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dc.contributor.authorCook, Julia Vanessa Fosketten
dc.date.accessioned2018-01-31T11:41:47Z
dc.date.available2018-01-31T11:41:47Z
dc.date.issued1996en
dc.identifier.urihttp://hdl.handle.net/1842/27825
dc.description.abstracten
dc.description.abstractMammalian reproduction is driven by gonadotrophin -releasing hormone (GnRH) - a decapeptide released from hypothalamic neurones into the pituitary portal blood vessels. The aim of this thesis was to study structure- function relationships of the Gprotein coupled GnRH receptor (GnRH -R), and has focused on the identification of key amino acids involved in GnRH ligand- receptor interactions as well as the role of putative disulphide bridge formation within the receptor itself. In addition, the role of disulphide bridge formation has also been explored in another G- protein coupled receptor (GPCR), the thyrotrophin- releasing hormone receptor (TRH -R). Comparative sequence analysis and computer molecular modelling approaches were used to target potentially important amino acids for site -directed mutagenesis study. Wild -type and receptor mutants were then expressed in mammalian cells, and receptor binding, expression, and activational properties compared between constructs.en
dc.description.abstractThe majority of GPCRs contain two conserved extracellular Cys residues which have been postulated to form a covalently linked disulphide bridge structure. In the GnRH -R, these Cys residues are positioned at Cys 114 and Cys 195 in the first and second extracellular loops respectively. In addition, the GnRH -R contains two non - conserved Cys residues at Cys 14 in the amino terminus and Cys 199 in the second extracellular loop. Substitution of these Cys residues to serine resulted in a loss of ligand binding. A comparative study in the TRH -R, substituting the conserved extracellular Cys residues, Cys98 and Cys 179, to either serine or alanine, confirmed these findings. This data suggests that extracellular Cys residues, through putative disulphide bridge formation, may maintain the tertiary extracellular structure of the receptor and therefore facilitate ligand- receptor binding. Further studies, using chemical modifying reagents, have indicated that Cys residues with free sulfhydryl groups may also be important in TRH-R binding.en
dc.description.abstractThe GnRH -R despite its structural homology to other GPCRs exhibits some unique features. These differences include the interchange of a highly conserved Asp and Asn residue in the transmembrane (TM) domains. Individual substitutions of Asn87 (in TM II) to Asp87 and Asp318 (in TM VII) to Asn318, revealed that Asn87 is important for GnRH agonist and antagonist binding whereas Asp318 is important for receptor activation. To investigate if the function as well as the position of these amino acids were transposed, a double mutation substituting both residues simultaneously was generated. However, this mutant receptor showed only a small degree of GnRH agonist binding, indicating that the functional role of these specific residues is not interchangeable.en
dc.description.abstractAmongst GPCRs, the GnRH -R is particularly suitable for three dimensional molecular modelling and computer aided simulations because of its short extracellular and intracellular domains. Using this approach, it has been possible to predict putative amino acids involved in ligand- receptor interactions. During this study, GnRH molecular models have evolved from a template predicted by the Baldwin model to a series of energy minimised computer generated three dimensional structures. To simulate GnRH ligand- receptor interactions, a model of the native GnRH peptide has also been constructed. The initial Baldwin model highlighted a series of TM located polar amino acids in TM II, TM III, TM IV and TM VII of the GnRH -R. Both the position and nature of these amino acids rendered them capable of interacting with the GnRH ligand. Mutations at these sites identified two residues, His305 located at the TM VII/extracellular interface and Asn314 in TM VII, that were potentially important for GnRH -R binding.en
dc.description.abstractFurther modelling studies, using GnRH ligand- receptor computer simulations, predicted that amino acids Phe312 in TM VII and Leu83 in TM II may interact with Trp3 and Leu7 in the GnRH ligand respectively. Substituting these amino acids to residues of either similar, different or neutral hydropathicity, showed that a hydrophobic amino acid was essential for GnRH -R binding at position 312 and for receptor activation at residue 83. Altogether, 15 sites within the GnRH -R have been experimentally modified and the information derived from site -directed mutagenesis studies has been utilised to redefine the structure of the molecular models.en
dc.description.abstractIn conclusion, the formation of a putative disulphide bond between extracellular cysteine residues in both the GnRH -R and TRH -R is important in maintaining tertiary protein structure. In addition, amino acids located in TM II and TM VII are essential for binding interactions between GnRH and its receptor. Analysis of structure -function relationships, particularly using this dual biochemical and molecular modelling approach, will greatly facilitate rational drug design. In the light of the enormous clinical applications of GnRH and its analogues, information regarding the mechanisms of hormone -receptor interactions will be of benefit in the development of new and novel drugs for clinical use in reproductive medicine.en
dc.publisherThe University of Edinburghen
dc.relation.isreferencedbyAlready catalogueden
dc.subjectAnnexe Thesis Digitisation Project 2017 Block 16en
dc.titleSite-directed mutagenesis studies of the GnRH and TRH receptors of the pituitary glanden
dc.typeThesis or Dissertationen
dc.type.qualificationlevelen
dc.type.qualificationnamePhD Doctor of Philosophyen


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