Cellular refractoriness to the heat-stable enterotoxin peptide
is associated with alterations in levels of the differentially
glycosylated forms of guanylyl cyclase C
Yashoda Ghanekar, Akhila Chandrashaker and Sandhya S. Visweswariah
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India
The heat-stable enterotoxin peptides (ST) produced by
enterotoxigenic Escherichia coli are one of the major causes
of transitory diarrhea in the developing world. Toxin bind-
ing to its receptor, guanylyl cyclase C (GC-C), results in
receptor activation and the production of high intracellular
levels of cGMP. GC-C is expressed in two differentially
glycosylated forms in intestinal epithelial cells. Prolonged
exposure of human colonic cell lines to ST peptides induces
cellular refractoriness to the ST peptide, in terms of intra-
cellular cGMP accumulation. We have investigated the
mechanism of cellular desensitization in human colonic
Caco2 cells, and observe that exposure of cells to ST leads to
a time and dose-dependent inability of cells to respond to the
peptide in terms of GC-C stimulation, both in whole cells
and membranes prepared from desensitized cells. This is
concomitant with a 50% reduction in ST-binding activity in
desensitized cells. Desensitization was correlated with a loss
of the plasma membrane-associated, hyperglycosylated
145 kDa form of GC-C, while the predominant 130 kDa
form, localized both on the plasma membrane and the
endoplasmic reticulum, continued to be present in ST-trea-
ted cells. Desensitized cells recovered ST-responsiveness on
removal of the ST peptide, which was correlated with a
reappearance of the 145 kDa form on the cell surface, fol-
lowing processing of the endoplasmic reticulum-associated
pool of the 130 kDa form. Selective internalization of the

We have been studying guanylyl cyclase C (GC-C), the
receptor for the guanylin/uroguanylin family of peptides.
GC-C is predominantly expressed in intestinal cells, where it
was initially described as being the mediator of the action of
the bacterial heat-stable enterotoxin peptides (ST) [4–6]. In
addition, robust GC-C expression is also observed in the
regenerating rat liver [7] and in extraintestinal tissues [8].
Ligand binding to GC-C leads to accumulation of intracel-
lular cGMP, followed by the activation of cyclic nucleotide-
dependent protein kinases resulting in the phosphorylation
of the cystic fibrosis transmembrane conductance regulator
[9,10]. Cystic fibrosis transmembrane conductance regulator
is a chloride channel and phosphorylation increases chloride
ion efflux resulting in loss of fluid from the cell and
characteristic watery diarrhea that is associated with the ST
peptides. Recently, GC-C has also been shown to be
involved in regulation of colonic cell proliferation [11] and
apoptosis [12], and modulation of a cGMP-gated ion
channel that then regulates DNA synthesis [13].
Correspondence to S. S. Visweswariah, Department of Molecular
Reproduction, Development and Genetics, Indian Institute of Science,
Bangalore 560012, India.
Fax: + 91 80 3600999, Tel.: + 91 80 3942542,
E-mail:
Abbreviations: ANP, atrial natriuretic peptide; GC-A, guanylyl cyclase
A; GC-C, guanylyl cyclase C; IBMX, isobutylmethyl xanthine;
PDE5, cGMP-binding, cGMP-specific phosphodiesterase;
PDZ, PSD-95, Disc-large, ZO-1; ST, stable toxin; STh, stable toxin
of the human variety; ST
Y72F

+
exchanger NHE3 [22] and apical
Cl
–
/OH
–
exchange activity by activation of protein
kinase G [23].
The transient nature of ST-induced diarrhea suggests
that the GC-C signaling pathway is modulated in vivo in
response to ligand. In T84 cells, 18 h ST treatment led to
cellular refractoriness to further ST stimulation and this
refractoriness was contributed by both down-regulation of
GC-C leading to reduced cGMP synthesis, as well as
activation of the type 5 phosphodiesterase (PDE5A) and
increased cGMP degradation [24]. On desensitization, there
was a decrease in the V
max
of the guanylyl cyclase catalytic
activity of GC-C with no change in the S
0.5
of the enzyme
for its substrate, MgGTP [25]. There did not appear to be an
appreciable change in the total receptor content in desen-
sitized cells, as measured by Scatchard analysis, which could
account for the reduction in catalytic activity.
In the current study, we have explored the phenomenon
of the induction of cellular refractoriness to the ST peptide
in Caco2 cells postdifferentiation, when GC-C levels are
relatively high and expressed at a uniform level over the

