| Summary: | 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 <i>in vivo</i>. 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 <i>in vitro</i> GST-fusion protein pull-downs and co-immunoprecipitation, that in CAS 7 cells, 14-3-3 associates with the M<sub>3</sub> 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 M<sub>3</sub> receptor. The implications of these interactions for physiological signalling by the M<sub>3</sub> receptor and PLD are discussed.
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