Báo cáo khoa học: Cellular refractoriness to the heat-stable enterotoxin peptide is associated with alterations in levels of the differentially glycosylated forms of guanylyl cyclase C pdf

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

145 kDa form of the receptor was required for cellular
desensitization, as ST-treatment of cells at 4 °C did not lead
to refractoriness. We therefore show a novel means of
regulation of cellular responsiveness to the ST peptide,
whereby altering cellular levels of the differentially glycos-
ylated forms of GC-C can lead to differential ligand-medi-
ated activation of the receptor.
Keywords: guanylyl cyclase C; desensitization; Caco2 cells;
heat-stable enterotoxin; glycosylation.
The regulation of any receptor-mediated signaling pathway
is integral for maintaining normal homeostasis of a cell.
Initial activation of a receptor by its ligand results in the
triggering of a cellular response, which is then usually
attenuated by various cellular mechanisms. These can
involve receptor internalization and/or modification, ligand
degradation, or the activation of cellular pathways that
counteract the initial response, and can lead to cellular
refractoriness. Membrane-associated forms of guanylyl
cyclase serve as receptors for a variety of peptide ligands
that mediate their response by increases in intracellular
cGMP levels [1]. Guanylyl cyclase A (GC-A), the receptor
for atrial natriuretic peptides (ANP), is basally phos-
phorylated in cells and a rapid dephosphorylation of
the receptor is correlated with a desensitization of the
receptor to its ligand, and contributes to the cellular
refractoriness that is observed in cells that are exposed to
ANP [2]. In addition, ligand-mediated internalization of
GC-A has been described, but only a fraction of the ligand-
bound receptor is directed for degradation, with a signifi-
cant amount is recycled to the plasma membrane surface [3].

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

, STh with tyrosine-19 replaced by
phenylalanine.
Enzyme: guanylyl cyclase (4.6.1.290)
(Received 8 May 2003, revised 17 July 2003,
accepted 4 August 2003)
Eur. J. Biochem. 270, 3848–3857 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03779.x
Several cell lines that express GC-C are available which
can be potentially used as a model to study GC-C signaling
[14]. These cell lines include T84, Caco2, HT29, NCI H508
and SW 116, all derived from different types of colonic
carcinomas. T84 cells are derived from lung metastasis of a
patient with colonic carcinoma and exhibit many charac-
teristics of polarized epithelial cells [15]. Caco2 cells, on
the other hand exhibit an enterocyte-like morphology,
although they were derived from a colonic adenocarcinoma
[16]. Upon reaching confluency, Caco2 cells spontaneously
differentiate in culture and resemble villus cells and thus
provide a good in vitro system to study regulation of
cellular pathways in differentiating enterocytes [17]. GC-C
has been cloned [18] and characterized from Caco2 cells
[19], and Caco2 cells express lower levels of GC-C in
comparison to T84 cells [14]. GC-C expression levels
increase after the cells differentiate in culture [19], and
unlike T84 cells, differentiated Caco2 cells also express
guanylin [20,21]. Recent studies indicate that Caco2 cells
express soluble guanylyl cyclase as well as protein kinase G
and activation of the soluble guanylyl cyclase leads to
inhibition of Na
+
/H

+
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

time period of the experiments as conducted here. As
shown below, alterations in the levels of the differentially
glycosylated forms of GC-C regulate the cellular response
to the ST-peptide, providing a novel means of inducing
desensitization, as well as suggesting that the glycosylation
state of GC-C determines its ability to be activated by its
ligands.
Materials and methods
Tissue culture media and all fine chemicals were from
Sigma-Aldrich, USA. Protein A agarose and ECL Plus
TM
Western blotting detection reagent were obtained from
Amersham Biosciences, UK.
125
Iodine and Western blot
chemiluminescence reagent were from NEN Life Science
Products, USA. Stable toxin of the human variety (STh)
and a mutant form of the STh peptide, ST
Y72F
,were
purified as described earlier [26]. Caco2 cells were obtained
from M. C. Rao, Department of Physiology and Biophys-
ics, University of Illinois at Chicago.
Culture and maintenance of cells
Caco2 cells were cultured in Dulbeccos’s modified Eagle’s
medium (DMEM) and Ham’s nutrient mixture F12 in the
ratio 1 : 1 (DMEM/F12) containing 10% fetal bovine
serum, nonessential amino acids, 120 mgÆL
)1
penicillin and

