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Metabolic Diseases Branch (G.F., P.K.G., R.C., A.M.S.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and NPS Pharmaceuticals (C.K.D., K.J.K., K.V.R.), Salt Lake City, Utah 84108
Address all correspondence and requests for reprints to: Allen M. Spiegel, Building 10, Room 9N-222, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892. E-mail: allens{at}amb.niddk.nih.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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600 residue)
amino-terminal extracellular domain contains 9 potential N-linked
glycosylation sites. Immunoblot of cell membranes derived from HEK-293
cells, stably transfected with the hCaR, showed two major
immunoreactive bands of approximately 150 and 130 kDa, respectively.
Complete digestion of the membranes with PN-glycosidase F yielded a
single major immunoreactive band of approximately 115 kDa, confirming
the presence of N-linked glycosylation. Treatment of these cells with
tunicamycin, which blocks N-linked glycosylation, inhibited signal
transduction in response to Ca2+. Flow cytometric analysis
showed decreased expression of the hCaR on the cell membrane in
tunicamycin-treated cells. Immunoblot of tunicamycin-treated cells
showed a reduction in the amount of the 150-kDa band and conversion of
the 130-kDa band to the presumptively nonglycosylated 115-kDa form.
Tunicamycin treatment of cells, transfected with a mutant hCaR
complementary DNA containing a nonsense codon at position 599 preceding
the 1st transmembrane domain, blocked the secretion of a 95-kDa
protein, representing the amino-terminal extracellular domain, into the
medium. These results demonstrate that N-linked glycosylation is
required for normal expression of the hCaR at the cell surface. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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600 residue) amino-terminal
extracellular domain (ECD), an integral membrane domain with 7
membrane-spanning alpha-helices, and an approximately 200-residue
carboxy-terminal intracellular domain (3). The CaR is a member of the
superfamily of G protein-coupled receptors (GPCR), most closely related
to the subfamily of metabotropic glutamate receptors (2). The putative
ECD of the hCaR contains 9 potential N-linked glycosylation sites,
suggesting that the CaR is a glycoprotein (3).
Protein glycosylation has been shown to be important for a variety of
functions, including normal protein folding, stability, intracellular
trafficking, cell surface expression, and secretion (4). The role of
N-linked glycosylation may differ for various membrane proteins. In the
GPCR superfamily, most members have relatively short N-terminal ECDs
with generally less than 3 N-linked glycosylation sites. The
glycoprotein hormone subfamily of GPCRs has a longer (400 residue)
ECD with as many as 3–6 putative N-linked glycosylation sites, a
subset of which has been shown to be critical for normal receptor
folding and cell surface expression (5, 6). The function of N-linked
glycosylation for the CaR or metabotropic glutamate receptors, with
their significantly larger ECDs and greater number of putative N-linked
glycosylation sites, is as yet unknown. In the present study, we used
an inhibitor of N-linked glycosylation, tunicamycin (7), to probe the
role of this covalent modification in the function and cell surface
expression of the hCaR. Our results indicate that N-linked
glycosylation is essential for cell surface expression of the hCaR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tunicamycin treatment
Transfected HEK-293 cells were cultured in supplemented DMEM,
with or without varying concentrations of tunicamycin (CalBiochem, San
Diego, CA), for 48 h. The cells were then harvested for various
analyses.
Membrane preparation
Crude cell membranes were prepared from the untransfected and
transfected HEK-293 cells as follows: cells were suspended in 10 ml
homogenization buffer (20 mM Tris, pH 7.2, containing 0.25
M sucrose, 1 mM EDTA, and protease inhibitors
(1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride, 10 µg/ml
bestatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml
calpain) and homogenized for 1 min using a Polytron homogenizer,
Brinkman Instruments (Westburg, NY). The homogenate was then spun at
500 x g for 30 min to remove nuclei and debris. The
supernatant was sedimented at 18,000 x g for 30 min.
The pellets were resuspended in homogenization buffer and stored at
-70 C.
