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Neurobiology Unit, St. Vincent’s Institute of Medical Research, Fitzroy 3065, Victoria, Australia
Address all correspondence and requests for reprints to: Patrick M. Sexton, Neurobiology Unit, St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy 3065, Victoria, Australia. E-mail: pms{at}rubens.its.unimelb.edu.au
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Abstract |
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58,000). The lower
Mr of the cloned pig CTR appeared to be due to absence of
N-terminal residues, but this did not impact on ligand-receptor
specificity when compared with the hypothalamic pig CTR. Cleavage under
nondenaturing conditions of N-linked sugars from the CTRs using
endoglycosidase F (Endo F), increased the electrophoretic mobility of
all receptors, except the pig CTRs, by 10 kDa. Under denaturing
conditions, electrophoretic mobilities increased by 30 kDa for the
rat C1a, rat C1b, and humanI1-ve (expressed in human
embryonic kidney-293 cells) CTRs and by 20 kDa for the cloned pig,
pig hypothalamic, and human CTR isoforms (expressed in baby hamster
kidney cells). Competition binding studies using glycosylated and
partially deglycosylated (nondenaturing conditions) receptor
preparations demonstrated no significant differences in binding
affinity or specificity. Thus the CTRs are N-linked glycoproteins whose
degree of glycosylation is both cell-type and species dependent. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, complementary DNAs (cDNAs) encoding CTRs from pig (5), rat
(6), and human (7, 8) have been cloned, with splice variation occurring
in the first intracellular (I1) and second extracellular (E2) domains
of the receptor (Fig. 1
). Comparison of the predicted
amino acid sequences indicates approximately 78% identity between rat
and human CTRs and approximately 67% between rat and pig CTRs, with
predicted native protein sizes of approximately 55 kDa (Fig. 1 and
Table 1). However, cross-linking studies in cells and
tissues endogenously expressing CTRs indicate relative molecular mass
(Mr) of 70,000–85,000 (9, 10, 11, 12, 13). Glycosylation may
contribute to the larger Mr seen in the endogenous
receptor, as the predicted amino acid sequences contain three to four
conserved potential N-linked glycosylation sites (Fig. 1).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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700
Ci/mmol. 125I-Bolton and Hunter-labeled rat amylin
(125I-rat amylin; specific activity, 2000 Ci/mmol) was
obtained from Amersham (Buckinghamshire, England). DMEM and G418
(Genticin) were from GIBCO (Grand Island, NY). FBS was from Trace
Biosciences (Sydney, Australia). The SDS-PAGE protein mol wt standards
phosphorylase b (97.4 kDa), BSA (66.2 kDa), ovalbumin (45
kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor
(21.5 kDa), and hen egg white lysozyme (14.4 kDa) and protein dye
reagent were from Bio-Rad (Hercules, CA). RNasin ribonuclease inhibitor
was from Promega (Madison, WI). The human CTR antibody was raised in
rabbits using a fusion protein of the C-terminus of the CTR and maltose
binding protein (21) and was a gift from Dr. E. Moore (Zymogenetics,
Seattle, WA).
Cell culture
Baby hamster kidney (BHK) and human embryonic kidney-293
(HEK-293) derived cell lines were maintained in DMEM containing 5%
(vol/vol) FBS and 200 µg/ml G418 at 37 C in a humidified atmosphere
containing 5% CO2 in air.
HEK-293 cells were stably transfected with either the cloned pig CTR (clonal cell line A7), the cloned rat C1a CTR (clonal cell line D11), or the cloned rat C1b CTR (clonal cell line B8-H10), as described previously (22). A truncated pig CTR (clonal cell line 5–1), which lacks 84 C-terminal amino acid residues, was also stably transfected in HEK-293 cells, as described previously (23). The human CTR, containing a 16-amino acid insert in the first intracellular domain (human CTRI1+ve) (clonal cell line BHK/RK28), and one lacking this insert (human CTRI1-ve) (clonal cell line Hollex 2) were both stably transfected into BHK cells as described previously (8, 24, respectively). The human CTRI1-ve cDNA was also stably transfected into HEK-293 cells as described for the rat CTRs (22) to compare expression between the two cell lines.
