Plasma membrane lipid microdomain and transmembrane domain self-association of neuronal membrane glycoprotein, M6a.

SCORTICATI, C; FERNANDEZ, ME; FUCHSOVA, B; FRASCH, AC.

Buzios, Brasil

Congreso; Neurolatam; 2008

International Brain Organization, SAN, SBN

We have previously identified M6a as stress/antidepressant-responsive gene in the hippocampus of different animal models.  This gene is down regulated in both physically and socially stressed animals. Antidepressant treatment can block these stress effects. M6a is a protein with four transmembrane domains (TMs), two extracellular loops and N- and C- terminal extensions toward the cell cytoplasm. M6a is involved in neuronal plasticity through an unknown mechanism. The self-association of membrane proteins and/or with  specific microdomains such as lipids rafts in the plasma membrane is important in the regulation of extra and intracellular signaling pathways and can give us a clue of how the protein exerts its function. Objectives: To study protein-protein and protein-lipid interactions of M6a in rat hippocampus and neuroblastoma cell line (N2A). Methods: To determine if M6a could be part of a lipid raft, a discontinuous sucrose gradient in the presence/absence of different non ionic detergents followed by ultracentrifugation and SDS-PAGE-WB technique in a rat hippocampus and N2A was developed. In addition, to establish if M6a is organized as a monomer or an oligomer with a homo/hetero-protein-protein interaction in the plasma membrane, a chemical crosslinking assay and an in vitro homodimerization of TMs using a TOXCAT system were performed.  Results: We identified the presence of M6a in detergent resistant membranes (DRMs) from both hippocampal tissue and N2A cell line. Also, we tested for the formation of stable M6a oligomers in the presence/absence of chemical cross-linkers and under increasing urea concentrations. We show that under non reducing conditions M6a form different stable multimers.  Same results were obtained when we used a chemical crosslinker, although the participation of other proteins cannot be excluded. Using a TOXCAT oligomerization assay for the four M6a-TMs we find self-interaction particulary in TM1 and TM2. Conclusion: We show that lipid raft microdomain association is an intrinsic property of M6a.  Furthermore, our data suggest a strong propensity to self-association of M6a-TMs that could be involved in the formation of oligomers.