Filopodia Formation driven by M6a depends upon M6a´s oligomerization

KARINA FORMOSO; MICAELA GARCIA; ALBERTO CC FRASCH; CAMILA SCORTICATI

Huerta Grande, Cordoba

Congreso; XXIX Congreso Anual de la Sociedad Argentina de Investigación en Neurociencias; 2014

M6a is a 278 aminoacids integral neuronal membrane glycoprotein that belongs to the proteolipid protein family (PLP). According to sequence prediction M6a has four transmembrane domains (TMDs) that result in an internal loop and two external loops and the N and C-tails facing to the cytoplasm. M6a function has been mostly related to neuronal plasticity. In our laboratory we showed that M6a induces filopodia formation and neurite extension, but the mechanism underlying this function remains unknown. Our hypothesis is that the mayor external loop of M6a interacts with an external stimuli that could produce homo- or hetero-associations of M6a, guided by its TMD, in specific membrane microdomains conferring its stabilization. Thereupon, the M6a auto/phosphorylation occurs in specific C-terminal residues leading to filopodia formation. Here we studied if M6a oligomerization and interaction through its TMDs domains are involved in M6a´s function. For this purpose, we determined by crosslinking assays that M6a forms oligomers in the neuronal membrane. We performed an assay designed to determine homotypic interactions between TMDs (TOXCAT assay) and demonstrated that the interactions between M6a´s TMDs might be aiding this oligomerization and that certain glycine residues present in TMD2 and TMD4 are necessary to drive this interaction. Moreover, we found that the homotypic interactions in M6a are needed for filopodia formation. We also studied three non synonymous SNPs from the NCBI database present in the sequence of GPM6a that correspond to the coding sequence of M6a´s TMDs and found that the presence of one of this SNPs impairs TMD2 interaction. We also found that all SNPs impair M6a induced filopodia formation. These results provide evidence that reinforce our model for a possible mechanism of action for M6a.