First published online 10 June 2003
doi: 10.1242/jcs.00626
The ectodomain shedding of angiotensin-converting enzyme is independent of its localisation in lipid rafts
Edward T. Parkin1,*,
Fulong Tan2,
Randal A. Skidgel2,
Anthony J. Turner1 and
Nigel M. Hooper1
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
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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 ).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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© The Company of Biologists Ltd 2003