270 mgÆL
)1
streptomycin. To allow differentiation of Caco2
cells into intestinal villus cells, cells were kept in culture for
7–10 days after they were confluent [19]. To confirm
differentiation, sucrase isomaltase gene expression was
monitored by reverse transcriptase and poymerase chain
reaction, using RNA prepared from 15-day-old Caco2 cells
[27] (data not shown).
Desensitization of Caco2 cells
In standard desensitization experiments, 14 to 20-day-old
Caco2 cells were washed with serum-free DMEM/F12 and
incubated with or without 10
)7
M
SThinDMEM/F12for
9 h. Monolayers were then washed and re-stimulated with
fresh STh (10
)7
M
) in the presence or absence of 500 l
M
isobutylmethyl xanthine (IBMX) for 30 min in serum-free
medium for 15 min. Cell monolayers were washed, and cells
lysedin0.1
M
citric acid or 0.1
M
HCl. Cyclic GMP in the
lysates was measured by radioimmunoassay as described

M
NaCl, 5 m
M
EDTA, 1 m
M
dithiothreitol,
5 lgÆmL
)1
soybean trypsin inhibitor, 5 lgÆmL
)1
leupeptin,
and 5 lgÆmL
)1
aprotinin). The cell lysate was sonicated and
centrifuged at 10 000 g for 1 h at 4 °C. The pellet obtained
was resuspended in buffer containing 50 m
M
Hepes,
pH 7.5, 5 lgÆmL
)1
soybean trypsin inhibitor, 5 lgÆmL
)1
leupeptin, 5 lgÆmL
)1
aprotinin and 100 l
M
sodium ortho-
vanadate. The protein was estimated by using a modifica-
tion of the Bradford protein assay [29].
In vitro

mediated activation of GC-C, 4 m
M
manganese and 1 m
M
GTP was used as substrate. In experiments performed to
monitor Lubrol-PX mediated activation, membranes were
incubated in 0.3% Lubrol-PX for 10 min at 37 °Cwith
4m
M
MgCl
2
and 1 m
M
GTP as substrate.
Receptor binding analysis
ST
Y72F
was iodinated using Na
125
I as described earlier [30]
and was available in the laboratory. Membrane protein
(100–200 lg) was incubated with increasing concentrations
of
125
I-labeled ST
Y72F
for 1 h at 37 °C in binding buffer
(50 m
M
Hepes, pH 7.5, 4 m

containing 4% paraformaldehyde for
20–30 min. Cells were washed and incubated with 2%
bovine serum albumin and 0.1% Triton X-100 in NaCl/P
i
for 1 h at room temperature to block nonspecific sites and
permeabilize cells. Cells were then incubated overnight with
5 lgÆmL
)1
GC-C:4D7, an antibody raised to the protein
kinase-like domain of GC-C [32] or with GC-C:4D7
antibody preadsorbed with a fusion protein of the kinase-
like domain of GC-C and glutathione S-transferase, in
blocking buffer [8]. After washing, FITC-conjugated anti-
mouse antibody (Life Technologies, USA) was added for
1 h at room temperature. Cells were washed and mounted
in Vectashield mounting medium (Vector Laboratories,
USA). Cells were visualized under a Zeiss fluorescence
microscope using standard filters for FITC and DAPI at
63 · magnification.
Immunoprecipitation of GC-C
Membranes prepared from Caco2 cells were solubilized at a
concentration of 1 mgÆmL
)1
in immunoprecipitation buffer
(20 m
M
Tris-Cl pH 7.5, 100 m
M
NaCl, 2 m
M

buffer with 0.1% SDS and 50 m
M
2-mercaptoethanol for 5
min. The reaction was cooled to room temperature and
NP-40 was added to a final concentration of 0.75%.
N-Glycosidase F (200 mU; Roche, Germany) was added
and the reaction incubated at 37 °C for 8 h. After incuba-
tion, the reaction was stopped by addition of Laemmli
buffer, boiled and subjected to SDS gel electrophoresis and
Western blot analysis.
For Endo H treatment of cells, GC-C was immunopre-
cipitated as described above and the immunoprecipitate
treated with Endo H (500 U; NEB, USA) as per the
manufacturer’s instructions. Incubation at 37 °Cwasper-
formed for 6 h and samples were then subjected to SDS gel
electrophoresis and Western blot analysis asdescribedabove.
Surface biotinylation of Caco2 cells
Cells were washed with NaCl/P
i
(pH 8.0) containing 1 m
M
CaCl
2
and 0.5 m
M
MgCl
2
(NaCl/P
i
-CM), and then incu-

