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Title: Characterisation of the tissue-specific expression, pharmacology and signalling cascades activated by chicken GnRH receptor subtypes suggested evolutionary specialisation of type III cGnRH receptor function
Author: Joseph, Nerine Theresa
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2010
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Variant GnRH ligand and receptor subtypes have been identified in a number of non-mammalian vertebrate species, however research into avian species GnRH systems is lacking. Two isoforms of GnRH are present in the domestic chicken, the evolutionarily conserved GnRH-II and diverged cGnRH-I. The expression of two GnRH ligands parallels the expression of two chicken GnRH receptor subtypes; cGnRH-R-I and the novel cGnRH-R-III. The occurrence of two isoforms of the receptor in the chicken raises questions about their specific biological functions and interactions with the two ligands. Differential roles for these molecules in regulating gonadotrophin secretion or other functions are currently unclear. To investigate this, cGnRH-R-III cDNA was cloned from a broiler chicken anterior pituitary gland and its structure and expression was compared with cGnRH-R-I. Expression profiling of cGnRH-R-III cDNA showed that it is predominantly expressed in the anterior pituitary, approximately 1400 times more abundantly than cGnRH-R-I suggesting that cGnRH-R-III is the predominant regulator of chicken gonadotrophin synthesis and secretion. Additionally, pronounced sex and age differences existed, with higher pituitary cGnRH-R-III mRNA levels in sexually mature females versus juvenile females. In contrast, higher mRNA expression levels occurred in juvenile males compared to sexually mature males. Determination of ligand-binding selectivity and the level of cGnRH-R-III activation in response to the endogenous ligands, cGnRH-I and GnRH-II, was anticipated as facilitating the elucidation of the physiological roles of the receptor subtypes. Additionally, the development of analogs that differentially promote or inhibit activation of the receptor subtypes may be valuable tools for determining the role of receptor types in the regulation of gonadotrophin production. To investigate this, pharmacological profiling of cGnRH-R-III in terms of ligand-binding selectivity and inositol phosphate production in response to GnRH analogs was determined in comparison with the pharmacological profile of cGnRH-R-I. Functional studies in COS-7 cells indicated that cGnRH-R-III has a higher binding affinity for GnRH-II than cGnRH-I (IC50: 0.57 v 19.8 nM) and more potent stimulation of inositol phosphate production (EC50: 0.8 v 4.38 nM). Similar results were found for cGnRH-R-I, (IC50: 0.51 v 10.8 nM) and (EC50: 0.7 v 2.8 nM). Mammalian receptor antagonist 27 distinguished between cGnRH-R-I and cGnRH-R-III (IC50: 2.3 v 351 nM), and application of this synthetic peptide may facilitate delineation of receptor subtype function either in-vitro or in-vivo. The length of the C-terminal tail of cGnRH-R-III is 8 residues longer than that of cGnRH-R-I and this observation stimulated investigation of differences in ligand-induced internalisation between the two receptor subtypes. The initial rate of receptor internalisation was faster for cGnRH-R-III than for cGnRH-R-I (26%.min-1 v 15.8%.min-1). Although proteins encoded by cGnRH-R-III splice variants do not bind GnRH ligands independently and mRNAs were not detectable by Northern blot analysis, cGnRH-R-III_SV2 significantly reduced maximum ligand-binding of cGnRH-R-III, suggesting that it may impair the function of the full-length type III cGnRH receptor. It was anticipated that the two cGnRH-R subtypes may have differential roles in the regulation of luteinising hormone (LH) and follicle stimulating hormone (FSH) gene transcription through the activation of differential second messenger pathways. Three putative Src homology domain 3 (SH3) binding motifs were identified in the type III cGnRH receptor cytoplasmic C-terminal tail domain which are not present in the type I cGnRH-R and suggested the potential for differential coupling to the Mitogen Activated Protein Kinase (MAPK) cascade. To investigate this possibility, activation of the MAPK cascade via cGnRH-R-III and cGnRH-R-I was determined by quantifying elevation of phosphorylated ERK (pERK 1/2) in response to GnRH. Studies performed in COS-7 cells showed a 4-6 fold increase in ERK 1/2 phosphorylation via the type I and type III receptors within 10 minutes of GnRH-I or GnRH-II stimulation, indicating that both receptors signal through the ERK 1/2 pathway in response to cGnRH-I or GnRH-II. The responses were dose-dependent at cGnRH-R-I and cGnRH-R-III. Effects of pre-treatment with PLC and c-Src inhibitors showed that both cGnRH-Rs may activate pERK 1/2 independently of PLC but dependently upon c-Src. However, it must be noted that 100% of the PLC activity was not inhibited by PLC inhibitor as measured by inositol phosphate production at 60 minutes, and the PLC inhibitor has not been shown to inhibit PLC in the same time frame used for the pERK experiments. Mutagenesis of the individual SH3 binding motifs of cGnRH-R-III were performed and the effects on pERK 1/2 levels quantified. The results indicated that the SH3 binding motifs of cGnRH-R-III do not contribute to additional MAPK activation when compared to the native cGnRH-R-III. Both cGnRH-R-I and cGnRH-R-III were HA epitope-tagged (HA-cGnRH-R-I and HA-cGnRH-R-III) and the methodology was optimised for HA-cGnRH-R-III immuno-precipitation. Several size forms of HA-cGnRH-R-III were detectable by immuno-precipitation, facilitating characterisation of the composition of the receptor protein-protein complexes formed using a western blot approach. In summary, the abundance of cGnRH-R-III expression compared to cGnRH-R-I suggests it is probably the major mediator of pituitary gonadotroph function, and that tissue-specific recruitment of cGnRH-R-isoforms has occurred in the avian pituitary during evolution. Pharmacological profiling demonstrated that cGnRH-R-III, like cGnRH-R-I, has a higher ligand-binding selectivity and induction of inositol phosphate production in response to GnRH-II than with cGnRH-I, although cGnRH-I is established as the physiological regulator of gonadotroph function. These results suggest that evolutionary recruitment of ligand-receptor pairing for particular physiological processes does not correlate with in-vitro properties such as highest ligand-binding affinity or efficacy of inositol phosphate production. Therefore evolutionary plasticity has occurred in the tissue-specific adoption of GnRH ligand and receptor subtypes for regulation of particular physiological functions in birds.
Supervisor: Dunn, Ian. ; Millar, Robert. ; Morgan, Kevin. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: chicken ; GnRH ; GnRH-receptor ; GPCR ; expression ; pharmacology ; signalling