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

previously [28].
To monitor the recovery of cellular responsiveness
following desensitization induced by ST, desensitized cells
were washed with serum-free DMEM/F12 and then incu-
bated with DMEM/F12, 10% fetal bovine serum and
nonessential amino acids for 12 h in the absence of ST
peptide, and in the presence of cycloheximide (100 lgÆmL
)1
)
or swainsonine (10 lgÆmL
)1
). Cells were either re-stimulated
with ST peptide, or membranes prepared for in vitro
guanylyl cyclase assays as described below. In some cases,
membranes were prepared from cells and taken for immuno-
precipitation and Western blot analysis as described
above.
Preparation of cell membranes
Membranes were prepared essentially as described previ-
ously [25]. Briefly, Caco2 cells were washed with chilled
phosphate buffered saline (NaCl/P
i
,10m
M
sodium phos-
phate buffer, pH 7.2, 0.9% sodium chloride) and scraped
into homogenization buffer (50 m
M
Hepes, pH 7.5,
100 m

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

guanylyl cyclase assays
Assays were carried out as described earlier [25]. Membrane
protein (20 lg) was incubated in presence or absence of
10
)7
M
ST in assay buffer consisting of 60 m
M
Tris-Cl,
pH 7.6, 500 l
M
IBMX, and a GTP regeneration system
Ó FEBS 2003 Glycosylation of GC-C and desensitization (Eur. J. Biochem. 270) 3849
consisting of 10 lg creatine kinase and 7.5 m
M
creatine
phosphate. The assay was initiated by adding 4 m
M
MgCl
2
and 1 m
M
GTP as substrate and incubated at 37 °C for 5–10
min. The reaction was terminated by addition of 400 lLof
50 m
M
sodium acetate buffer, pH 4.6. The samples were
boiled in a water bath, centrifuged at 10 000 g for 5 min
and cGMP in the supernatant was assayed by radioimmuno-
assay, as described earlier [28]. To measure the manganese-

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

M
MgCl
2
, 0.1% bovine serum
albumin, 10 lgÆmL
)1
leupeptin, 10 lgÆmL
)1
aprotinin).
Following incubation, the reactions were filtered through
GF-C filters, filters dried and associated radioactivity
monitored in an LKB gamma counter. The data was
analyzed using
GRAPHPAD PRISM
(San Diego, CA, USA).
Immunodetection of GC-C
Western blot analysis was performed with 50 lgofmem-
brane protein and monoclonal antibody GCC:C8
(500 ngÆmL
)1
) raised to the protein kinase like domain of
GC-C, as described earlier [25]. Bound antibody was
detected by enhanced chemiluminescence according to the
manufacturer’s instructions.
Immunofluorescence of Caco2 cells
Immunocytochemistry was carried out as described earlier
[31]. Cells were plated on coverslips, washed with NaCl/P
i
and fixed in NaCl/P
i

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

EDTA, 1%
Triton X-100, 5 lgÆmL
)1
soybean trypsin inhibitor,
5 lgÆmL
)1
aprotinin, 5 lgÆmL
)1
leupeptin, and 100 l
M
sodium orthovanadate) for 1 h at 4 °C. The soluble fraction
was incubated overnight with a polyclonal antibody raised
to the C-terminal domain of GC-C (CTD antibody) [32] at a
concentration of 20 lgÆmL
)1
. Ten microliters of protein A
agarose was added to collect the immunocomplex. The
immunoprecipitate was washed thrice with immunoprecip-
itation buffer, boiled in sample buffer and subjected to
polyacrylamide gel electrophoresis and Western blot ana-
lysis, as described earlier.
For complete deglycosylation of GC-C with PNGaseF,
approximately 300 lg of membrane protein was solubilized
and immunoprecipitated as described above. The immuno-
precipitate was washed twice with 50 m
M
sodium phosphate
buffer, pH 7.2 and then boiled in 50 m
M
sodium phosphate