Immunoblotting
Proteins were separated on 5–15% gradient SDS-acrylamide gels,
as described by Laemmli (8). After transfer to nitrocellulose
membranes, the proteins were probed with 2 µg/ml protein A-purified
monoclonal anti-hCaR antibody, ADD, (raised against a synthetic peptide
corresponding to residues 214–235 of hCaR) overnight at room
temperature. Blots were washed 3 times with Tris-buffered saline with
Tween-20 (50 mM Tris-HCl, 500 mM NaCl, 0.1%
Tween-20, pH 8.0). Subsequently, the membranes were incubated with 1
µg/ml goat antimouse IgG(
) antibody conjugated to horseradish
peroxidase (Kierkegaard and Perry Laboratories, Inc., Gaithersburg, MD)
for 2 h at room temperature. Membranes were then washed with
Tris-buffered saline with Tween-20 and developed using 4-chloronaphthol
as substrate (9).
Peptide N-glycosidase F (PNGase F) treatment
Membrane proteins were denatured by 1-h incubation at room
temperature in 50 µl 0.5 M sodium phosphate (pH 8.0)
containing 0.5% SDS and 50 mM ß-mercaptoethanol. A
20-µl aliquot of this solution was mixed with 10 µl 7.5% NP-40 and
10 µl enzyme-containing solution. The mixture was incubated in a vol
of 40 µl (final membrane protein concentration = 1.5 µg/µl)
with various concentrations of PNGase F (Genzyme, Cambridge, MA)
for 18 h at 37 C. The reaction was stopped by adding an equal
volume of SDS-PAGE loading buffer, and samples were subsequently
analyzed by immunoblotting.
Measurement of PI breakdown
Cells grown in supplemented DMEM were plated in 24-well culture
plates at a density of 500,000 cells per well. After culturing in the
absence or presence of tunicamycin for 24 h, cells were then
incubated with 3.0 µCi/ml H3-myoinositol (New England
Nuclear, Boston, MA) in DMEM, with or without tunicamycin, for another
24 h, followed by 30-min preincubation with PI buffer (119
mM NaCl, 5 mM KCl, 5.6 mM glucose,
0.4 mM MgCl2, 20 mM LiCl in 25
mM PIPES buffer, pH 7.2) containing 0.5 mM
Ca2+. After removal of PI buffer, cells were incubated for
an additional 30 min with 6 mM Ca2+ or 20
mM F-. The reaction was terminated by the
addition of 1 ml acid methanol (167 µL HCl in 120 ml methanol). Total
inositol phosphates were purified by chromatography on Dowex-1-X8, as
described (10).
Preparation of clone 32-cell lysates and culture medium
Forty-eight hours after treatment with or without tunicamycin,
clone 32 cells and culture media were separated by centrifugation.
Cells were lysed in homogenization buffer containing 1% (wt/vol)
Triton X-100 on ice for 10 min. Insoluble material was removed by
centrifugation at 35,000 x g for 30 min at 4 C. Culture media
were concentrated by ultrafiltration over amicon YM-50 membranes.
Flow cytometric analysis
One million transfected or untransfected 293 cells were
incubated with 0.5 ml Dulbecco’s PBS (DPBS) containing 1% BSA and 20
µg/ml monoclonal anti-hCaR antibody LRG (raised against a synthetic
peptide corresponding to residues 374–391 of hCaR) at 4 C for 60 min
in conical polystyrene tubes. Cells were washed with DPBS three times
and then incubated with 5 µg/ml fluorescein-labeled goat antimouse
IgG() (Kierkegaard and Perry Laboratories, Inc.) in DPBS, 1% BSA
for 60 min at 4 C. After three washes with DPBS, samples were analyzed
with a FACS flow cytometer (Becton-Dickinson, San Jose, CA) using an
argon-ion laser tuned to 488 nm. Per cent cells gated (% Gated in
Table 1
) represents the percentage of cells showing
fluorescence above an arbitrarily defined gating value. Cells incubated
only with second antibody (see under Buffer in Table 1) were used as
controls for gating. The mean fluorescence, a measure of amount of
antibody bound/cell, also is recorded.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows an immunoblot of membranes from
untransfected and from clone 7 cells using a monoclonal antibody, ADD,
raised against a synthetic peptide corresponding to a sequence within
the ECD of the CaR. Membranes from untransfected 293 cells show no
immunoreactivity. Membranes from clone 7 cells show two predominant
bands at approximately 150 and 130 kDa.
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). With increasing enzyme concentration, both
bands were converted to a predominant species of approximately 115 kDa
and a series of fainter, slower migrating species. The latter
persisted, even at 10 and 20 units/ml (the highest enzyme
concentrations tested) (Fig. 2, lanes 9 and 10).
