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First published online 10 June 2003
doi: 10.1242/jcs.00626

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The ectodomain shedding of angiotensin-converting enzyme is independent of its localisation in lipid rafts

¹ Proteolysis Research Group, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
² Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA

View larger version (21K): [in a new window]   Fig. 1. Schematic diagram of WT-ACE and GPI-ACE. The domain structures of human somatic ACE (WT-ACE) and the GPI-anchored ACE (GPI-ACE) construct are shown. WT-ACE is a type I transmembrane protein with a C-terminal transmembrane domain (bold). The site of cleavage by the secretase is indicated. In GPI-ACE, the C-terminal 64 residues, including the transmembrane and cytosolic domains, were replaced with the 24 residue GPI anchor signal sequence (underlined) of human carboxypeptidase M (CPM). The site of GPI anchor attachment is indicated (*) (Tan et al., 2003).

View larger version (40K): [in a new window]   Fig. 2. Characterisation of WT-ACE and GPI-ACE expression in CHO and SH-SY5Y cells. CHO and SH-SY5Y cells were transfected with either empty pIRESneo vector (mock), WT-ACE cDNA or GPI-ACE cDNA. Cell lysates were prepared as described in Materials and Methods. (A) Lysates were immunoblotted using the ACE antibody, RP183. (B) Lysates were immunoblotted using a monoclonal ß-actin antibody. (C) Lysates were assayed for ACE activity with BzGly-His-Leu as substrate. Results are the mean ± s.d. of three experiments.

View larger version (24K): [in a new window]   Fig. 3. Distribution of WT-ACE and GPI-ACE in lipid rafts. CHO and SH-SY5Y cells expressing either WT-ACE or GPI-ACE were used to prepare lipid rafts by buoyant sucrose density-gradient centrifugation in the presence of Triton X-100 as described in Materials and Methods. The sucrose gradients were fractionated in 0.5 ml aliquots (0, insoluble pellet; 1, bottom of tube; 9, top of tube). (A) Distribution of total protein in sucrose gradient fractions. (B) Distribution of caveolin (CHO cells) and flotillin (SH-SY5Y cells) in sucrose gradient fractions. (C) Distribution of ACE activity in sucrose gradient fractions. The results are representative of triplicate experiments.

View larger version (90K): [in a new window]   Fig. 4. Immunocytochemistry of CHO cells expressing WT-ACE or GPI-ACE. (A) and (B) CHO cells transfected with WT-ACE and GPI-ACE, respectively were subjected to immunocytochemistry using the polyclonal anti-ACE antibody RP183 as described in Materials and Methods. (C and D) CHO cells transfected with WT-ACE and GPI-ACE, respectively, were incubated for 20 minutes at 4°C with 2% (v/v) Triton X-100 prior to paraformaldehyde fixation and subsequent immunocytochemistry using the polyclonal anti-ACE antibody RP183, as described in Materials and Methods.

View larger version (18K): [in a new window]   Fig. 5. Phorbol ester stimulates the shedding of WT-ACE and GPI-ACE. (A) CHO and (B) SH-SY5Y cells transfected with either WT-ACE or GPI-ACE were incubated for 7 hours in the absence (control) or the presence of either PMA (1 µM), PMA (1 µM) and batimastat (10 µM), carbachol (30 µM), or carbachol (30 µM) and batimastat (10 µM) as indicated. The medium was then harvested and concentrated as described in Materials and Methods. The samples were assayed for ACE activity with BzGly-His-Leu as substrate. Results are the mean ± s.d. of three experiments.

View larger version (26K): [in a new window]   Fig. 6. Inhibition of the regulated shedding of WT-ACE and GPI-ACE by a range of hydroxamate-based inhibitors. CHO cells transfected with either WT-ACE or GPI-ACE were incubated for 7 hours in the presence of 1 µM PMA and 10 µM concentrations of a range of structurally variant hydroxamate-based inhibitors [numbers as in Parkin et al. (Parkin et al., 2002)]. The medium was then harvested and concentrated and samples were assayed for ACE activity with BzGly-His-Leu as substrate. Results are the mean ± s.d. of three experiments.

View larger version (30K): [in a new window]   Fig. 7. Lack of effect of filipin on the shedding of GPI-ACE. CHO cells transfected with either WT-ACE or GPI-ACE were incubated for 1 hour in the presence of PMA (1 µM) with or without filipin (10 nM). The conditioned medium was harvested and concentrated and the cells then fixed in paraformaldehyde and subjected to immunocytochemistry using the polyclonal anti-ACE antibody RP179 as described in Materials and Methods. (A) WT-ACE-transfected CHO cells incubated in the presence of PMA alone. (B) GPI-ACE-transfected CHO cells incubated in the presence of PMA alone. (C) WT-ACE-transfected CHO cells incubated in the presence of PMA and filipin. (D) GPI-ACE-transfected CHO cells incubated in the presence of PMA and filipin. (E) Conditioned media samples were assayed for ACE activity with BzGly-His-Leu as substrate. Results are the mean ± s.d. of three experiments.

View larger version (20K): [in a new window]   Fig. 8. Effect of PMA on the membrane compartmentalisation of WT-ACE and GPI-ACE. CHO cells transfected with either WT-ACE or GPI-ACE were incubated for 7 hours in the presence of batimastat (10 µM) with or without PMA (1 µM). Lipid rafts were prepared by buoyant sucrose density-gradient centrifugation in the presence of batimastat (10 µM) as described in Materials and Methods. The sucrose gradients were fractionated in 0.5 ml aliquots (0, insoluble pellet; 1, bottom of tube; 9, top of tube) and each fraction was assayed for ACE activity with BzGly-His-Leu as substrate. (A) Distribution of WT-ACE activity in sucrose gradient fractions. (B) Distribution of GPI-ACE activity in sucrose gradient fractions. Results are the mean ± s.d. of three experiments.