Decoding the Role of Receptor Dimerization in Plexin-Semaphorin Signaling

Abstract

Plexin-semaphorin signaling plays an important role in the formation of neural networks by acting as the guidance cue for axon growth or retraction. The plexin receptor is a single-pass transmembrane protein with a sema domain at the extracellular N-terminus and a GTP-ase activating protein domain on the cytoplasmic C-terminus. Binding of semaphorin is known to activate the plexin receptor, but the mechanism of how that binding event leads to activation is not understood. We will present our recent work using pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to measure plexin protein-protein interactions in live cells. PIE-FCCS transforms fluctuations in fluorescence intensity (arising mainly from diffusion) into information about a protein's mobility and concentration. By cross-correlating the fluorescence fluctuations in two color channels, PIE-FCCS also quantifies co-localization and co-diffusion with high accuracy. This method has been used to resolve the mechanism of epidermal growth factor receptor (EGFR) activation and inhibition, as well as the dimerization constant of the opsin G protein-coupled receptor. In this work we measure the ligand-free dimerization of plexin receptors in live cell membranes and the response of the receptor to semaphorin-induced activation.