Filopodia formation driven by membrane glycoprotein M6a depends on the interaction of its transmembrane domains

Abstract

Membrane glycoprotein M6a, which belongs to the tetraspan proteolipid protein family, promotes structural plasticity in neurons and cell lines by unknown mechanisms. This glycoprotein is encoded by Gpm6a, a stress-regulated gene. The hippocampus of animals chronically stressed by either psychosocial or physical stressors shows decreased M6a expression. Stressed Gpm6a-null mice develop a claustrophobia-like phenotype. In humans, de novo duplication of GPM6A results in learning/behavioral abnormalities, and two single-nucleotide polymorphisms (SNPs) in the non-coding region are linked to mood disorders. Here, we studied M6a dimerization in neuronal membranes and its functional relevance. We showed that the self-interaction of M6a transmembrane domains (TMDs) might be driving M6a dimerization, which is required to induce filopodia formation. Glycine mutants located in TMD2 and TMD4 of M6a affected its dimerization, thus preventing M6a-induced filopodia formation in neurons. In silico analysis of three non-synonymous SNPs located in the coding region of TMDs suggested that these mutations induce protein instability. Indeed, these SNPs prevented M6a from being functional in neurons, owing to decreased stability, dimerization or improper folding. Interestingly, SNP3 (W141R), which caused endoplasmic reticulum retention, is equivalent to that mutated in PLP1, W161L, which causes demyelinating Pelizaeus-Merzbacher disease.

The Membrane Glycoprotein M6a Endocytic/Recycling Pathway Involves Clathrin-Mediated Endocytosis and Affects Neuronal Synapses

“…In neurons, we immunodetected the total amount of endogenous M6a at T0, T1, and WO (20 h after medium replacement) by Western blot using a polyclonal antibody against the C-tail of M6a. M6a can be displayed as a single or a double band of approximately 32–35 kDa as previously described ( Fuchsova et al, 2009 ; Scorticati et al, 2011 ; Formoso et al, 2015b ). In HEK293 cells, the replacement medium (WO) was supplemented with cycloheximide and maintained for 4 h to prevent the amount of M6a per cell from being modified by the new synthesis.…”

Identification of Potential Interacting Proteins With the Extracellular Loops of the Neuronal Glycoprotein M6a by TMT/MS

“…Apart from GPM6A (here, 6,9/3,0 average.top3/log 2 FC, Supplementary Table S2 ), none of them were confirmed by our approach. Nevertheless, the oligomerization of M6a in cell surface from rat hippocampi and cultured neurons has been already proven (Formoso et al, 2015b ). However, when (Honda et al, 2017 ) co-immunoprecipitated Triton X-100 extracts from mice neuronal growth cone membrane fractions using M6a-mAb; they identified and confirmed by immunostaining the endogenous association between M6a and Rufy3.…”

“…M1 and M2 estimate the fluorescence intensity of the two colors that overlap in a pixel from the PLP or TfR image to M6a and vice versa, respectively. A coefficient close to one indicates a high overlap of the signals (Formoso et al, 2015b ). Data are expressed as the average of Pearson’s coefficient ± SEM.…”

“…The membrane glycoprotein M6a—together with proteolipid protein PLP1, DM20, and M6b—belongs to the tetraspan PLP family (Schweitzer et al, 2006 ). M6a is a neuronal surface protein that promotes neuronal stem cell differentiation, migration, neurite outgrowth, filopodia/spine induction, and synapse formation in primary neuronal cultures and (non-) neuronal cell lines (Alfonso et al, 2005 ; Michibata et al, 2008 ; Zhao et al, 2008 ; Michibata et al, 2009 ; Formoso et al, 2015a , b , 2016 ; Garcia et al, 2017 ; Honda et al, 2017 ). In humans, alterations in M6a levels or polymorphisms in GPM6A , are associated with depression, schizophrenia, claustrophobia, bipolar disorders, and learning disabilities (Boks et al, 2008 ; Greenwood et al, 2012 ; El-Kordi et al, 2013 ; Gregor et al, 2014 ; Fuchsova et al, 2015 ).…”

Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay

“…Plant receptor-like kinases: CR4 and ACR4 [79,80] Cytokine receptors: thrombopoietin receptor [81], p75 neurotrophin receptor [82] Cellular adhesion: integrin αIIbβ3 [83][84][85], syndecan family [86], E-cadherin [87], Na,K-ATPase subunit β [88], Lselectin [89], GP Ib-IX complex [90,91] Neurological proteins: M6a [92], sigma-1 receptor [67], myelin proteolipid protein [93], myelin protein zero [94], p75 neurotrophin receptor [82], synaptobrevin [95], drosophila Wnt receptor [96] Mitochondrial proteins: UbiB protein kinase-like ADCK3 [69], rat carnitine palmitoyltransferase 1A [97,98], Bcl-2 family apoptosis regulator BNIP3 [35,99] Amyloid forming proteins: pigment cell-specific protein PMEL [100], amyloid precursor protein [101] Immunological: major histocompatibility complex (MHC) 1 class II-associated invariant chain [102] Transporters: ABC transporter ABCG2 (TM helix 1) [103] GPCRs: Mam2 (TM helix 1) [104] Chaperones: yeast ER chaperone Rot1 [105] Channels: gap junction protein connexin 26 (TM helix 1) [106], voltage-dependent potassium channel BK (TM helix 1) [107], Epithelial Na + channel EnaC subunit α (TM helix 1) [108] Hematopoietic proteins: mediator of receptor activation DAP12 [109], spleen focus forming virus gp55-A and gp55-P [110], thrombopoietin receptor [81] Bacterial proteins: cell division proteins FtsB…”