in preparing test sample treatments, the reference physiological activator PDGF-BB, Luciferase Detection Reagents, and a cell culture-ready assay plate. [INDIGO Item Catalog Nos. IB23001, IB23001-32, IB23002]

WARNING This product is not for human or veterinary use.