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-

bated in NaCl/P
i
-CM containing 500 lgÆmL
)1
sulfo-NHS-
biotin (Sigma) for 30 min at room temperature. Excess
biotin was quenched by incubation with 50 m
M
Tris-Cl,
pH 7.5, for 5 min. Cells were briefly washed with NaCl/P
i
and then scraped in homogenization buffer and membranes
were prepared. Membrane protein was solubilized and then
immunoprecipitated as described above. Beads were washed
three times with immunoprecipitation buffer containing
Triton X-100 and twice with immunoprecipitation buffer
without Triton X-100 and proteins were resolved on 7.5%
SDS, transferred onto poly(vinylidene difluoride) mem-
brane and subjected to Western blot analysis with strept-
avidin–peroxidase.
Results
Homologous desensitization of GC-C in Caco2 cells
We studied this phenomenon of GC-C desensitization in
Caco2 cells by treating cells with ST for 18 h, following
which the cells were washed and restimulated with 10
)7
M
ST. Significant cGMP production was elicited by ST
application to control cells as a consequence of GC-C
activation. In cells that were preincubated with ST for 18 h,

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,

as addition of 8-Br cGMP to Caco2 cells did not result in
cellular refractoriness to ST (data not shown).
In vitro
guanylyl cyclase assay after ST-induced
desensitization
In vitro guanylyl cyclase assays were performed with
membranes prepared from control and ST-treated Caco2
cells. Consistent with the results seen in whole cells, there
was a 15-fold stimulation of guanylyl cyclase activity in
membranes prepared from control cells on addition of
ST (Fig. 2A). In contrast, there was a dramatic loss of
ST-mediated activation of GC-C in the membranes pre-
pared from cells that had been exposed to ST earlier.
Interestingly, receptor-binding analysis performed with
membranes prepared from control and ST-treated cells
showed only a 50% loss in receptor content (Fig. 2B). These
results indicate that although ST binding sites were present
in the desensitized cells, ligand binding to these sites was not
coupled to cGMP production, as significant loss of ST-
mediated activation of GC-C was observed in desensitized
cells.
We monitored guanylyl cyclase activity in membranes
prepared from control and ST-treated cells using MnGTP
as a substrate, where guanylyl cyclase activity can be
observed even in the absence of ligand. Again, in contrast to
the significant loss of ST-mediated activation of guanylyl
cyclase activity, 50% of the activity seen in control cells was
still observed, representing the amount of GC-C detected by
ST binding (Fig. 2C). Therefore, the fraction of GC-C
present in the membranes of desensitized cells possessed

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.

Cells were lysed in 0.1
M
citric acid and intracellular cGMP was
measured by radioimmunoassay. (B) Cells were treated with 10
)7
M
ST
for the indicated times. At the end of incubation, cells were washed and
restimulated with 10
)7
M
ST in the presence of 500 l
M
IBMX for
15 min. (C) Caco2 cells were treated with varying concentrations of ST
for 9 h. Following incubation, cells were washed and restimulated with
10
)7
M
ST for 15 min in the presence of IBMX. Values represent
mean ± SEM of duplicate determinations with each experiment
performed at least twice.
Ó FEBS 2003 Glycosylation of GC-C and desensitization (Eur. J. Biochem. 270) 3851
the high mannose containing form of GC-C. It was likely
therefore that the predominant 130 kDa form was residing
in the endoplasmic reticulum. Indeed, immunofluorescence
with GC-C:D7 monoclonal antibody showed that a signi-
ficant fraction of GC-C was present inside the cell,
presumably in the endoplasmic reticulum (Fig. 3B). This
large fraction of GC-C present intracellularly appears to be