Membrane preparations from transfected cells and pig
hypothalamus
Transfected cells were grown to confluence in 175
cm2 flasks and placed on ice 30 min before the preparation.
Media was then poured off and 10 ml of ice-cold lysis buffer [25
mM Tris/HCl, pH 7.3, 5 mM EDTA, 0.05%
bacitracin, 0.5 mg/100 ml leupeptin, 0.5 mM
phenylmethylsulfonyl fluoride (PMSF)] added to each flask. Cells were
then scraped from the flask surface and homogenized in an Ultra-Turrax
(Kinematica, Lucerne, Switzerland) (13,000 rev/min) for 30 sec. The
homogenate was centrifuged at 120 x g for 10 min. The
supernatant was then centrifuged at 40,000 x g for 15
min, and the pellets retained. The pellet was resuspended in lysis
buffer (5 ml/flask) with three strokes of a motorized glass-Teflon
homogenizer and washed twice by recentrifugation and homogenization.
The final pellet was resuspended in lysis buffer (1–2 mg protein/ml)
and stored at -80 C until use.
Pig brains were collected fresh from the abattoir and transported on
ice before dissection (30 min). The hypothalamus was dissected free
from surrounding tissue, blended for 30 sec in lysis buffer (25
mM Tris/HCl, pH 7.3, 5 mM EDTA, 0.05%
bacitracin, 0.5 mM PMSF, 5 ml/g tissue) and then
homogenized in an Ultra-Turrax (13,000 rev/min) for a further 2 min.
The homogenate was centrifuged at 160 x g for 10 min,
and the supernatant retained. The pellet was rehomogenized in an equal
volume of lysis buffer, recentrifuged at 160 x g for
10 min, and the supernatant retained. The low-speed supernatants were
then centrifuged at 40,000 x g for 20 min, and the
pellets retained. The pellets were resuspended in lysis buffer (1 ml/g
tissue) in an Ultra-Turrax (13,000 rev/min), then centrifuged again at
40,000 x g for 20 min. Pellets were resuspended in
lysis buffer (14–15 mg protein/ml) and stored at -80 C until use. All
procedures were carried out at 4 C. Protein concentration was
determined by the method of Bradford (25) using 
-globulin as the
protein standard.
Covalent cross-linking in membrane preparations and transfected
cells
Membrane preparations were thawed, microfuged for 10 min, and
resuspended in 0.5 ml buffer A [0.8% NaCl, 0.2% KCl, 8
mM NaH2PO4, 1.5 mM
KH2PO4, pH 7.3 (PBS) containing 0.1% BSA,
0.05% bacitracin]. The membranes (0.1–0.5 mg protein) were
subsequently incubated for 1 h at 20 C with 4–9 nM
125I-sCT in the presence or absence of unlabeled sCT (1
µM). Membranes were then centrifuged and washed once with
PBS. Cross-linking was initiated by resuspension in 0.5 ml of 1
mM BS3 in PBS and allowed to proceed for 20 min
on ice. Reactions were then centrifuged for 10 min and terminated by
resuspension in 0.5 ml of 0.1 M Tris/HCl, pH 7.4,
containing 10 mM EDTA. Membranes were then either subjected
to endoglycosidase treatment or solubilization in 50 mM
Tris/HCl, pH 6.8, containing 2% (wt/vol) SDS, 0.1% bromophenol blue,
10% (vol/vol) glycerol, 100 mM dithiothreitol (sample
buffer). Extracts were boiled for 3 min, centrifuged at 100,000 x
g for 30 min at 4 C, and the supernatants analyzed on 10%
(wt/vol) SDS-PAGE by the method of Laemmli (26). Following
electrophoresis, gels were stained with Coomassie blue R-250,
destained, dried, and exposed to phosphor screens (Molecular Dynamics,
Sunnyvale, CA). The Mr of the labeled bands was determined
from a standard curve generated from the electrophoretic mobility of
mol wt markers that were coelectrophoresed with the samples.
