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First published online 29 August 2006
doi: 10.1242/jcs.03106

Journal of Cell Science 119, 3799-3810 (2006)
Published by The Company of Biologists 2006
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Helix-1 of the cAMP-specific phosphodiesterase PDE4A1 regulates its phospholipase-D-dependent redistribution in response to release of Ca2+

Elaine Huston1, Irene Gall1, Thomas M. Houslay2 and Miles D. Houslay1,*

1 Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences
2 Bioinformatics Research Centre, Davidson Building, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK


Figure 1
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  		Fig. 1. Schematic representation of the constructs used in this study. Full-length PDE4A1 is shown with nomenclature of the N-terminal deletions and amino acid substitutions in detail below. Mutations are in bold and the hinge region is in italics.

 

Figure 2
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  		Fig. 2. (1-25)-4A1-GFP localises to the trans-Golgi network and this localisation requires the presence of both helix-1 and helix-2. (A) (1-25)-4A1-GFP (green) was transfected into Cos-1 cells. Cells were viewed live (top row) or fixed with 4% paraformaldehyde (middle and bottom rows) and co-stained with either the general Golgi stain, BODIPY ceramide (red), the cis-Golgi marker GM130 or the trans-Golgi marker, TGN46 before visualising, using an Alexa Fluor 594-conjugated secondary antibody (red). (B) Cos1 cells were transfected with constructs encoding (1-25)-4A1-GFP with either helix-1 [{Delta}2-7(1-25)-4A1-GFP, green, top panel] or helix-2 [{Delta}14-25(1-25)-4A1-GFP, green, lower panel] deleted. Where indicated, cells were stained with the Golgi marker BODIPY ceramide or the live cell mitochondrial probe, Mitotracker (red). Cells were then visualised live. Merged images are presented as marked and any co-localisation is indicated in yellow. Bars, 10 µm. (C) Quantification of the imaging examples shown in B. Images were analysed using the co-localisation tool of Metamorph Imaging as detailed in the Materials and Methods (n=31 cells; values are mean ± s.e.m.).

 

Figure 3
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  		Fig. 3. More than one binding site is required for localisation of PDE4A1 in the intact cell. (A) Cos1 cells were transfected with engineered constructs formed from either substitution of helix-1 with helix-2 (H1-H1-GFP, green), or by substitution of helix-2 with helix-1 (H2-H2-GFP, green). Cells were stained with BODIPY ceramide (red) or treated with Mitotracker (red) and visualised. (B) Percentage (± s.e.m.) co-localisation of H2-H2-GFP with markers of the Golgi apparatus (BODIPY ceramide), ER (ER tracker) or mitochondria (Mitotracker) (n=25 cells). (C) Cos1 cells were transfected with a construct that encodes (1-25)-4A1-GFP with deletion of the mobile hinge region [{Delta}8-13 (1-25)-4A1-GFP, green] and co-stained with BODIPY ceramide (red) or treated with Mitotracker (red) and visualised. Merged images are shown and co-localisation is indicated in yellow. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Helix-1 contains a distinct motif that facilitates Golgi targeting. (A) Cos1 cells were transfected with a construct in which the Asp5 in (1-25)-4A1-GFP was mutated to Ala [D5A(1-25)-4A1-GFP, green]. Cells were then fixed and co-stained with the Golgi marker, BODIPY ceramide, the ER probes, ERtracker, the lysosomal probe, Lysotracker, the mitochondrial probe, Mitotracker or the Early endosome marker, EEA1 (all red). Merged images are shown in final column. (B) Mean percentage (± s.e.m.) co-localisation between D5A(1-25)-4A1-GFP and the various organelle probes used (n=25 cells). (C) Cells were transfected with the constructs L3AV4A(1-25)-4A1-GFP and F5AF6A(1-25)-4A1-GFP (green), co-stained with BODIPY ceramide (red) and then visualised live. Merged images are shown where indicated. (D) Cells were transfected with the constructs L3A(1-25)-4A1-GFP, V4A(1-25)-4A1-GFP, F5A(1-25)-4A1-GFP or F6A(1-25)-4A1-GFP (green), probed with the Golgi marker BODIPY ceramide (red) and then visualised live. Merged images only are shown. Co-localisation is indicated in yellow. Bars, 10 µm. (E) Percentage co-localisation (± s.e.m.) of the constructs described in D with the Golgi probe BODIPY ceramide (n=25 cells).

 

Figure 5
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  		Fig. 5. Phosphatidic acid generation by PLD influences the distribution of Asp5Ala(1-25)-4A1-GFP. (A) Cos1 cells were transfected with either (1-25)-4A1-GFP or the mutated construct D5A(1-25)-4A1-GFP. Cells were then treated with 0.3% (40 mM) butan-1-ol or 0.3% (40 mM) butan-2-ol or calphostin-c (1 µM) for 10 minutes at 37°C. (B) Cos1 cells were transfected with a construct of full-length PDE4A1, which was mutated at Asp5 to Ala. Cells were then treated with 0.3% (40 mM) butan-1-ol or 0.3% (40 mM) butan-2-ol for 10 minutes at 37°C, fixed in 4% paraformaldehyde, permeabilised and then stained for PDE4A. Cells were visualised using Alexa Fluor 594.

 

Figure 6
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  		Fig. 6. Mobilisation of intracellular calcium stores by thapsigargin causes redistribution of PDE4A1 from the Golgi in a PLD-dependent manner. (A) Cos1 cells were transfected with either (1-25)-4A1-GFP (top row), D5A(1-25)-4A1-GFP (middle row) or full-length PDE4A1 (bottom row). Cells were then treated with 2 µM thapsigargin for 30 minutes at 37°C. Where indicated cells were pre-incubated for 10 minutes before thapsigargin treatment with 0.3% (40 mM) butan-1-ol, 0.3% (40 mM) butan-2-ol or calphostin-c (1 µM) and then visualised. Bars, 10 µm. (B) Various organelles were visualised following the treatment of (1-25)-4A1-GFP-transfected Cos1 cells with thapsigargin (2 µM) for 30 minutes at 37°C. The Golgi apparatus was visualised using BODIPY ceramide, the ER using ERtracker, lysosomes using ERtracker and mitochondria using Mitotracker.

 

Figure 7
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  		Fig. 7. Functional surfaces on the two helices forming the unique N-terminal region of PDE4A1. Shown in N- to C-terminal projections are three different visual orientations of the unique 25 amino acid N-terminal region of PDE4A1. These surface projections were generated in RasMol using the co-ordinates from the known structure derived by 1H-NMR (Brookhaven database pdb file 1LOI). In helix-2 the bilayer insertion module Trp19-Trp20 is indicated (W19:W20), together with the Ca2+-binding Asp21. In helix-1 the hydrophobic pocket formed from Leu3, Phe6 and Phe7 are displayed, together with the Ca2+-binding Asp5.

 

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© The Company of Biologists Ltd 2006