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Title: The role of receptor phosphorylation in M3 receptor signalling
Author: Spragg, Elizabeth
ISNI:       0000 0001 3475 5581
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2006
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GPCRs are rapidly phosphorylated in response to stimulation and this regulates receptor function and signalling. This process is widely considered to be mediated by the GRK family of protein kinases. In the current thesis we extend this paradigm by examining the role of the casein kinases in the phosphorylation of the M3-muscarinic receptor. Investigation of a phosphorylation deficient mutant of the M 3-receptor (mutant 6), where 17 putative serine phospho-acceptor sites had been substituted, demonstrated that receptor internalisation and efficient coupling to the phospholipase C pathway are dependent on receptor phosphorylation. However, this receptor mutant coupled normally to the ERK and JNK kinase pathway and mediated calcium entry similar to the wild type receptor. The importance of the specific sites of phosphorylation in driving defined receptor processes was illustrated by the use of another phosphorylation deficient mutant of the M3-receptor where amino acids between Lys 370-Ser425 inclusive were removed. Specific reduction of CK1alpha and CK2 protein expression levels with siRNA resulted in decreased agonist-induced receptor phosphorylation and had no effect on receptor internalisation, indicating that phosphorylation by kinase/s other than the casein kinases (probably the GRKs) are required for internalisation. Following inhibition of CK2-mediated receptor phosphorylation, receptor stimulated Ins(1,4,5)P3 production and ERK activation occurred as normal. In contrast, siRNAs against CK1alpha reduced the ability of the receptor to stimulate Ins(1,4,5)P3 production whereas coupling to the ERK pathway was unaffected. The data presented here support the hypothesis that phosphorylation at specific sites is mediated by a range of receptor kinases that include CK1alpha, CK2 and the GRKs. It appears that site specific phosphorylation by particular kinases regulates defined receptor processes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available