Is the integrity of Gpm6a s transmembrane domains important in filopodium formation?

KARINA FORMOSO; ALBERTO C C FRASCH; CAMILA SCORTICATI

Vancouver

Congreso; 6th Annual Canadian Neuroscience Meeting; 2012

IBRO

Neuronal glycoprotein M6a is involved in hippocampal plasticity through still unknown mechanisms. Although it is not classified within the Tetraspanin protein family, M6a bears striking structural similarities with this family. It is well known that the ability to self-associate and interact with different membrane proteinsand lipids through specific alpha-helix residues is what allows tetraspanins to carry out their functions. Previous results have determined the existence of M6a-oligomers in neuronal membranes and an in vitro assay demonstrated the self-association of M6a-transmembrane domains (TM).The TMs of membrane proteins are a frequent target of disease causing mutations that impair such interactions. In this sense, our aim is to assess the association between genetic variants in GPM6A‘s TMs and filopodium induction in neurons. Three nonsynonymous polymorphisms were found in the TMs coding regions of the GPM6A gene (SNP1 (F93C) and SNP2 (197S) for TM2 and SNP3 (W141R) for TM3). To study the ability of M6a to induce Filopodium formation, the different mutants were overexpressed in primary hippocampal neurons and in neuroblastoma cell line, N2a. In neurons overexpressing all mutants display reduced filopodia density. On the contrary, in N2a cells only M6a-SNP3 mutant failed to induce filopodia formation. Moreover, in non-permeabilized neurons only M6a-SNP3 clone was not recognized by monoclonal antibody directed to a major extracellular loop of M6a and prevented the cell surface expression of M6a which was retained in the endoplasmic reticulum showing a colocalization pattern with calnexin. Altogether, in this work we provided evidence that all SNPs impaired M6a plasticity in neurons. In the case of SNP3 this could be due to the fact that M6a-TM3 mutation impaired the proper assembly required for surface expression.In the case of SNP1 and SNP2 further studies are needed to analyze a possible link between TM2-M6a mutants and reduced hippocampal plasticity.