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The management of G protein-coupled receptor trafficking is essential for the normal function of these receptors. The P2Y12 purinoceptor plays an essential role in the platelet aggregation response, and has been shown to desensitise and rapidly resensitise as a result of agonist stimulation. These processes are tightly regulated, involving receptor kinases, arrest ins and clathrin. Recent studies have suggested that as well as this tightly regulated agonist driven pathway of receptor trafficking there is also an agonist-independent, constitutive pathway. This thesis aims to identify this constitutive pathway, and to discern the mechanisms involved. P2Y12 internalisation was assessed III human platelets by a novel immunofluorescence approach using an anti-P2Y12 antibody. This study found that P2Y12 internalised both in response to ADP but also in the absence of ADP. This observation was also confirmed through the expression of RA-tagged P2Y12 receptor in CRG cells. P2Y12 receptors were found to localise in Rab 4 and Rab 11 positive endosomes, suggesting that the receptor recycles back the membrane post internalisation. This was confirmed through the use of a novel acid strip ELISA protocol. This study found that the mechanisms that regulate the constitutive traffic of P2Y 12 are broadly similar to those that regulate agonist driven traffic. The presence of the C-terminal PDZ binding motif has been shown to be essential for agonist-induced internalisation of P2Y 12, yet constitutive internalisation was unaffected by disruption of this motif. This led to the identification of two amino acid motifs within the C-terrninus that play a significant role in receptor regulation. The importance of these regulatory mechanisms was confirmed through the use of lentiviral P2Y12 constructs in murine bone marrow derived megakaryocytes as a model for platelets. In conclusion, this thesis presents for the first time evidence that the P2Y 12 receptor undergoes constitutive agonist-independent internalisation, and dissects some of the molecular mechanisms that underlie this process. 11 |