Transfected cells in six-well plates were analyzed identically, except that cells were incubated with 125I-sCT in DMEM containing 0.1% BSA, 0.05% bacitracin for 1 h at 37 C in a humidified atmosphere containing 5% CO2 in air before cross-linking.
Endoglycosidase treatment
After cross-linking, membranes were recovered by microfugation,
and treated with Endo F under reducing and denaturing conditions,
nonreducing and nondenaturing conditions, or following solubilization
and reduction. In some experiments Endo F was omitted to monitor the
stability of the cross-linked receptor during the incubation period.
For treatment under reducing and denaturing conditions, membranes were
resuspended in sample buffer, heated at 50 C for 6 min, diluted 1:1
with distilled water to lower the SDS concentration, and then incubated
overnight at 37 C with 1.25% (vol/vol) Nonidet P-40 and 2 U/ml Endo F.
Extracts were then concentrated by a chloroform/methanol protein
precipitation protocol (27) and resuspended in sample buffer. For
treatment under nonreducing and nondenaturing conditions, membranes
were resuspended in 50 mM Tris/HCl (pH 6.8) and incubated
overnight at 20 C with 4 U/ml Endo F. Membranes were then pelleted by
centrifugation and resuspended in sample buffer. For experiments in
which the receptors were solubilized but not denatured, membranes were
resuspended in 100 mM Tris/HCl, pH 8.6, 10 mM
EDTA, 0.25% (wt/vol) SDS, 1% (wt/vol) octyl-ß-glucoside, 0.1%
2-mercaptoethanol, 2 mM PMSF, 0.5 mg/100 ml leupeptin, 0.5
mg/ml pepstatin, and 10 mM benzamidine and then incubated
overnight at 20 C with 4 U/ml Endo F. Extracts were then resuspended in
sample buffer and heated at 50 C for 6 min.
Receptor binding assays
Membranes were microfuged for 10 min at 4 C and resuspended in
buffer A. Aliquots of membrane (0.15 mg of protein/ml) were
incubated with 125I-sCT (0.18 nM) for 1 h
at 20 C in the presence or absence of increasing concentrations of
unlabeled peptide. In some experiments, membranes were first incubated
overnight at 20 C in lysis buffer with 0.4 units/ml of Endo F. To check
the success of deglycosylation, aliquots from each experiment were
cross-linked and analyzed as described above. Reactions were terminated
by centrifugation in a Beckman (Palo Alto, CA, USA) micro-centrifuge at
12,000 g for 4 min at 4 C and the supernatants removed. The
pellets were overlaid with ice-cold PBS and centrifuged again for 4
min. The pellets were counted for radioactivity in a Packard
-radiation counter at approximately 70% efficiency.
In vitro transcription/translation
DNA (0.5 µg) from pcDNA1Neo (Invitrogen, San
Diego, CA) constructs containing either the pig or rat C1a CTR (6) were
transcribed and translated in vitro using the TNT T7 coupled
reticulocyte lysate system (Promega). Control reactions were also
performed using the vector DNA without an insert, insert-containing DNA
without T7 RNA polymerase, and a reaction without template to identify
nonspecific bands. Proteins were labeled with 60 µCi of
[35S]methionine (Redivue L-[35S]methionine;
>1000Ci/mmol; 1 mCi>37TBq/mmol, Amersham) in the translation
reaction, separated by SDS-PAGE, and visualized by autoradiography as
described above.
