IIBIO   27936
INSTITUTO DE INVESTIGACIONES BIOTECNOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Analysis of the key functional residues within the C-terminal cytoplasmic tail of Gpm6a critical for filopodium outgrowth
Autor/es:
FRASCH, A. C.; ROSAS, N. M.; FUCHSOVA, B.
Lugar:
Córdoba
Reunión:
Congreso; XXXIII Congreso Anual de la Sociedad Argentina de Investigación en Neurociencias (SAN) 2018; 2018
Institución organizadora:
SAN
Resumen:
Analysis of the key functional residues within the C-terminal cytoplasmic tail of Gpm6a critical for filopodium outgrowthRosas N.M., Frasch A.C. and Fuchsova B.Instituto de Investigaciones Biotecnológicas (IIB-INTECH, UNSAM, CONICET), San Martin, Buenos Aires, Argentina Gpm6a is a neuronal membrane glycoprotein with four transmembrane domains and the N- and C-terminal ends facing the cytoplasm. It functions in the processes of neuronal development and its overexpression leads to the extensive formation of filopodia. However, the mechanism of action of Gpm6a is not clearly understood.Previously, we mapped the regulatory effect of Gpm6a in filopodium formation to its C- but not the N-terminal cytoplasmic end. Following alanine scanning mutagenesis of the C-terminal cytosolic end identified K250, K255, and E258 as the key functional residues. Subsequent bioinformatic analysis revealed that K250, K255, and E258 are predicted as part of sorting signals of transmembrane proteins.Here, we use flow cytometry analysis to show that total expression levels of truncation mutants do not differ from the wt Gpm6a, but the amount of both truncated proteins on cell surface is lower. Our colocalization assay shows that deletion of the C- but not the N-terminus diminishes the association of Gpm6a with clathrin implying involvement of clathrin-mediated trafficking events. Substitution of K250, K255, and E258 with alanine also diminishes the amount Gpm6a on cell surface and in case of K255 and E258 also leads to the lower amount of total expressed protein. Subsequent subcellular localization studies using confocal microscopy reveal that mutant forms of Gpm6a that failt o induce filopodia formation display preferential localization to Lamp1-positive structures.<!-- /* Font Definitions */@font-face{font-family:"MS 明朝";mso-font-charset:78;mso-generic-font-family:auto;mso-font-pitch:variable;mso-font-signature:-536870145 1791491579 18 0 131231 0;}@font-face{font-family:"Cambria Math";panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:auto;mso-font-pitch:variable;mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;mso-font-charset:0;mso-generic-font-family:auto;mso-font-pitch:variable;mso-font-signature:-520092929 1073786111 9 0 415 0;}@font-face{font-family:ArialMT;panose-1:0 0 0 0 0 0 0 0 0 0;mso-font-alt:Arial;mso-font-charset:77;mso-generic-font-family:swiss;mso-font-format:other;mso-font-pitch:auto;mso-font-signature:3 0 0 0 1 0;} /* Style Definitions */p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin-top:0cm;margin-right:0cm;margin-bottom:10.0pt;margin-left:0cm;line-height:115%;mso-pagination:widow-orphan;font-size:11.0pt;font-family:Calibri;mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-ansi-language:ES;mso-fareast-language:EN-US;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-size:11.0pt;mso-ansi-font-size:11.0pt;mso-bidi-font-size:11.0pt;font-family:Calibri;mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-ansi-language:ES;mso-fareast-language:EN-US;}.MsoPapDefault{mso-style-type:export-only;margin-bottom:10.0pt;line-height:115%;}@page WordSection1{size:612.0pt 792.0pt;margin:70.85pt 3.0cm 70.85pt 3.0cm;mso-header-margin:36.0pt;mso-footer-margin:36.0pt;mso-paper-source:0;}div.WordSection1{page:WordSection1;}-->