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

12 Citations (Scopus)

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

Fingerprint

Semaphorins
Dimerization
Spectrum Analysis
Fluorescence
Protein Multimerization
Cell Membrane
Ligands
plexin
Mutant Proteins
Fluorescence Microscopy
Color
Proteins

ASJC Scopus subject areas

  • Biophysics

Cite this

Marita, M., Wang, Y., Kaliszewski, M. J., Skinner, K. C., Comar, W. D., Shi, 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

Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface. / Marita, Morgan; Wang, Yuxiao; Kaliszewski, Megan J.; Skinner, Kevin C.; Comar, William D.; Shi, Xiaojun; Dasari, Pranathi; Zhang, Xuewu; Smith, Adam W.

In: Biophysical Journal, Vol. 109, No. 9, 03.11.2015, p. 1937-1945.

Research output: Contribution to journalArticle

Marita, M, Wang, Y, Kaliszewski, MJ, Skinner, KC, Comar, WD, Shi, X, Dasari, P, Zhang, X & Smith, AW 2015, 'Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface', Biophysical Journal, vol. 109, no. 9, pp. 1937-1945. https://doi.org/10.1016/j.bpj.2015.04.043
Marita M, Wang Y, Kaliszewski MJ, Skinner KC, Comar WD, Shi X et al. Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface. Biophysical Journal. 2015 Nov 3;109(9):1937-1945. https://doi.org/10.1016/j.bpj.2015.04.043
Marita, Morgan ; Wang, Yuxiao ; Kaliszewski, Megan J. ; Skinner, Kevin C. ; Comar, William D. ; Shi, Xiaojun ; Dasari, Pranathi ; Zhang, Xuewu ; Smith, Adam W. / Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface. In: Biophysical Journal. 2015 ; Vol. 109, No. 9. pp. 1937-1945.
@article{c8776ad2ea604797809d56a339bdd7a6,
title = "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.",
author = "Morgan Marita and Yuxiao Wang and Kaliszewski, {Megan J.} and Skinner, {Kevin C.} and Comar, {William D.} and Xiaojun Shi and Pranathi Dasari and Xuewu Zhang and Smith, {Adam W.}",
year = "2015",
month = "11",
day = "3",
doi = "10.1016/j.bpj.2015.04.043",
language = "English (US)",
volume = "109",
pages = "1937--1945",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "9",

}

TY - JOUR

T1 - Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface

AU - Marita, Morgan

AU - Wang, Yuxiao

AU - Kaliszewski, Megan J.

AU - Skinner, Kevin C.

AU - Comar, William D.

AU - Shi, Xiaojun

AU - Dasari, Pranathi

AU - Zhang, Xuewu

AU - Smith, Adam W.

PY - 2015/11/3

Y1 - 2015/11/3

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84947547293&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84947547293&partnerID=8YFLogxK

U2 - 10.1016/j.bpj.2015.04.043

DO - 10.1016/j.bpj.2015.04.043

M3 - Article

C2 - 26536270

AN - SCOPUS:84947547293

VL - 109

SP - 1937

EP - 1945

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 9

ER -