Immunoblot analysis
Following SDS-PAGE, extracts of human CTR from both cross-linked
and non-cross-linked cells were transferred to nitrocellulose filters
(0.2 µm), incubated with a C-terminally directed antihuman CTR
antibody (1:500), and processed using a chemiluminescence Western
blotting kit according to the manufacturer’s protocol (Boehringer
Mannheim). Sheep antirabbit IgG coupled to horseradish peroxidase
(1:10,000) was employed as the second antibody, and immunoreactive
proteins visualized by exposure to x-ray film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cross-linking of 125I-sCT to whole cells expressing the rat
C1a CTR revealed a major, broad, specific band of Mr
79,000 (Fig. 2A and Table 2). In cells
expressing the rat C1b CTR, a major band of Mr 78,000
was observed, but minor bands of Mr 53,000 and 32,000
were also visualized (Fig. 2B and Table 2). Broad, labeled bands of
Mr 73,000 and 75,000 were observed for BHK cells
expressing the human CTRI1-ve and human
CTRI1+ve receptors, respectively (Fig. 2, C and D,
respectively, and Table 2). The electrophoretic mobility of the human
CTRI1-ve expressed in the HEK-293 cell line was slightly
higher, migrating at a Mr of 76,000 (Fig. 2, E
vs. C; Table 2). In contrast to the other CTRs, which
migrated at Mrs well above the predicted mol wt of the
native protein (55 kDa), cross-linking of 125I-sCT to
the HEK-293/pig CTR revealed a Mr of only 57,000 (Fig. 2F and Table 2), which is similar to the mol wt of the predicted native
protein {52,500 + 3,557 (125I-sCT) + 572
(BS3) = 56,600 Da}. In some experiments, specific
bands of higher Mr were also seen that are likely due to
cross-linking of receptor complexes to each other by the bifunctional
cross-liking agent.
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, A-E (i), compare lanes T with E],
except for the pig CTR [Fig. 2F (i)]. These results are summarized in
Table 2. The difference in Mrs of glycosylated and
deglycosylated receptor-ligand complexes for the rat and human
receptors ranged between 10,000 and 13,000 (Table 2). The two receptor
isoforms of the rat and human CTRs were each deglycosylated to a
similar extent, however the Mrs for these deglycosylated
receptors remained in excess of the predicted mol wts of the native
proteins (55 kDa). Similar results were obtained with solubilized
receptors (data not shown).
Full deglycosylation of some receptor proteins requires reduction and
denaturation of the protein (28). Under reducing and denaturing
conditions, Endo F digestion produced marked increases in the
electrophoretic mobility of all CTRs. Deglycosylation of the two rat
and two human CTR-ligand complexes under reducing and denaturing
conditions produced broad, specific bands that are similar in size to
the mol wt of the predicted native proteins (Table 1 vs.
Table 2). The two receptor isoforms of the rat CTR and the
HEK-293/human CTRI1-ve were deglycosylated to a similar
extent; differences in the Mrs of these glycosylated and
deglycosylated receptor-ligand complexes ranged between 29,000 and
33,000 (Table 2). Deglycosylation of the two human CTR isoforms
expressed in BHK cells under reducing and denaturing conditions
increased their electrophoretic mobility by 19,000 to 21,000 (Table 2).
Minor bands of Mr 43,000 and 44,000 were also
observed for endoglycosidase-treated BHK cells expressing the human
CTRI1-ve and human CTRI1+ve respectively [Fig. 2, C (ii) and D (ii), respectively]. These lower Mr bands
are likely due to proteolysis during the procedure in this cell line,
because cross-linking to membrane preparations containing protease
inhibitors yielded only the higher Mr product (not shown).
Western blot analysis using a C-terminally directed antihuman CTR
antibody indicated that the cleavage site was located in the C-terminal
region of the receptor (not shown). Interestingly, the Mr
of the HEK-293/pig CTR was also reduced by this treatment, migrating at
a Mr of 39,000, below the predicted mol wt of the native
protein [Fig. 2F (ii)].
Characterization of the pig CTR
The low apparent Mr of the cloned pig CTR expressed in
the HEK-293 cell line differs considerably from both the other cloned
CTRs and the reported Mrs of endogenously expressed CTRs in
cells and tissues. Consequently we examined whether pig CTR,
endogenously expressed in pig hypothalamic membranes, also exhibits a
low Mr. BS3 cross-linking of
125I-sCT to pig hypothalamic CTRs revealed a specific band
of Mr 69,000. Cross-linking to HEK-293/pig CTR
membranes, carried out in parallel, again showed a single broad band of
Mr 60,000 (Fig. 3). Treatment of the
hypothalamic pig CTR-ligand complex with Endo F under reducing and
denaturing conditions increased its electrophoretic mobility by
18,000, generating a broad specific band of Mr 52,000
(Fig. 3A), which is similar to the mol wt of the predicted native
protein (Table 1). However, like the cloned pig CTR, Endo F digestion
under nonreducing and nondenaturing conditions did not alter the
electrophoretic mobility of the hypothalamic pig CTR (results not
shown).
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80 residues from the C-terminus of the protein
(23). If the size difference was due to cleavage of the C-terminus of
the native receptor then no change in Mr of the protein
should be observed between the engineered truncated receptor and the
intact receptor. Mechanistically this suggested that the cloned pig CTR
expressed in HEK-293 cells either undergoes altered posttranslational
processing or undergoes translation from an AUG distal to the predicted
start site of translation. To address the latter possibility we carried
out in vitro transcription/translation of the pig CTR. Both
the pig CTR and the rat C1a CTR DNAs generated proteins of 50 kDa
consistent with the protein mass of full-length receptor (Fig. 4). The increased size of the pig CTR, however, was not
due to receptor glycosylation because Endo F treatment did not alter
migration of the proteins. Thus the smaller size of the pig CTR
expressed in HEK-293 cells is likely to arise from cell-specific
posttranslational processing of the receptor.
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100-fold less potent. Rat CT had little or no effect on binding
(Fig. 5A). A similar profile was seen in pig
hypothalamic membranes (Fig. 5B). In competition for
125I-amylin, the relative specificities were sCT >
rat amylin > pCT > rCT (Fig. 5, C and D). Again, no
significant differences in relative specificity were observed between
the endogenously expressed pig CTR preparation and that of the cloned
pig CTR (Fig. 5, C vs. D). However, for both
125I-amylin and 125I-sCT binding competition
the absolute affinity of peptides appeared higher in the HEK-293/pig
CTR preparation.
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) and human CTRI1-ve (Fig. 6B),
respectively. Partial deglycosylation reduced the Mrs to
71,673 ± 1,119 (n = 6) and 63,313 ± 1,295 (n = 4)
for the rat C1a CTR and the human CTRI1-ve, respectively
(Fig. 6). For the C1a CTR, a band of Mr 71,000 was also
seen in control membranes (Fig. 6A). suggesting the presence of
endogenous glycosidase activity. In 125I-sCT competition
binding studies, no significant differences were observed in peptide
binding affinity or relative specificity for either the rat C1a CTR
(Fig. 7) or human CTRI1-ve (Fig. 8) between partially deglycosylated (Figs. 7C and 8C) and control membranes (Figs. 7, A and B; 8, A and
B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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55 kDa, of which the N-terminal 2.5–3 kDa
constitutes a putative, cleavable, hydrophobic signal peptide sequence
(Table 1). These mol wts for native receptor proteins are significantly
lower than the Mrs observed for mature CTRs, indicating
that the CTR undergoes posttranslational processing. Previous studies
on endogenously expressed CTRs have indicated the presence of at least
two forms of the receptor. Both human placenta (10) and rat osteoclasts
(11) contain two binding species of Mr 88,000 and
71,000, whereas UMR 106–06 (11), BEN and T47D (13) cells display
the higher Mr form of the receptor. A single binding
protein (Mr 72,000) corresponding to the lower
Mr form of the receptor has been identified in sheep
diencephalon (9), whereas in rat kidney a minor band of Mr
70,000 was observed along with lower Mr species of
40,000 and 33,000 (29). In the present study, molecular
characterization of several cloned CTRs indicated broad bands of
125I-sCT cross-linked protein, suggesting that the extent
of glycosylation is heterogeneous. Although the Mr values
of the rat and human CTRs described here are similar to previous
results, the Mr of the pig CTR (57,000) is much smaller
than previously described CTRs. It should also be noted, however, that
migration of glycoproteins on SDS-PAGE is affected by the concentration
of cross-linker in the gel (30) and as such, the absolute contribution
of carbohydrate to the Mr may differ from these estimates.
Indeed, differences in the percentage of cross-linker in SDS-PAGE
analysis of CTRs may account for some of the variations observed in
Mrs within the literature.
Analysis of the predicted amino acid sequences of the cloned receptors
reveals four conserved, potential N-linked glycosylation sites, with
the pig CTR lacking the first of these sites (Fig. 1). Deglycosylation
with Endo F after reduction and denaturation (which cleaves N-linked
oligosaccharides at the protein backbone), produced a marked reduction
in the Mrs of all CTRs, providing the first direct evidence
for the glycoprotein nature of the CTR. With the exception of the
cloned pig CTR, all receptors exhibited Mrs consistent with
the predicted mol wts of native receptor proteins (Table 1
vs. Table 2), suggesting that all carbohydrate residues were
removed when treated under denaturing conditions. Further evidence for
this comes from the observation that the cell-free translation product
of the rat C1a CTR migrated with almost identical electrophoretic
mobility (48,000) compared with the transfected, Endo F-treated form
(47,000). This is consistent with other G-protein-coupled receptors
where there is little evidence for O-linked glycosylation (14, 16, 31).
The extent of glycosylation fell into two classes. The rat and human
receptors expressed in HEK-293 cells had a carbohydrate component of
30 kDa (Table 2), whereas the human CTRs expressed in BHK cells and
the pig CTRs had a carbohydrate component of 20 kDa (Table 2). The
difference in the Mr of the human CTR expressed
alternatively in HEK-293 and BHK cells is indicative of cell-dependent
processing of the receptor. Both receptors undergo similar shifts in
electrophoretic mobility following deglycosylation under nondenaturing
conditions (10 kDa; Table 2). Thus the difference in Mrs
between receptors is likely to arise from differences in glycosylation
at sites that are inaccessible to Endo F under nondenaturing
conditions.
Endo F treatment, either under nondenaturing conditions or following
solubilization and reduction, resulted in only partial deglycosylation
of the rat and human CTRs (10 kDa) and no change in the pig CTRs.
Full deglycosylation required denaturation of the receptor, suggesting
that the enzyme-resistant carbohydrate is conformationally protected
and consequently may be oriented towards the interior of the
receptor.
The exact role of N-linked carbohydrates in the function of G-protein-
coupled receptors is varied. In the ß-adrenergic and histamine
H2 receptors, prevention of glycosylation by tunicamycin
treatment or mutation of glycosylation sites has no effect on either
cell surface expression of the receptor or receptor function (31, 32).
In the FSH receptor, glycosylation of at least one site is essential
for correct folding and cell surface expression of the receptor (14).
However, in the mature receptor, the carbohydrate could be
enzymatically removed without loss of high affinity FSH binding,
indicating that it did not play a direct role in ligand-receptor
binding (14). In contrast, enzymatic removal of components of N-linked
carbohydrate (particularly sialic acid residues) from rat brain
somatostatin receptors or AT-20 cells, reduced high affinity agonist
binding, indicating a potential role for the carbohydrate in agonist
recognition (16). Similarly, alteration to the terminal glycosylation
of the VIP receptor decreases VIP binding affinity without affecting
cell surface receptor number (17), although prevention of
N-glycosylation prevents presentation of the receptor at the cell
surface (17). However, it is unclear whether changes in the relative
affinity of the VIP or somatostatin receptors for other ligands is
changed. In the current study, removal of 10 kDa of carbohydrate
under nondenaturing conditions had no effect on either binding affinity
or specificity for both the rat C1a and human CTRI1-ve
receptors, indicating that this component of the carbohydrate plays no
role in ligand recognition. However, we were unable to assess the role
of the enzyme-resistant carbohydrate in receptor binding affinity and
specificity.
