eprintid: 10103480
rev_number: 9
eprint_status: archive
userid: 695
dir: disk0/10/10/34/80
datestamp: 2020-07-01 14:45:39
lastmod: 2020-07-01 14:45:39
status_changed: 2020-07-01 14:45:39
type: thesis
metadata_visibility: show
creators_name: Ojla, Gurmail Singh
title: GRIF-1, a novel neuronal trafficking protein: Molecular and cellular characterisation
ispublished: unpub
keywords: (UMI)AAI10104303; Biological sciences; GABA receptors
note: Thesis digitised by ProQuest.
abstract: GABAA receptor interacting factor-1 (GRIF-1) is a novel protein that has been proposed to function in the trafficking of GABAA receptors and/or organelles. Evidence of in vitro association of GRIF-1 with GABAA receptor β2 subunits implicates a role of GRIF-1 in the regulation of receptor number at synapses in adult brain. GRIF-1 also shares 45% amino acid sequence identity with the human protein, β-O-linked N-acetyl glucosamine transferase (OGT) interacting protein, OIP106. Both GRIF-1 and OIP106 contain predicted coiled-coil domains in their N-terminal regions. It is thought that they constitute a novel coiled-coil domain gene family. GRJF-1 may be a species homologue of Milton, a Drosophila protein thought to function as a mitochondrial transport protein, via the microtubule-based molecular motor protein kinesin. GRIF-1 and OIP106 both interact with OGT and kinesin. Thus GRIF-1 may play a role in the anterograde transport of both GABAA receptors and/or organelles in neurons. To gain further insight into the function of GRIF-1, the quaternary structure was characterised using epitope-tagged GRIF-1 constructs in an immunoprecipitation strategy. Results showed that GRIF-1 is a soluble protein that forms disulphide-linked homo-oligomers. Size determination of recombinant GRIF-1 under native conditions using sucrose density gradient sedimentation demonstrated that GRIF-1 exists as Mr species with S20W values = 10 ± 0.8 and 13 ± 1 representing dimeric and higher molecular weight GRIF-1 complexes. The localisation of GRIF-1 was studied in primary cultures of hippocampal pyramidal neurons. Expression of exogenous GRIF-1 was investigated using an enhanced green fluorescent protein (EGFP)-tagged GRIF-1 fusion protein (EGFP-GRIF-1). Characterisation of the expression of EGFP-GRIF-1 in HEK 293 cells showed that it behaved as wild-type GRIF-1 in that it formed disulphide-linked homodimers. EGFP-GRIF-1 was transfected using Nucleofector technology. Expression of EGFP-GRIF-1 was detected over 1- 3 DIV and was colocalised with the mitochondrial marker, Mitotracker Far red 633, at the growth cones. Investigation of endogenous GRIF-1 using anti-GRIF-1 antibodies showed that endogenous GRIF-1 colocalised with the mitochondrial marker, Mitotracker™ red. Therefore GRIF-1 may play a role in the neuronal intracellular trafficking of mitochondria and/or GABAA receptors.
date: 2006
oa_status: green
full_text_type: other
thesis_class: doctoral_open
thesis_award: Ph.D.
language: eng
primo: open
primo_central: open_green
verified: verified_manual
full_text_status: public
pages: 204
institution: University College London (United Kingdom)
thesis_type: Doctoral
citation:        Ojla, Gurmail Singh;      (2006)    GRIF-1, a novel neuronal trafficking protein: Molecular and cellular characterisation.                   Doctoral thesis  (Ph.D.), University College London (United Kingdom).     Green open access   
 
document_url: https://discovery.ucl.ac.uk/id/eprint/10103480/1/GRIF-1%2C_a_novel_neuronal_traff.pdf