Charged amino acid residues K250, D253, K255, E258 and E259A within the C-terminal cytoplasmic tail of Gpm6a mediate its function in filopodium formation

ANABEL ALVAREZ JULIÁ; BEATA FUCHSOVA; NICOLÁS M. ROSAS; ALBERTO C. FRASCH

Mar del Plata

Congreso; XXXII CONGRESO ANUAL SAN 2017; 2017

Sociedad Argentina de Neurociencias

Neuronal filopodia underlie many major morphogenetic events ranging from the initiation, extension, guidance and branching of neuronal processes to the formation of synapsis. A conserved cellular function in filopodium formation has been described for a neuronal membrane glycoproteinGpm6a. It displays structural similarity to tetraspanins with four transmembrane domains, a small extracellular loop (EC1), a short intracellular loop, and a large extracellular loop (EC2), flanked by N- and C-terminal cytoplasmic tails. In our previous study we have showed that Gpm6a lacking C- but not N-terminal cytoplasmic tail fails to induce filopodium formation in hippocampal neurons. Here we used charged-to-alanine scanning mutagenesis to identify functionally critical residues within the C-terminus of Gpm6a. We show that neurons expressing K250A, D253A/K255A or E258A/E259A mutants display decreased filopodium number. Their recognition by a function-blocking monoclonal antibody directed to the EC2 and the accessibility to digestion with the extracellular proteinase K demonstrate surface exposure of these mutant proteins. When D253, K255, E258 and E259 were mutated individually, the effect on filopodium formation was lost suggesting that these residues function in synergy. Subsequent bioinformatic analysis revealed that the residues D253, E258 and E259 form a part of the internalization motifs pointing to the functional significance of the active membrane turnover in filopodial dynamics.