only a slight increase in cGMP synthesis was observed after
fresh ST stimulation (Fig. 1A), indicating that similar to
T84 cells, Caco2 cells also showed cellular refractoriness to
3850 Y. Ghanekar et al. (Eur. J. Biochem. 270) Ó FEBS 2003
ST. Interestingly, even when we inhibited PDE activity in
cells by the addition of a phosphodiesterase inhibitor to
desensitized cells, increased cGMP accumulation was not
observed, in contrast to our results with T84 cells (Fig. 1A).
PDE5 is expressed in Caco2 cells, and as we have reported
earlier in T84 cells [24], PDE5 activation was observed as a
consequence of increased cGMP accumulation in Caco2
cells during the initial ST application (unpublished obser-
vations). This suggested that the refractoriness to the ST
peptide observed in Caco2 cells must be attributed to down-
regulation of GC-C activity on ligand addition.
ST was applied to cells for varying times and we
measured the ability of these cells to respond to ST on
fresh stimulation. As seen in Fig. 1B, desensitization was
observed after 3 h ST treatment and at least 6 h ST
treatment was required for maximum down-regulation of
GC-C activity. This requirement for prolonged treatment of
cells to ST to observe desensitization, is in contrast to the
rapid inactivation that is seen for other members of the
guanylyl cyclase receptors, such as GC-A and the sea urchin
sperm receptor [2,33,34].
As shown in Fig. 1C, high concentrations of ST were
required to induce desensitization, suggesting that the
mechanism of desensitization appeared to be coupled to a
high occupancy of the receptor by the ligand. Increases in
intracellular cGMP alone could not trigger desensitization,

guanylyl cyclase activity, but this form of the receptor could
not respond to ligand stimulation.
Expression of differentially glycosylated forms
of GC-C in Caco2 cells
Western blot analysis with a monoclonal antibody to GC-C
using membranes prepared from control Caco2 cells
revealed the presence of two immunoreactive bands of 145
and 130 kDa in size. Treatment of immunoprecipitated
GC-C with protein N-glycosidase F resulted in the genera-
tion of an immunoreactive band of M
r
120 kDa, a size
predicted from the cDNA sequence of GC-C without
glycosylation, indicating that the two forms of GC-C
represented alternately glycosylated forms of the receptor
(Fig. 3A). EndoH treatment of the immunoprecipitate led
to a reduction in size of the 130 kDa form and not the
145 kDa form, showing that the 130 kDa form represented
Fig. 1. Prolonged ST treatment leads to cellular refractoriness to further
ST-stimulation in Caco2 cells. (A) Caco2 monolayers were treated with
10
)7
M
ST for 18 h, monolayers were washed and restimulated with
10
)7
M
ST for 15 min in the presence or absence of 500 l
M
IBMX.

a property of all cells that express the receptor, as has been
reported earlier [31,35,36]. No fluorescence was observed
with cells incubated with GC-C:4D7 antibody preadsorbed
with the fusion protein comprising the kinase-like domain of
GC-C and glutathione S-transferase, as has been reported
earlier (Fig. 3B).
Western blot analysis was carried out using membrane
protein prepared from control and desensitized cells. Most
interestingly, while both the 130 and 145 kDa forms were
detected in control cells, only the 130 kDa form of GC-C
was detected in desensitized Caco2 cells (Fig. 4A). As
shown earlier, membranes prepared from desensitized cells
did not show ligand-stimulated activation, even thought
they were able to bind the ST peptide (Fig. 2). Therefore,
there appeared to be a correlation between the presence of
the 145 kDa form of GC-C, which represents the mature
glycosylated form of GC-C, and the ability of GC-C to be
stimulated by ST.
Cells regain their ability to be stimulated by ST following
the reappearance of the 145 kDa form of GC-C. Cells were
cultured for 18 h in the presence of ST, ST was then
removed and cells fed with serum-containing medium
without ST. At various times after renewal of the medium,
cells were harvested and membranes subjected to Western
blot analysis and stimulation with ST peptide. As shown in
Fig. 4B, loss of ST-induced stimulation was correlated with
the absence of the 145 kDa form. Following ST removal,
Fig. 2. GC-C activity and content in desensitized cells. (A) Twenty
micrograms of membrane protein prepared from control and
ST-treated Caco2 cells were incubated with or without 10

