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.
authors.affiliation: Department of Chemistry, University of Akron, Akron, Ohio.
authors.forename: Morgan
authors.initials: M
authors.lastname: Marita
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California.
authors.forename: Yuxiao
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Akron, Akron, Ohio.
authors.forename: Megan J
authors.initials: MJ
authors.lastname: Kaliszewski
authors.affiliation: Department of Chemistry, University of Akron, Akron, Ohio.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Skinner
authors.affiliation: Department of Chemistry, University of Akron, Akron, Ohio.
authors.forename: William D
authors.initials: WD
authors.lastname: Comar
authors.affiliation: Department of Chemistry, University of Akron, Akron, Ohio.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Shi
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas.
authors.forename: Pranathi
authors.initials: P
authors.lastname: Dasari
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas.
authors.forename: Xuewu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, University of Akron, Akron, Ohio. Electronic address: awsmith5@uakron.edu.
authors.forename: Adam W
authors.initials: AW
authors.lastname: Smith
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D061848:Optical Imaging; D055503:Protein Multimerization; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D039961:Semaphorins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; C492110:Plxna4 protein, mouse; D011956:Receptors, Cell Surface; D039961:Semaphorins