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

Frontiers in Molecular Neuroscience
Nicolás M RosasBeata Fuchsova

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 for...Continue Reading

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Citations

Aug 11, 2019·Journal of Neuropathology and Experimental Neurology·Raymond A SobelJulian R Hinojoza
Apr 7, 2020·Journal of Biomolecular Structure & Dynamics·Zoya Khalid, Osman Ugur Sezerman
May 22, 2021·Frontiers in Synaptic Neuroscience·Antonella LeónCamila Scorticati

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Methods Mentioned

BETA
PCR
flow cytometry
transfection
scraping
glycosylation
protein folding
fluorescence microscopy
ubiquitination
confocal microscopy
GTPase

Software Mentioned

ConSurf
PhosphositePlus
ASW
FlowJo
ImageJ
UniProtKB
Ensembl
Prosite
FV10

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