Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases

Author:

Koh Duk-Su1,Stratiievska Anastasiia1,Jana Subhashis2ORCID,Otto Shauna C.1,Swanson Teresa M.1,Nhim Anthony1,Carlson Sara1,Raza Marium1,Naves Lígia Araujo1,Senning Eric N.3,Mehl Ryan2,Gordon Sharona E.1ORCID

Affiliation:

1. University of Washington, Department of Physiology & Biophysics

2. Department of Biochemistry and Biophysics, Oregon State University

3. Department of Neuroscience, University of Texas at Austin

Abstract

Ligands such as insulin, epidermal growth factor, platelet derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

Publisher

eLife Sciences Publications, Ltd

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