Cysteine residues in the large extracellular loop (EC2) are essential for the function of the stress-regulated glycoprotein M6a.
Gpm6a was identified as a stress-responsive gene in the hippocampal formation. This gene is down-regulated in the hippocampus of both socially and physically stressed animals, and this effect can be reversed by antidepressant treatment. Previously we showed that the stress-regulated protein M6a is a key modulator for neurite outgrowth and filopodium/spine formation. In the present work, mutational analysis was used to characterize the action of M6a at the molecular level. We show that four cysteines 162, 174, 192, and 202 within EC2 are functionally crucial sites. The presence of cysteines 162 and 202 is essential for the efficient cell surface expression of the M6a protein. In contrast, cysteines 174 and 192, which form a disulfide bridge as shown by biochemical analysis, are not required for the efficient surface expression of M6a. Their mutation to alanine does not interfere with the localization of M6a to filopodial protrusions in primary hippocampal neurons. The neurons expressing C174A and/or C192A mutants display decreased filopodia number. In non-permeabilized cells, these mutant proteins are not recognized by a function-blocking monoclonal antibody directed to M6a. Moreover, neurons in contact with axons expressing C174A/C192A mutant display significantly lower density of presynaptic clusters over their dendrites. Taken together, this study demonstrates that cysteines in the EC2 domain are critical for the role of M6a in filopodium outgrowth and synaptogenesis.
Fuchsova
Beata
B
Instituto de Investigaciones Biotecnológicas-INTECH, Universidad Nacional de San Martín, Consejo Nacional de Investigaciones Científicas y Técnicas, 1650 San Martin, Argentina. beata@iib.unsam.edu.ar
Fernández
María E
ME
Alfonso
Julieta
J
Frasch
Alberto C
AC
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar / Q000196:embryology; D013312:Stress, Physiological
C484159:Gpm6a protein, rat; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D003545:Cysteine