and 1 m
M
GTP) as a substrate. Reaction was carried out for
5 min at 37 °C,andcGMPsynthesizedwasmonitoredbyRIA.Values
represent mean ± SEM of duplicate determinations with each
experiment performed at least twice.
Fig. 3. Expression of differentially glycosylated forms of GC-C in
Caco2 cells. (A) GC-C was immunoprecipiated from Caco2 cells using
the CTD antibody. The immunoprecipitate was incubated with or
without PNGase F or Endo H, and separated by 6% SDS/PAGE. (B)
Immunocytochemistry of Caco2 cells. Cells cultured on coverslips were
blocked, permeabilized and incubated with 10 lgÆmL
)1
GC-C:4D7 or
with normal mouse IgG (data not shown) and then with FITC-tagged
anti-(mouse IgG). The cells were mounted in Vectashield mounting
medium and visualized using a standard filter for FITC at 63 · mag-
nification.
3852 Y. Ghanekar et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the 145 kDa form reappeared, and along with that,
ST-induced stimulation was restored. These results there-
fore show that the extent of glycosylation of GC-C
determines its ability to be ligand-stimulated, and respon-
siveness of cells to the ST peptide can be controlled by the
presence or absence of differentially glycosylated forms of
GC-C.
It is possible that the reappearance of the 145 kDa form
following removal of ST peptide was a consequence of
further glycosylation of the ER-associated 130 kDa form,
and subsequent transport to the plasma membrane. There-

had occurred, as we do not detect any low molecular weight
Fig. 4. Alterations of differentially glycosylated forms of GC-C in
Caco2 cells. (A) One hundred micrograms of membrane protein from
control and desensitized cells was subjected to Western blot analysis
with GC-C:C8 antibody. (B) GC-C was immunoprecipitated using the
CTD antibody and immunoprecipitates were subjected to Western
blot analysis using GC-C:C8 antibody. Lane 1, control cells; lane 2,
desensitized cells; lane 3, recovery. (C) Desensitized Caco2 cells were
washed and incubated without ST for 12 h in culture medium in the
absence or presence of cycloheximide. Membranes were prepared from
these cells. Twenty micrograms of membrane protein was incubated
with MgGTP (4 : 1 m
M
) in the presence or absence of 100 n
M
ST for
10 min. Values represent mean ± SEM of duplicate determinations
with each experiment performed at least twice. (D) Desensitized cells
were incubated in medium containing 10% serum and swainsonine as
indicated, for 12 h. Monolayers were then washed and restimulated
with ST peptide for 15 min and cGMP produced monitored by radi-
oimmunoassay. Values represent the mean ± SEM of duplicate
determinations with the experiment performed twice. In addition,
membrane protein prepared from control or swainsonine treated cells
(200 lg) was solubilized and taken for immunoprecipitation and
Western blot analysis. Lane 1, desensitized cell membrane; lane 2,
membrane after recovery; lane 3, membrane after recovery in the
presence of swainsonine.
Ó FEBS 2003 Glycosylation of GC-C and desensitization (Eur. J. Biochem. 270) 3853
fragment of GC-C in desensitized cells, using multiple

specific down-regulation of the 145 kDa form of GC-C on
prolonged ligand treatment, indicating that only the ligand-
responsive form of the receptor is perhaps routed to the
lysosomal compartment for degradation. The continued
presence on the surface of cells of the 130 kDa form, even
on ST-treatment, indicates that this form is clearly ligand-
unresponsive, and is either not internalized, or recycled
efficiently to the surface.
Discussion
The studies described in this report suggest that regulation
of the glycosylation of GC-C can act as a means of
controlling the ability of cells to respond to the ST peptide.
Desensitization studies carried out so far in various
members of the receptor guanylyl cyclase family have
shown that some of these receptors are down-regulated by
rapid dephosphorylation after short treatment with the
ligand. For example, the sea urchin sperm receptor guanylyl
cyclases are dephosphorylated rapidly upon ligand binding,
leading to receptor desensitization [33,34]. Studies carried
out with the receptors for natriuretic peptides, GC-A and
GC-B, also showed that dephosphorylation is the mechan-
ism of desensitization of these receptors. GC-A is phos-
phorylated on six serine and threonine residues present in
the protein kinase-like domain in the basal state [2].
Mutation of any of these sites to alanine led to a decrease
in ANP-mediated activation and simultaneous mutations in
all the sites resulted in a complete loss of ANP-mediated
activation [38]. Recent studies have indicated that GC-A is
possibly dephosphorylated by two phosphatases, a micro-
cystin inhibited phosphatase and another phosphatase that

