Receptor Tyrosine Kinase Assays
Cell-based Assays to Analyze RTK Dimerization, Phosphorylation, Internalization, and SH2-Recruitment
Receptor tyrosine kinases (RTKs) play important roles in a variety of cellular processes, including growth, motility, differentiation, and metabolism. Recent large-scale genomic studies have revealed the presence of various alterations in the genes encoding RTKs, such as EGFR, HER2/ErbB2, and MET, among many others. Dysregulation of RTK signaling leads to an assortment of human diseases, most notably numerous cancers. Abnormal RTK activation in human cancers is mediated by four principal mechanisms: gain-of-function mutations, genomic amplification, chromosomal rearrangements, and/or autocrine activation.
Eurofins DiscoverX® offers PathHunter® cell-based assays for RTK and cytokine receptors associated with cytosolic tyrosine kinases (CTK). These innovative assays provide cellular context to the activation of these receptors, as well as for the identification of novel inhibitors and therapeutic antibodies. Utilize the PathHunter assays to analyze RTK and cytokine receptor functional activity by analyzing their dimerization, phosphorylation, internalization, and SH2-recruitment.
Product Highlights
- Broadly Applicable - Identify various ligands, including anti-receptor, anti-ligand, or activating antibodies; non-ATP pocket binders (allosteric modulators); ligand binding inhibitors (ATP-competitors); or dimerization inhibitors
- High Specificity - Tagged, tyrosine kinase eliminates background from endogenous tyrosine kinases
- Biologically Relevant - MOA-reflective, functional assays for monitoring RTK and cytokine receptor activation and dimerization for characterization through screening applications
Eurofins DiscoverX offers a variety of cell-based assays in the form of cell lines and ready-to-use eXpress™ kits for functional activity, dimerization, SH2 recruitment, characterization, and screening of RTKs and CTKs associated with cytokine receptors. Please contact our custom development team for additional assays.
Cell Lines
- All RTK cell lines
- RTKs and CTKs activity and functional cell lines
- RTK internalization cell line
- Engineered Parental Cell Lines for the development of SH2-recruitment assays
Kinase Assay Ready Kits including eXpress assays and bioassays
- All RTK assay ready kits
- RTKs and CTKs activity and functional assay kits
PathHunter receptor tyrosine and cytosolic tyrosine kinase assays are whole cell, high-throughput assays that utilize the enzyme fragment complementation technology based on a split β-galactosidase (β-gal) enzyme system to measure receptor functional activity, dimerization, SH2 recruitment, characterization, and screening.
PathHunter receptor tyrosine and cytosolic tyrosine kinase functional and activity assay principle. For both assay types, a small inactive peptide fragment of the β-gal enzyme called ProLink™ (PK) is expressed recombinantly on the intracellular C-terminus of the RTK (left image) or cytokine receptor (right image). One of the many different partner proteins containing SH2 domains is co-expressed with a larger fragment of β-gal, termed enzyme acceptor (EA). Ligand-induced receptor activation causes either homo- or hetero-dimerization of the receptor resulting in cross-phosphorylation. The SH2-EA fusion protein then binds the phosphorylated receptor forcing complementation of the PK and EA fragment. This interaction generates an active β-gal enzyme that can be detected using a chemiluminescent substrate.
Create Your Own SH2-Protein Recruitment Cell-Based Assays
Generate your own PathHunter SH2-protein recruitment assay using the enzyme fragment complementation (EFC) technology. Create a plasmid vector with your target protein of choice (e.g. RTK in this example) tagged with the small enzyme donor, ProLink™ (PK), of the EFC β-galactosidase (β-gal). Simply transfect this plasmid into a PathHunter SH2-recuitment parental cell line containing an SH2 protein tagged with the large enzyme acceptor (EA) and perform a kinase functional or activity assay in the presence of a ligand.
Evaluate Dimerization and Functional Activity
Measure dimerization and functional activity of RTKs and cytokine receptors associated with CTKs. A. The insulin receptor is constitutively dimerized, but undergoes a conformational change to become active in the presence of insulin (agonist) and this can be inhibited by the small molecule antagonists staurosporine and HNMPA. B. The colony stimulating factor 3 receptor (CSF3R-JAK1) cell line was preincubated with antagonists staurosporine, lestaurtinib and pyridone 6, P6, DBI (a Jak inhibitor 1) and then challenged with EC80 of agonist G-CSF. A dose-dependent inhibition was observed with the antagonist, indicating that the assay can be used effectively to profile or screen inhibitors against the cytosolic tyrosine kinase JAK1.
Monitor RTK Internalization through Ligand or Receptor Antibody Activation
Monitor Receptor Tyrosine Kinase (RTK) internalization through ligand or receptor antibody activation. A. Insulin receptor b (INSRb) internalization in response to incubation with increasing insulin concentrations. B. Epidermal growth factor B2 (ErbB2) receptor internalization is detected when cells are incubated with two different ErbB2 antibodies, including the therapeutic antibody trastuzumab (Herceptin®). Similar robust ErbB2 internalization was observed when cells were incubated with Herceptin and pertuzumab (Perjeta®; data not shown). Herceptin and Perjeta are registered trademarks of Genentech USA, Inc.
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