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To determine whether N-glycosylation plays a role in hCaR expression
and function, we treated clone 7 cells with increasing concentrations
of tunicamycin for 48 h and assessed Ca2+-stimulated
PI breakdown. Tunicamycin treatment inhibited
Ca2+-stimulated PI breakdown in a dose-dependent manner.
Significant inhibition was seen at 0.1 µg/ml tunicamycin and maximal
inhibition (75%) at 0.8 µg/ml (Fig. 3). Further
increases in tunicamycin concentration had no significant effect. To
establish whether this inhibition is related directly to inhibition by
tunicamycin of glycosylation of the hCaR or to some nonspecific effect,
we tested the effect of tunicamycin treatment on stimulation of PI
hydrolysis by F-, which stimulates PI hydrolysis by
directly activating the G protein linked to phospholipase C
stimulation. As seen in Fig. 3, F- stimulation of PI
hydrolysis is not inhibited by tunicamycin treatment at any
concentration tested.
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), whereas untransfected cells showed minimal antibody
binding. When treated with tunicamycin, the transfected cells showed a
concentration-dependent decrease in antibody binding, as evidenced
by reductions in both the percent of cells gated and the mean
fluorescence (Table 1).
Next we used immunoblots of clone 7 cell membranes to assess the
effect of tunicamycin treatment on synthesis of the hCaR (Fig. 4). Both 0.1 and 1 µg/ml tunicamycin reduced the
amount of the upper (150 kDa) immunoreactive band. At 0.1 µg/ml,
the amount of the lower (130 kDa) band also was reduced, and several
more rapidly migrating bands were generated. At 1 µg/ml, the 130-kDa
band was lost and at least two more rapidly migrating species
generated, of which the lowest was approximately 115 kDa.
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, lane 1). Tunicamycin treatment of clone
32 cells blocked secretion of the ECD into the medium, with 1 µg/ml
being more effective than 0.1 µg/ml (Fig. 5, lanes 2 and 3). An
immunoblot of a lysate of untreated clone 32 cells revealed a single
major immunoreactive band that migrated somewhat more rapidly (85
kDa) than the secreted ECD (Fig. 6, lane 1). Tunicamycin
treatment caused a major reduction in the amount of this immunoreactive
species. At 0.1 µg/ml of tunicamycin, a series of additional
immunoreactive bands were generated in the clone 32 cell lysate,
ranging from approximately 80–60 kDa (Fig. 6, lane 2); at 1 µg/ml,
essentially all of these species were converted to the 60-kDa form
(Fig. 6, lane 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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150 and 130 kDa.
Our results on 293 cells, stably expressing the hCaR, are similar to
those of Bai et al. (12), who performed immunoblots with a
different antibody on membranes from 293 cells transiently transfected
with hCaR cDNA. Previous evidence already suggested that the CaR is a
glycoprotein (2, 12). We digested membranes from clone 7 cells with
increasing concentrations of PNGase F and noted a progressive reduction
in size of both immunoreactive bands with increasing enzyme until 10
units/ml. At higher enzyme concentrations (Fig. 2, see lanes 7–10),
there is a suggestion of a ladder of immunoreactive species above the
predominant (115 kDa) deglycosylated form. This could reflect
sequential cleavage of N-linked sugar from multiple (>4) sites on the
hCaR, but as suggested by Bai et al. (12), the presence of
other types of posttranslational modifications cannot be excluded.
Bai et al. examined the effects of digestion with Endo H and
of tunicamycin, an inhibitor of N-linked glycosylation (7), on
immunoreactivity of the hCaR in transiently transfected 293 cells (12).
They suggest that the higher molecular mass immunoreactive band
(corresponding to our 150 kDa) represents the mature, fully
processed form of the hCaR and that the lower form (corresponding to
our 130 kDa) represents an incompletely processed, high mannose form
of the hCaR. Our results at 1.0 µg/ml tunicamycin agree with those of
Bai et al. Note that even at this higher concentration of
tunicamycin, the band of approximately 150 kDa, although significantly
diminished in amount, is still detectable. In contrast, the form of
approximately 130 kDa essentially disappears and is converted to lower
molecular mass forms at or slightly above 115 kDa. At 0.1 µg/ml
tunicamycin, some approximately 130-kDa immunoreactivity is still
detectable, but the majority is converted to forms intermediately
between the approximately 130- and 115-kDa forms. We noted also that
total CaR immunoreactivity was reduced by tunicamycin treatment (1.0
µg/ml > 0.1 µg/ml). For some proteins, N-linked glycosylation
is critical for normal folding in the endoplasmic reticulum (4).
