Identification of Endophilins 1 and 3 as Selective

Authors: Stephanie De Gois Elisabeth Jeanclos Marie Morris Sukhjeevan Grewal Helene Varoqui Jeffrey D Erickson

Publish Date: 2006/05/19

Volume: 26, Issue: 4-6, Pages: 677-691

PDF Link

Abstract

1 Selective protein–protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission We screened a yeast twohybrid library with bait containing the Cterminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3 proteins considered to play an integral role in glutamatergic vesicle formation2 Using a modified yeast plasmid vector to enable more costeffective screens we analyzed the selectivity and specificity of this interaction Endophilins 1 and 3 selectively recognize only VGLUT1 as the Cterminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs Pro1 PPAPPP and Pro2 PPRPPPP found only in VGLUT1 and two conserved stretches SEEK SYGAT found also in VGLUT2 and VGLUT3 The absence of the VGLUT conserved regions does not affect VGLUT1–endophilin association Of the two polyproline stretches only one Pro2 is required for binding specificity to both endophilin 1 and endophilin 33 We also show that endophilin 1 and endophilin 3 colocalize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate

Keywords: