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Title: An investigation of the putative 3-phosphorylated inositide receptors GAP1IP4BP and GAP1m
Author: Bhatti, F.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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The project describes the setting up of a permeabilised L1210 cell system, a model system for investigating the role of Ins(1,3,4,5)P4 and its putative receptors in Ca2+ mobilisation. In particular, an attempt was made to design a protocol so that multiple replicates could be easily performed. The difficulties encountered with setting up such a system, such as achieving reproducible cell permeabilisation and removing contaminating Ca2+, are discussed. In an attempt to elucidate the preferred endogenous small G-protein substrate(s) for GAP1IP4BP and GAP1m the characterisation and expression of activation-specific hooks for R-Ras, Rap and Ras are described in some detail. The use of these probes to assay small G-protein activation in cells transiently overexpressing GAP1IP4BP and GAP1m is also detailed. In addition, the synthesis of an inducible cell line for GAP1IP4BP and GAP1m, and the various problems encountered, are described. Finally, the technique of fluorescence-recovery after photobleaching (FRAP) has been utilized to explore the mobility of GAP1IP4BP and GAP1m when they are either cytoplasmic or attached to the plasma membrane. The PH-domain of PI-PLCδ1 and ICAM were used as representatives of respectively another PtdIns(4,5)P2-binding protein, and a single-pass transmembrane protein,. The data from GAP1m and the PI-PLCδ1PH domain show for the first time that when proteins associate with inositols lipids in the plasma membrane they retain a mobility similar to that in the cytoplasm, and probably also similar to the inositols lipid to which they are attached. In contrast, the plasma membrane mobility of GAP1IP4BP was much lower than the soluble form and membrane-associated GAP1m, suggesting that it interacts with some other abundant cellular component. Moreover, GAP1IP4BP mobility was not detectably altered by the generation of Ins(1,3,4,5)P4.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available