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

)7
M
ST in
the presence of Mg-GTP (4 : 1 m
M
) for 10 min and the cGMP
synthesized was measured by radioimmunoassay. Values represent
mean ± SEM of duplicate determinations with the experiment
performed at least twice. (B) Receptor binding analysis of control and
desensitized cells. Membrane protein prepared from control (right
panel) and desensitized cells (left panel) were subjected to
125
I-labeled
ST binding. Membrane protein was incubated with increasing
amounts of
125
I-labeled ST
Y72F
at 37 °C for 1 h. At the end of incu-
bation, the reaction was filtered through GF-C filters, filters were dried
and radioactivity associated with the filter was measured. Data was
analyzed using
GRAPHPAD PRISM
. The experiment was performed at
least twice and the values shown represent data from a single experi-
ment. (C) Five micrograms of membrane protein prepared from
control and desensitized cells were incubated with MnGTP (4 m
M
MnCl
2

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-

fore, cells were allowed to recover in the absence and
presence of cycloheximide. As shown in Fig. 4C, the
presence of cycloheximide did not hinder the reappearance
of the 145 kDa form of GC-C, nor prevent restoration of
ST-responsiveness in cells, indicating that de novo protein
synthesis was not required, and a pool of ER-associated
GC-C is available to replenish the ligand-activable form of
the receptor, lost from the cell surface on ST addition. The
levels of cGMP accumulation achieved during recovery in
the presence of cycloheximide was slightly greater than in its
absence across (P < 0.05), suggesting that the synthesis of a
factor that could destabilize GC-C expression in cells was
inhibited in cycloheximide-treated cells.
To determine whether inhibition of glycosylation could
prevent recovery, desensitized cells were treated with
swainsonine, an inhibitor of a-mannosidase II, during the
recovery process, and then stimulated with ST. As shown in
Fig. 4D, swainsonine inhibited the reacquisition of respon-
siveness by 50%. This shows that modification of the a-1,6
arm of the mannose residues was essential to allow
formation of the 145 kDa form of GC-C and ligand
responsiveness. Western blot analysis of cells cultured in the
presence of swainsonine showed the presence of a band of
136 kDa, representing the partially glycosylated form of
GC-C.
Desensitization requires GC-C internalization
and receptor activation
Removal of the 145 kDa form from the surface of cells
could be either through selective proteolysis or internaliza-
tion and degradation. It is unlikely that selective proteolysis

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

monoclonal and polyclonal antibodies (data not shown). If
internalization and subsequent degradation of the 145 kDa
form of GC-C was the cause for the desensitization,
prolonged exposure of cells to ST may be required. We
performed desensitization experiments at 4 °C, and as
shown in Fig. 5A, no receptor desensitization was observed,
and no loss of the 145 kDa fraction of GC-C was seen in
cells treated at 4 °C with ST (Fig. 5B). These results
therefore suggest that internalization of the 145 kDa form
followed by degradation may account for the inability of
cells to respond to the ST peptide, and this process is
inhibited at 4 °C.
We surface biotinylated control and desensitized cells,
immunoprecipitated the receptor using a GC-C antibody
and probed immunoprecipitates with streptavidin peroxi-
dase. As shown in Fig. 5C, interestingly, a significant
amount of 130 kDa protein was localized on the surface of
the cells, along with the 145 kDa form of GC-C, perhaps
representing a form of the receptor that had reached the
surface in a Golgi-independent manner [37]. The presence of
a biotinylated 130 kDa form of the receptor was not due to
biotinylation of the intracellular 130 kDa protein as a
consequence of leaky or damaged cells during the biotiny-
lation reaction. This was judged by trypan blue exclusion,
where more than 99% of the cells were viable following
biotinylation (data not shown). Interestingly, on ST treat-
ment of cells, the 130 kDa form was retained on the cell
surface, as monitored by immunoprecipitation of GC-C
using the CTD antibody followed by Western blot analysis
with streptavidin-peroxidase (Fig. 5C). Therefore, there is a