Western blot analysis using GC-C:4D7 antibody. Lane 1, control cells
incubated at 4 °C; lane 2, cells incubated with ST at 4 °C; lane 3, cells
incubated at 37 °C; lane 4, cells incubated with ST at 37 °C. (C)
Control and desensitized cells (ST treated for 9 h) were surface bio-
tinylated and membrane protein prepared. GC-C was immunopre-
cipitated from equal amounts of solubilized membrane protein with
CTD antibody, and immunoprecipitates analyzed by Western blot
analysis using streptavidin–peroxidase conjugate.
3854 Y. Ghanekar et al. (Eur. J. Biochem. 270) Ó FEBS 2003
ligand exposure, while that of GC-C takes many hours, it
was likely that distinct regulatory mechanisms are operative
in the two receptors, as indeed is the case, and shown in the
studies described here. Until date, the role of glycosylation
in either GC-A or GC-B signaling has not been studied, but
may be worthwhile to pursue now, in light of the
observations described here, given the possible similarity
in the overall structure of the extracellular domains of the
receptors [37].
Using [
125
I]ANP binding assays, GC-A has also been
reported to undergo ligand-mediated internalization with a
t
1/2
of 8 min in HEK293 cells [3]. Forty to fifty per cent of
the internalized receptor is recycled back to the surface and
the rest is directed to the degradation pathway. GC-B and
NPR-C, the clearance receptor for atrial natriuretic factor,
also undergo ligand-mediated internalization and are recy-
cled back to the surface in PC12 cells [42]. GC-C has been

T84 and Caco2 cells, that selectively allows the degradation
of the 145 kDa form. Recently a PSD-95, Disc-large, ZO-1
(PDZ) domain protein which interacts with GC-C was
identified in a yeast two hybrid screen [44]. This protein
named Ôintestine and kidney-enriched PDZ proteinÕ
(IKEPP) interacts with GC-C through one of its PDZ
domains. In the presence of IKEPP, the EC
50
of GC-C for
ST increased 10-fold, suggesting that IKEPP regulates
ligand-mediated activation of GC-C. Interestingly, IKEPP
is expressed in T84 and Caco2 cells but not in HEK293 cells,
and therefore could be a possible candidate protein involved
in selective down-regulation of GC-C [44].
Our studies carried out by treating cells with ST at 4 °C
suggest that down-regulation is brought about by selective
internalization of the 145 kDa form through endocytosis
and subsequent degradation. The mechanisms involved in
this process are not yet identified. It is possible that the
activation of the cyclase domain of the 145 kDa form upon
ligand binding leads to a conformational change, exposing a
signal that promotes internalization and exposure of a
ubiquitination signal, leading to selective degradation of the
145 kDa form. Alternatively the sugar residues present on
the 145 kDa form could act as a signal for internalization
and/or degradation. Indeed, glycosylation-based recogni-
tion motifs are involved in internalization as well as
degradation of proteins. N-Linked glycosylation of Edg-1,
which is a G-protein-coupled receptor, is essential for
targeting the receptor to caveolin-rich domains in the

130 kDa form of the receptor is unresponsive to the ST
peptide, even though it can bind the ligand with an affinity
similar to the hyperglycosylated form, and remains present
on the surface of cells even after desensitization, one can
suggest that the 130 kDa form of GC-C can act as a ÔsinkÕ
for its ligands, when present on the plasma membrane of
intestinal cells. This may partly account for the differential
responsiveness of various regions of the intestine to the
guanylin/uroguanylin family of peptides [6], and also
regulate GC-C signaling in extraintestinal tissues where
GC-C and its ligands are expressed.
Acknowledgements
This work was funded by the Department of Biotechnology, Govern-
ment of India. YG is supported by the Indian Council of Medical
Research, and AC by the Department of Atomic Energy, Government
of India. We would like to thank Ms. Vani Iyer for the purification and
radioiodination of ST peptide.
Ó FEBS 2003 Glycosylation of GC-C and desensitization (Eur. J. Biochem. 270) 3855
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