Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface

Abstract

Plexins are single-pass transmembrane receptors that bind the axon guidance molecules semaphorins. Single-pass transmembrane proteins are an important class of receptors that display a wide variety of activation mechanisms, often involving ligand-dependent dimerization or conformational changes. Resolving the activation mechanism and dimerization state of these receptors is extremely challenging, especially in a live-cell environment. Here, we report on the dimerization state of PlexinA4 and its response to activation by semaphorin binding. Semaphorins are dimeric molecules that activate plexin by binding two copies of plexin simultaneously and inducing formation of a specific active dimer of plexin. An open question is whether there are preexisting plexin dimers that could act as autoinhibitory complexes. We address these questions with pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a two-color fluorescence microscopy method that is directly sensitive to protein dimerization in a live-cell environment. With PIE-FCCS, we show that inactive PlexinA4 is dimerized in the live-cell plasma membrane. By comparing the cross correlation of full-length PlexinA4 to control proteins and plexin mutants, we show that dimerization of inactive PlexinA4 requires the Sema domain, but not the cytoplasmic domain. Ligand stimulation with Sema6A does not change the degree of cross correlation, indicating that plexin activation does not lead to higher-order oligomerization. Together, the results suggest that semaphorin activates plexin by disrupting an inhibitory plexin dimer and inducing the active dimer.

A G Protein-Coupled Receptor Dimerization Interface in Human Cone Opsins

“…Average molecular brightness values for green and blue cone opsins were 451 ± 19 and 461 ± 25 cpsm, respectively (Figure 1D), consistent with monomeric lipid-anchored eGFP measured on the same instrument under similar conditions (errors reported here and below are standard errors of the mean). 45,51 For red cone opsin, the average molecular brightness was 738 ± 51 cpsm (Figure 1D), a value similar to that obtained from a dimer control protein consisting of a fluorescent protein fused to a leucine zipper dimerization motif and a lipid-anchored peptide. 45,51 Our brightness data indicate that red cone opsin is dimeric in the plasma membrane.…”

“…45,51 For red cone opsin, the average molecular brightness was 738 ± 51 cpsm (Figure 1D), a value similar to that obtained from a dimer control protein consisting of a fluorescent protein fused to a leucine zipper dimerization motif and a lipid-anchored peptide. 45,51 Our brightness data indicate that red cone opsin is dimeric in the plasma membrane. The molecular brightness is not quite double the monomer brightness, which could indicate that the protein is distributed into a mixture of monomer, dimer, and oligomer states.…”

“…45,51 In this instrument, 488 and 561 nm laser light was selected from a pulsed white light source and directed through fibers of different lengths before being focused at the same point within the sample. This configuration introduced a delay of ~50 ns such that individual photons arriving at the detectors could be assigned to an excitation source.…”

“…TCSPC files were processed as described previously to produce the fluorescence correlation curves and fluorescence lifetime histograms. 45,51 …”

Secondary PDZ domain-binding site on class B plexins enhances the affinity for PDZ–RhoGEF

“…Before activation, plexin exists as an inactive monomer or inhibitory dimer (3,9,36). Semaphorin binding induces the formation of the active dimer of plexin (2,(4)(5)(6)(7).…”