Alanine Scanning Mutagenesis of the C-Terminal Cytosolic End of Gpm6a Identifies Key Residues Essential for the Formation of Filopodia

Abstract

Neuronal membrane glycoprotein M6a (Gpm6a) is a protein with four transmembrane regions and the N- and the C-ends facing the cytosol. It functions in processes of neuronal development, outgrowth of neurites, and formation of filopodia, spines, and synapsis. Molecular mechanisms by which Gpm6a acts in these processes are not fully comprehended. Structural similarities of Gpm6a with tetraspanins led us to hypothesize that, similarly to tetraspanins, the cytoplasmic tails function as connections with cytoskeletal and/or signaling proteins. Here, we demonstrate that the C- but not the N-terminal cytosolic end of Gpm6a is required for the formation of filopodia by Gpm6a in cultured neurons from rat hippocampus and in neuroblastoma cells N2a. Further immunofluorescence microcopy and flow cytometry analysis show that deletion of neither the N- nor the C-terminal intracellular domains interferes with the recognition of Gpm6a by the function-blocking antibody directed against the extracellular part of Gpm6a. Expression levels of both truncation mutants were not affected but we observed decrease in the amount of both truncated proteins on cell surface suggesting that the incapacity of the Gpm6a lacking C-terminus to induce filopodium formation is not due to the lower amount of Gpm6a on cell surface. Following colocalization assays shows that deletion of the C- but not the N-terminus diminishes the association of Gpm6a with clathrin implying involvement of clathrin-mediated trafficking events. Next, using comprehensive alanine scanning mutagenesis of the C-terminus we identify K250, K255, and E258 as the key residues for the formation of filopodia by Gpm6a. Substitution of these charged residues with alanine also diminishes the amount of Gpm6a on cell surface and in case of K255 and E258 leads to the lower amount of total expressed protein. Subsequent bioinformatic analysis of Gpm6a amino acid sequence reveals that highly conserved and functional residues cluster preferentially within the C- and not within the N-terminus and that K250, K255, and E258 are predicted as part of sorting signals of transmembrane proteins. Altogether, our results provide evidence that filopodium outgrowth induced by Gpm6a requires functionally critical residues within the C-terminal cytoplasmic tail.

Glycoprotein M6A (GPM6A) is over-expressed in brain metastatic breast cancer.

“…GPM6A spans 274 amino acids in length and is a membrane glycoprotein with expression originally reported as specific to the brain 4,5 . Structural features of GPM6A include four transmembrane domains, two extracellular loops, and a small intracellular loop 7 . In human embryonic stem cells, GPM6A ectopic expression results in neuroectoderm-associated genes, including Pax2 and Sox2, as well increased numbers of hES cell-derived neural stem cells 8 .…”

Glycoprotein M6A is differentially expressed in non-small cell lung cancer and associates with patient survival.

“…Human GPM6A is a membrane glycoprotein of 274 amino acids and was originally described to possess specific expression in the brain 7,8 . GPM6A contains four transmembrane domains, two extracellular loops, and a small intracellular loop 9 . Ectopic expression of GPM6A in a human embryonic stem cell line (hES) results in expression of genes associated with the neuroectoderm, like Pax2 and Sox2, as well as an increase in the number of neural stem cells derived from hES cells 10 .…”

Neuronal Glycoprotein M6a: An Emerging Molecule in Chemical Synapse Formation and Dysfunction

“…On the contrary, neurons subjected to siRNA depletion of M6a and neurons overexpressing a truncated form of M6a at the EC2 loop exhibited a decreased number of synaptophysin puncta (Alfonso et al, 2005;Fuchsova et al, 2009;Formoso et al, 2016). Moreover, M6a internalizes and recycles back to the cell membrane via clathrin-mediated endocytosis, and localizes in Rab5, Rab7, and Rab11 positive endosomes, just like the case of SVs recycling pathways (Wu et al, 2007;Formoso et al, 2016;Garcia et al, 2017;Rosas et al, 2018). Indeed, inducing M6a acute internalization correlated with a decrease of both the number of synaptophysin puncta and synapses (Garcia et al, 2017), suggesting that M6a might be playing an active role in SV/neurotransmitter release.…”

“…M6a has been widely described to be involved in filopodia/spine formation in different cell culture models ( Alfonso et al, 2005 ; Sato et al, 2011b ; Scorticati et al, 2011 ; Formoso et al, 2015b ; Alvarez Julia et al, 2016 ; Formoso et al, 2016 ; Rosas et al, 2018 ). Two pathways have been implicated by which M6a increases filopodia density.…”

“…Likewise, signaling pathways that include Rac1 and Pak1 activation through coronin1A facilitate M6a-induced filopodium formation ( Alvarez Julia et al, 2016 ). There are key domains required for M6a to induce filopodia/spine formation ( Figure 1D ): (i) TMDs homotypic-interactions, especially commanded by particular glycine residues at TMD2 and TMD4 ( Formoso et al, 2015b , 2016 ), (ii) a disulfide bridge at EC2 loop formed between cysteine residues C174 and C192 ( Fuchsova et al, 2009 ), and (iii) C-terminal domain residues K250/K255/E258 ( Rosas et al, 2018 ). Although the N-terminus is not involved in filopodia induction ( Mita et al, 2015 ; Rosas et al, 2018 ), phosphorylation of certain serine/threonine residues at N-and C-termini, T10/S256/S267/T268, proved to be necessary for filopodial motility ( Brocco et al, 2010 ).…”