Inhibition of hCaR glycosylation by tunicamycin may block normal
folding, leading to retention and degradation in the endoplasmic
reticulum. One would expect tunicamycin to affect only hCaR protein
that is being newly synthesized in the endoplasmic reticulum, not
mature hCaR already present at the plasma membrane. Because our studies
were performed on 293 cells, stably expressing high amounts of the
hCaR, in contrast to those of Bai et al. on 293 cells
transiently transfected with the hCaR cDNA, it is not surprising that
even at the highest tunicamycin concentration tested, we observe
persistent immunoreactivity at approximately 150 kDa, reflecting the
mature form of the hCaR. The diminution in the amount of the
approximately 150-kDa band during the 48 h of tunicamycin
treatment presumably reflects the turnover of the mature, cell surface
receptor. We interpret the reduction in size and eventual disappearance
of the approximately 130-kDa immunoreactive band to reflect increasing
inhibition by tunicamycin of normal processing of newly synthesized
hCaR protein.
Our results, as well as those of Bai et al. (12), indicate that the hCaR, in fact, contains N-linked sugars and that tunicamycin treatment causes significant alterations in the forms of the CaR detected by immunoblots. The functional implications of this modification of the hCaR had not previously been explored. To assess the functional significance of N-linked glycosylation of the hCaR, we first studied the effect of tunicamycin treatment of clone 7 cells on signal transduction. Tunicamycin caused a dose-dependent inhibition of [Ca2+]o but not fluoride-stimulated PI hydrolysis, consistent with a critical role for N-linked glycosylation in hCaR expression and/or function. The maximal inhibition observed was approximately 75%. Failure to achieve complete inhibition likely reflects persistence of mature hCaR, synthesized before tunicamycin treatment in clone 7 cells, stably expressing this receptor, as discussed above.
To distinguish between a role for N-glycosylation in the function of the hCaR vs. a role in expression at the cell surface, we studied the effect of tunicamycin treatment on cell surface expression of the hCaR by flow cytometry using a monoclonal antibody that binds specifically to the ECD of the native hCaR. We found that tunicamycin significantly reduced cell surface expression of the hCaR, as reflected in a reduction in the number of cells expressing detectable receptor at the cell surface (% cells gated) and in the amount of antibody bound to cells still expressing receptor (mean fluorescence). Although theoretically it is possible that the reduction in LRG antibody binding on flow cytometry analysis of tunicamycin-treated cells reflects inability of the antibody to bind to a nonglycosylated receptor that does, in fact, reach the cell surface, we consider this extremely unlikely. The LRG monoclonal antibody, like the ADD antibody used for immunoblots in the present study, was raised against a synthetic peptide (residues 374–391 of the hCaR), rather than the native, glycosylated receptor protein. LRG’s ability to react, not only with the native receptor on flow cytometric analysis, but also with the denatured, deglycosylated hCaR and ECD on immunoblot (not shown), support our interpretation that reduction in LRG binding on flow cytometry analysis of tunicamycin-treated cells, in fact, reflects decreased cell surface expression of receptor.
Although the intact hCaR is not normally secreted, both the intact hCaR
and the truncated form expressed by clone 32 cells are likely to be
processed and transported identically, with the difference that the ECD
in the intact receptor remains tethered to the cell surface by the
integral seven membrane-spanning domain, rather than being secreted
into the medium. Secretion of the hCaR ECD into the extracellular
medium by clone 32 cells thus gave us another way of studying the role
of glycosylation in this process. Culture medium collected from clone
32 cells contains an approximately 95-kDa broad band that reacts
specifically with a series of antibodies raised against different
portions of the hCaR ECD (Fig. 5 and unpublished observations). Based
on the size of the protein encoded by the cDNA up to the artificially
engineered stop codon and allowing for removal of a putative signal
peptide (2, 3), a band of approximately 60 kDa would have been
expected. The increased size and diffuse nature of the approximately
95-kDa band presumptively reflects N-linked glycosylation at multiple
sites. This is confirmed by PNGase F treatment of this protein
(unpublished observations). Tunicamycin treatment of clone 32 cells
caused a significant reduction in immunoreactive, approximately 95 kDa
protein detected in the culture medium, consistent with a critical role
for N-linked glycosylation in the normal processing and secretion of
this protein. Immunoblot analysis of cell lysates from clone 32 cells
revealed a single major band, slightly lower in molecular mass than the
secreted, approximately 95-kDa protein. Tunicamycin treatment caused a
significant decrease in amount but not complete disappearance of this
protein. Again, persistence of some of this protein likely reflects
material synthesized before tunicamycin treatment. The majority of
immunoreactivity is converted with 1 µg/ml tunicamycin treatment into
a sharp band at approximately 60 kDa, corresponding to the
nonglycosylated form of the protein. After 0.1 µg/ml tunicamycin
treatment, a ladder of immunoreactive bands, comprising a minimum of 4
discrete species between 85 and 60 kDa, is seen. We interpret this
result as reflecting generation of multiple variably N-glycosylated
forms of the ECD at intermediate tunicamycin concentrations.
