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Title: Membrane re-insertion of myristoyl-peptidyl and glycophosphatidyl inositol anchored domain growth hormone receptors
Author: Jarrett, Caroline
ISNI:       0000 0001 3589 2780
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2007
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Growth honnone (GH) is an anabolic hormone important for childhood growth and the maintenance of nonnal adult body composition. GH acts through the growth hormone receptor (GHR) which is a member of the type I cytokine receptor family. ~ese receptors possess in common a single transmembrane domain, lack intrinsic kinase activity and signal through a phosphorylation cascade. In the case of GH it binds to a prefonned GHR dimer and ligand binding generates a conformational change that triggers receptor signalling. Truncated GHRs that have a transmembrane domain but no intracellular domain exist at low . levels in nonnal human tissues. If over expressed these truncated extracellular domain receptors act to block GHR signalling through heterodimerisation with the full length GHR. The hypothesis of this thesis was that synthetic truncated GHRs could be generated and if anchored in the cell membrane used to create novel GH antagonists. In humans there are a variety of different mechanisms by which proteins attach to cell surface membranes. A common mechanism is through post-translational modification with a glycophosphatidyl inositol (GPI) anchor and an alternative mechanism is achieved via a cotranslational acylation process such as myristoylation. Myristoylation has been exploited as a therapeutic to develop synthetic myristoylated peptides that can be inserted into cell membranes. This thesis has examined whether truncated GHR could be lipid anchored either through the addition of a GPI moiety or through myristoylation and whether anchored truncated GHR could modulate GH signalling. A truncated GHR with a GPI anchor (GHRed-OPI) was cloned, expressed and purified from the slime mould Dictyostelium discoideum. The extracellular domain of GHR was cloned and expressed in Escherichia coli. A truncated form (1-241 amino acids) was then purified and myristoylated (GHR-mp) through the addition of a synthetic myristoyl-peptidyl tail (synthetic peptide APT542) to the free C-terminal cysteine (amino acid 241). Both the GHRed-OPI and GHR-mp bound OH and inserted into cell membranes. The GHR-mp was examined in a bioassay and shown to partially inhibit OH signalling.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available