Development of a Luciferase Reporter Gene Assay for Artemin‐induced RET Signaling

Abstract

RET is a receptor tyrosine kinase that mediates the response of cells to stimulation with members of the glial cell‐line derived neurotrophic factor (GDNF) family of neurotrophins, which are important in the development and maintenance of a subset of neuronal cells as well as in other cell types and tissues. Improved methods to measure RET receptor activation and function are sought after to support the development of GDNF family ligands as potential drugs. Reporter gene assays are valuable tools to measure transcriptional and signaling pathway changes. Here, we report the development and validation of a stable luciferase reporter cell‐line for activation of RET by the GDNF family neurotrophin artemin (ART), using a previously established murine NB41A3 cell‐line expressing the GFRα3 co‐receptor. We observed a robust, dose‐dependent luciferase response upon stimulation of the cells with ART, which was quantitatively consistent with RET activation in the same cells measured using our previously reported RET Kinase Receptor Activation (KIRA) ELISA, or by measuring levels of activated ERK or Akt. Through inhibitor studies, we validated that RET activation of our reporter cell‐line is directly related to transcriptional changes induced by activation of the ERK/MAPK pathway. The reporter gene assay can be used to quantify the activation of RET by ART in a sensitive, convenient and robust way. We additionally report the use of this assay to investigate signaling dynamics and the efficiency of receptor‐effector coupling when RET is activated by a variety of different agonist proteins.