It is not possible for us to make a precise quantitative comparison between the results obtained in the several assays we used, but with each assay [1) immunoblot detection of the mature, approximately 150-kDa form of the receptor; 2) Ca2+-stimulated PI hydrolysis; 3) flow cytometric analysis of cell surface hCaR expression; and 4) immunoblot detection of the 95-kDa ECD secreted into the medium], tunicamycin led to significant, dose-dependent inhibition. Studies on a variety of secreted and membrane proteins have revealed the importance of N-linked glycosylation for normal protein folding, stability, solubility, trafficking, secretion, and biological function (4, 5). Different roles for this modification have been identified for different proteins (13). Even within the superfamily of GPCR, the importance and role of N-glycosylation seem to vary (5, 6, 14, 15, 16, 17). Our studies with tunicamycin have revealed a critical role for N-linked glycosylation in the normal expression of the hCaR at the cell surface (and in the secretion of a truncated form of the receptor). Reduced cell surface expression likely reflects abnormal folding of the nonglycosylated receptor protein, reduced stability, and resultant reduction in net synthesis; glycosylation may, in addition, be important for normal transport of the hCaR to the cell surface, but the present data do not permit a definite conclusion on this point. Our results, showing inhibition of CaR signaling by tunicamycin, could be explained simply as failure of the hCaR to reach the plasma membrane (as documented by flow cytometry), but we cannot, at present, exclude an additional role for N-linked glycosylation in CaR function.
N-linked glycosylation of the hCaR may have pathophysiologic relevance, considering that certain inactivating mutations of the receptor identified in subjects with familial hypocalciuric hypercalcemia seem to compromise normal receptor synthesis and processing in an in vitro assay (12). Further studies are now required to answer questions concerning the number and location of N-glycosylation sites and their specific role in biosynthesis, trafficking, and function of the hCaR. Biochemical studies on the secreted ECD, expression studies on CaR cDNAs mutated at putative glycosylation sites, and immunocytochemistry and biosynthetic labeling studies on cells expressing the CaR are underway to address these questions.
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Acknowledgments |
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Received November 29, 1996.
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K. Ray, G.-F. Fan, P. K. Goldsmith, and A. M. Spiegel The Carboxyl Terminus of the Human Calcium Receptor. REQUIREMENTS FOR CELL-SURFACE EXPRESSION AND SIGNAL TRANSDUCTION J. Biol. Chem., December 12, 1997; 272(50): 31355 - 31361. [Abstract] [Full Text] [PDF]
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M. E. Handlogten, C. Huang, N. Shiraishi, H. Awata, and R. T. Miller The Ca2+-sensing Receptor Activates Cytosolic Phospholipase A2 via a Gqalpha -dependent ERK-independent Pathway J. Biol. Chem., April 20, 2001; 276(17): 13941 - 13948. [Abstract] [Full Text] [PDF]
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L. Canaff, J.-L. Petit, M. Kisiel, P. H. Watson, M. Gascon-Barre, and G. N. Hendy Extracellular Calcium-sensing Receptor Is Expressed in Rat Hepatocytes. COUPLING TO INTRACELLULAR CALCIUM MOBILIZATION AND STIMULATION OF BILE FLOW J. Biol. Chem., February 2, 2001; 276(6): 4070 - 4079. [Abstract] [Full Text] [PDF]
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G. Reyes-Cruz, J. Hu, P. K. Goldsmith, P. J. Steinbach, and A. M. Spiegel Human Ca2+ Receptor Extracellular Domain. ANALYSIS OF FUNCTION OF LOBE I LOOP DELETION MUTANTS J. Biol. Chem., August 17, 2001; 276(34): 32145 - 32151. [Abstract] [Full Text] [PDF]
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