2015 Beckman Symposium   

Mikayla Huestis

Presentation Date:
August-6-2015

Presenter:
Mikayla Huestis

Title:
The Dynamics of RET Signaling and Commitment to Biological Outcomes

Institution:
Boston University

Department:
Chemistry

Description:
RET is a receptor tyrosine kinase that is activated by ligands of the glial cell-line derived neurotrophic factor (GDNF) family, and is important in kidney development and in the development and maintenance of the peripheral nervous system. In addition to a GDNF family ligand, activation of RET requires a membrane-associated co-receptor from the GFRα family. RET signals through the Raf/MEK/ERK and Akt pathways, among others. We used a lentiviral vector to infect a murine neuroblastoma cell-line expressing RET and GFRα3, to introduce the gene for firefly luciferase under the control of the serum response element (SRE), a promoter that responds to ERK pathway activation. We subcloned the transfected cells to generate a monoclonal reporter cell line that provides a quantitative, convenient, and robust method to measure RET activation by Artemin (ART), the GDNF family ligand that uses GFRα3. We used this reporter gene assay to investigate how RET activation is coupled to nuclear signaling events, and how these in turn drive the biological responses of cell survival and differentiation, quantified by cell titer blue and neurite outgrowth experiments. We found that RET activation and intracellular signaling peak at ten minutes, however full commitment to luciferase expression requires continuous ART stimulation for at least one hour.


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