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Title: Studies on the C-terminal tail of Vasopressin 1a receptor
Author: Azam, Maria Tahir
ISNI:       0000 0004 7432 2942
Awarding Body: University of Birmingham
Current Institution: University of Birmingham
Date of Award: 2017
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Site-directed mutagenesis and fluorescent-protein based techniques were used to evaluate the role of the C-terminal tail of human V\(_1\)\(_a\) vasopressin receptor (V\(_1\)\(_a\)R). Mutants engineered with the C-tail truncations were characterised with respect to cell-surface expression by enzyme linked immunosorbent assay (ELISA), agonist binding by competition radioligand binding assay and signalling capability by inositol phosphates (InsP-InsP\(_3\)) accumulation assay. A series of Ser/Thr mutations disrupted phosphorylation sites and identified a putative G-protein-coupled receptor kinase 2 (GRK2) consensus site contributing to V\(_1\)\(_a\)R internalisation rate and desensitisation in response to vasopressin. GRK2 and GRK2 constructs mutated at functional domains were used to identify the role of GRK2 in V\(_1\)\(_a\)R internalisation. A V\(_1\)\(_a\)R-mCherry fusion, V\(_1\)\(_a\)R-vYC and A2AR-vYC were generated and likewise functionally characterised. Addition of mCherry or vYC at the C-terminus of V\(_1\)\(_a\)R did not affect the binding affinity of V\(_1\)\(_a\)R, cell-surface expression and ability to signal via Gq/11 coupling. Similarly, A\(_2\)\(_A\)R-vYC displayed WT like binding profile and receptor internalisation. Total internal reflection fluorescence microscopy (TIRF-M) and confocal imaging were performed to monitor V\(_1\)\(_a\)R-mCherry internalisation upon agonist stimulation. Overall, results presented in this thesis provide insight into the role of the C-terminal tail domain in V\(_1\)\(_a\)R internalisation and desensitisation and identify functionally important residues including a putative GRK2 regulatory site.
Supervisor: Not available Sponsor: Charles Wallace Pakistan Trust ; Commonwealth Scholarship Commission
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General)