Includes abstract. / Includes bibliographical references. / Includes bibliographical references (leaves 74-81). / Over-expression of β-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a β-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is β-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for β~arrestin dependent internalisation, chimeric receptors were created. / The mammalian type II GnRH receptor possesses an intracellular C-terminal tail that is known to play a role in desensitisation, internalisation and overall signalling in GPCRs. On the other hand, the mammalian type I GnRH receptor, which lacks a C-terminal tail, does not readily desensitise and undergoes slow internalisation compared to the mammalian type II GnRH receptor. Over-expression of ß-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a ß-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is ß-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for ß-arrestin dependent internalisation, chimeric receptors were created. Firstly, a chimera in which the full length type II GnRH receptor C-terminal tail was added to the tail-less type I GnRH receptor (TI/T2tail) was created. This chimera internalised in a ß-arrestin and GRK dependent manner, demonstrating that the type II GnRH receptor C-terminal tail confers ß-arrestin JGRK dependent internalisation on the originally ß-arrestin/GRK insensitive GnRH receptor. Mutating the putative GRK and casein kinase phosphorylation sites (serines 338 and 339) on the C-terminal tail of TI/T2tail to alanine residues did not abolish ß-arrestin dependent internalisation but eliminated GRK dependent internalisation, suggesting that other regions on the C-terminal tail are required for ß-arrestin dependent internalisation. A second chimera, in which the whole third intracellular loop of the type II GnRH receptor was replaced with that of the type I GnRH receptor (T2/TIICL3), was created. This chimera could not utilise ß-arrestin in its internalisation, indicating that the third intracellular loop of the type II GnRH receptor is required for ß-arrestin dependent internalisation. An alignment of the amino acid sequences of the two mammalian GnRH receptor third intracellular loops identified a basic residue rich area (R234, R236 and K237) on the type II GnRH receptor that was absent on the type I GnRH receptor. Interestingly, the triple mutant (R234,236,K237 A) still internalised in a ß-arrestin dependent manner, however, truncation of the C-terminal tail of R234,236,K237A abolished the ability of the receptor to internalise in a ß-arrestin dependent manner. This result indicated that the C-terminal tail of the type II GnRH receptor was compensating for the absence of the three basic residues. To summarise, this thesis demonstrates that the C-terminal tail of the type II GnRH receptor can confer ß-arrestin dependent intemalisation on the type I GnRH receptor. Furthermore, the third intracellular loop, and more specifically, basic residues R234, R236 and K237 on the mammalian type II GnRH receptor are required for ß-arrestin dependent intemalisation.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/3178 |
| Date | January 2006 |
| Creators | Nkwayana, Nonhlanhla |
| Contributors | Katz, Arieh, Katz, Arieh |
| Publisher | University of Cape Town, Faculty of Health Sciences, Department of Clinical Laboratory Sciences |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Master Thesis, Masters, MSc |
| Format | application/pdf |
Page generated in 0.0154 seconds