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Title: Selective peptide binding determinants on the N-terminal domain of parathyroid hormone receptors
Author: Mann, Rosalind Jane
ISNI:       0000 0001 3618 1817
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2007
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The focus of this PhD work was to elucidate the mechanisms by which PTH2R mediates its ligand binding selectivity. The approach taken was to create a chimeric PTH creceptor with the aim of removing the selectivity filter from the PTH2R so that it would bind PTHrP(1-36). As residue 23 from PTHrP (shown to be responsible for its binding selectivity) has been cross linked to the extreme N-terminal region of the PTH1R, residues 1-41 from this receptor were introduced into the PTH2R. As both receptors bind PTH(1-34), it was expected that this chimeric receptor would also bind the ligand normally but have altered affinity for PTHrP(1-36). However this chimera had drastically reduced affinity for all ligands. Alternatively, the reverse chimera, consisting of the PTH1R with residues 1-43 from the PTH2R was found to be functional and had over a 10-fold increase in selectivity for binding PTH(1-34) over PTHrP(1-36) compared to the PTH1R. Through creation of double and then single mutant receptors, the residue responsible for this increase in ligand binding selectivity was identified as residue 41 (Leu in PTH1R and Val in PTH2R). This residue was found to mediate ligand binding selectivity through interaction with ligand at residue 23. A molecular model of the N-terminal domain (NTD) of PTH1R was created using the crystal structure of the NTD of the gastric inhibitory polypeptide receptor (GIPR) as a template. This model, in combination with mutagenesis, demonstrated that the way the PTH2R mediates its ligand binding selectivity is through the size of residue at position 41. A larger residue, such as Leu41 in PTH1R can interact with either PTH (containing Trp23) or PTHrP (containing Phe23). However, the smaller Val41 in PTH2R cannot interact with ligand when there is a Phe at residue 23 (such as in PTHrP) as there is not enough bulk in the binding pocket, although it can interact with the larger Trp in PTH. This model will facilitate further experiments to elucidate how the ligands interact with the PTH1R.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available