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

Morgan Marita, Yuxiao Wang, Megan J. Kaliszewski, Kevin C. Skinner, William D. Comar, Xiaojun Shi, Pranathi Dasari, Xuewu Zhang, Adam W. Smith

Research output: Contribution to journalArticle

15 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1937-1945
Number of pages9
JournalBiophysical journal
Volume109
Issue number9
DOIs
StatePublished - Nov 3 2015

ASJC Scopus subject areas

  • Biophysics

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    Marita, M., Wang, Y., Kaliszewski, M. J., Skinner, K. C., Comar, W. D., Shi, X., Dasari, P., Zhang, X., & Smith, A. W. (2015). Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface. Biophysical journal, 109(9), 1937-1945. https://doi.org/10.1016/j.bpj.2015.04.043