Subcellular receptor redistribution and enhanced microspike formation by a Ret receptor preferentially recruiting Dok

Ret is a receptor tyrosine kinase for the GDNF family of ligands and plays important roles during nervous system development for cell proliferation, cell migration and neurite growth. Signaling initiated from intracellular tyrosine 1062, by recruitment of several different phosphotyrosine binding (PTB) proteins (i.e. Shc, Frs2 and Dok), is important for these biological effects. By a single amino acid substitution in the PTB domain binding sequence of Ret, we have rewired the receptor such that it preferentially recruits Dok (Ret Dok+ ) with little or no remaining interactions with Shc and Frs2. Ret Dok+ displays a sustained MAP kinase activation and a loss of Akt signaling compared to Ret WT. We show that early events after ligand stimulation of Ret Dok+ include massive formation of fine microspikes that are believed to be priming structures for neurite growth from the cell soma. The Ret Dok+ receptors relocated in the membrane compartment into focal clusters at the tip of the microspikes, which was associated with Cdc42 activation. These results suggest that engagement of different adaptor proteins by Ret results in very different downstream signaling and functions within neurons and that Dok recruitment leads to a rapid receptor relocation and formation of microspikes.