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Title: Functional interactions of 14-3-3 proteins with phospholipase D and the M₃ muscarinic receptor
Author: Collins, Daniel M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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14-3-3 proteins are a family of small, acidic, scaffolding and adaptor proteins, which have been implicated in cell cycle regulation, apoptosis and signal transduction mechanisms. There are seven isoforms of 14-3-3 (β, η, γ, ε, τ/θ, σ, and ζ) that form hetero-and homodimers in vivo. Recently, 14-3-3 has been shown to associate with members of the heptahelical, plasma membrane spanning G-protein coupled receptor (GPCR) superfamily. GPCRs mediate neurotransmitter and other extracellular agonist-evoked activation of intracellular effectors and signalling cascades. Some of these effector mechanisms lead to the activation of phospholipase D (PLD). Mammalian PLD isoforms catalyse the hydrolysis of phosphatidylcholine, forming choline and phosphatidic acid, a novel second messenger molecule. 14-3-3 dimers associate with other proteins containing specific target motifs, including an RSxpSxP motif (where pS is phosphoserine), or an unphosphorylated WLDLE/DALDL motif. We recognised that the former motif is present in mammalian PLD1 at residues 712-717 and therefore have investigated whether 14-3-3 isoforms associate with PLD and GPCRs to provide a functional role in intracellular signalling.  It was shown, using in vitro GST-fusion protein pull-downs and co-immunoprecipitation, that in CAS 7 cells, 14-3-3 associates with the M3 muscarinic receptor. 14-3-3 was also demonstrated to associate with PLD1, and to a lesser extend PLD2, in an isoform-dependent manner. The effect of PLD activation by protein kinase C (PKC) on this interaction was investigated using the aforementioned techniques and confocal microscopy. Furthermore, in whole cell signalling assays, the overexpression of different 14-3-3 isoforms selectively modified PKC or GPCR-mediated activation of PLD. In addition, PLD was found to physically associate with the M3 receptor. The implications of these interactions for physiological signalling by the M3 receptor and PLD are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available