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

is activated by magnesium and manganese [39]. Activation
of GC-B by CNP also leads to desensitization of GC-B after
brief treatment with ligand, and here again, this is accom-
panied by a decrease in the phosphate content of GC-B [40].
Mutational analysis has shown that there are five serine/
threonine phosphorylation sites in GC-B and mutation of
all these sites together led to a loss of CNP-dependent
activity [41].
The sites for phosphorylation in GC-A or GC-B are not
conserved in GC-C. In our initial studies, we had investi-
gated the phosphorylation status of GC-C in Caco2 cells,
and found that while there is a basal level of phosphory-
lation in the receptor on serine residues, there is no
alteration in the phosphorylation status on ligand addition
(unpublished observations). Given the contrasting rates of
desensitization observed between GC-A and GC-C, with
inactivation of GC-A occurring in a few minutes following
Fig. 5. Desensitization of Caco2 cells to ST peptide requires GC-C
internalization. Caco2 cells were treated with 100 n
M
ST for 9 h at 4 °C
to inhibit internalization. Another set was incubated with or without
ST at 37 °C and membranes were prepared. (A) Twenty micrograms
of membrane was incubated with MgGTP (4 : 1 m
M
) in the presence
or absence of ST for 10 min cGMP was measured by RIA. Values
represent mean ± SEM of duplicate determinations with each
experiment performed at least twice. (B) GC-C was immunoprecipi-
tated using CTD antibody and the immunoprecipitate was subjected to

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

shown to be internalized and recycled in T84 cells following
ST treatment, but those experiments were conducted with
periods of ST treatment for 3 h or less, during which time
we observe only a slight desensitization in either T84 or
Caco2 cells [43]. Moreover, earlier studies monitored ST
binding to monolayers of cells in culture, and not ligand-
stimulatable activity of surface-localized GC-C. As shown
in the studies described here, the recycling of the 145 kDa
form of GC-C does not appear to occur on long-term
treatment of cells with ST. As the 130 kDa form of GC-C is
still on the surface after prolonged ST treatment, it is not
clear at this time whether the 130 kDa form alone is
recycled, or not internalized at all.
GC-C down-regulation apparently occurs by differential
expression of the two glycosylated forms of GC-C, and a
strong correlation between the presence of the 145 kDa
form and ST-stimulatabilty is seen, indicating that the
145 kDa form is the ligand activated form of GC-C. Our
earlier studies have shown that GC-C desensitization is
modulated in a cell specific manner [25]. GC-C desensitiza-
tion is observed only in cells that endogenously express
GC-C, such as T84 and Caco2 but not in HEK293 cells
stably transfected with GC-C, HEK293GC-C cells. Inter-
estingly, there is no loss of the 145 kDa form in
HEK293GC-C cells on ST treatment, providing an explan-
ation for the continuous ability of these cells to respond to
ST, even on prolonged prior exposure [25]. The absence of
GC-C desensitization (which is correlated with removal of
the 145 kDa form of GC-C from cells) in HEK293-GC-C
cells could be due to lack of a cellular factor that is present in

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

plasma membrane [45]. Glycosylation of the b-adrenergic
receptor is also important for its internalization and a single
nucleotide polymorphism which leads to mutation of serine
49 to glycine leads to loss of glycosylation and enhances
internalization of the b-adrenergic receptor [46]. Recently,
glycosylation-based recognition motifs have also been
recognized in substrates for the endoplasmic reticulum-
associated degradation (ERAD) pathway. One of the
proteins associated with the E3 ubiquitin ligase complex,
Fbx2, specifically binds N-glycosylated substrates through
mannose residues on the glycocalyx [47]. Although glyco-
sylation-based recognition signals have not yet been iden-
tified for degradation of cell surface receptors, the possibility
of such a mechanism operating at the plasma membrane
cannot be ruled out, and the two forms of GC-C are
perhaps targeted to different endocytic routes based on
glycosylation sorting motifs at the plasma membrane.
Alternatively, glycosylation-dependent sorting of the two
forms could also take place intracellularly after they are
endocytosed and not at the plasma membrane. Surface
localized receptors such as EGF receptor are carried to the
endoplasmic reticulum before being targeted to the degra-
dation pathways [48] and it is possible that GC-C is also
carried to the endoplasmic reticulum where the 145 kDa
form is targeted to the degradation pathway and the
130 kDa form can be recycled back to the surface.
Therefore in this study, we have described a novel means
of regulation of a member of the guanylyl cyclase receptor
family, by controlling the amounts of differentially glycosy-
lated forms of GC-C in a cell. Given the fact that 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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