Ojla, Gurmail Singh;
(2006)
GRIF-1, a novel neuronal trafficking protein: Molecular and cellular characterisation.
Doctoral thesis (Ph.D.), University College London (United Kingdom).
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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.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D. |
| Title: | GRIF-1, a novel neuronal trafficking protein: Molecular and cellular characterisation |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. |
| Keywords: | (UMI)AAI10104303; Biological sciences; GABA receptors |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10103480 |
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