Studies on the C-terminal tail of Vasopressin 1a receptor

Azam, Maria Tahir (2017). Studies on the C-terminal tail of Vasopressin 1a receptor. University of Birmingham. Ph.D.

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Abstract

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.