Mutational analysis of the role of potential N-linked glycosylation sites in the VIP receptor, a member of the CT-subfamily of G-protein- coupled receptors, demonstrated that glycosylation at only one site (either N58 or N69) was required for cell surface expression of the receptor and maintenance of high affinity VIP binding (33). Like the FSH receptor, prevention of glycosylation by mutation (33) or tunicamycin treatment (17) prevented cell surface expression of the receptor. Similarly, early studies in human breast cancer-derived T47D cells showed that treatment with tunicamycin abolished binding to CTR (34), indicating that the CTR also requires glycosylation of at least one site to be expressed at the cell surface.
In the human VIP (33) and GRF receptors (27), 10 kDa of carbohydrate
is associated with each glycosylation site. Our current data is
consistent with a similar degree of carbohydrate modification assuming
that two to three of the potential glycosylation sites are actually
glycosylated in the various receptors studied.
In contrast to the rat and human CTR isoforms, the Mr of
the fully deglycosylated cloned expressed pig CTR was substantially
below the mol wt predicted from its cDNA sequence (52,000, Table 1).
Our gel mobility for this fully deglycosylated receptor implies a mass
of 35,000 after adjusting for the coupled 125I-sCT (3557
Da) and cross-linking reagent (572 Da). In addition, the Mr
of the cloned expressed pig CTR was smaller than that of the
hypothalamic pig CTR. This size heterogeneity does not seem to be due
to differences in the amount of N-linked carbohydrate, because both are
deglycosylated by a similar extent following Endo F digestion.
The pig CTR exhibits an unusual phenotype in that it has a relatively high affinity for amylin and low affinity for rat/human CT when expressed in either HEK-293 or COS cells (19). Furthermore, recent work has suggested that the phenotype of the cloned pig CTR may alter depending on the host cell used for heterologous expression (35). Given the Mr differences between the hypothalamic and the cloned (LLC-PK1 derived) receptors, we investigated whether there was a concomitant change in receptor phenotype. However, no significant differences in relative binding specificity were found between the hypothalamic and cloned pig CTR preparations.
Although the data from the human CTR expressed in BHK cells suggested
the potential for proteolytic cleavage of the C-terminus of CTRs, the
further increase in electrophoretic mobility of C-terminally truncated
mutants of the pig CTR indicated that the C-terminus of the receptor
was intact. Thus the lower Mr of the cloned receptor is
likely due to removal (or lack) of N-terminal amino acid residues. Of
note, despite the absence of 10 kDa of the receptor N-terminus, the
cloned pig CTR exhibited similar ligand binding and specificity to the
hypothalamic receptor, which had an intact N-terminus. This implies
that much of the receptor N-terminus is not required for high affinity
ligand binding and specificity. In support of this, characterization of
a novel splice variant of the human CTR, encompassing functional
deletion of the N-terminal 47 amino acid residues, displayed similar
properties to the nondeleted form of this receptor (36). The
mechanistic basis of the low Mr pig CTR was further
investigated through in vitro transcription and translation
of the receptor cDNA to determine whether sequence-specific elements in
the pig CTR cDNA directed translation from an AUG downstream of the
predicted start site. In this system, the pig CTR generated a protein
of 50 kDa consistent with translation from the first-in frame AUG,
suggesting that a further cell-specific posttranslational processing of
the receptor occurs.
In conclusion, we have provided direct evidence for the glycoprotein nature of the CTR, and that the extent and pattern of glycosylation is dependent on both the host cell and the receptor under investigation. The sites of glycosylation differ in their susceptibility to enzymatic removal of the carbohydrate with the carbohydrate accessible under nondenaturing conditions unlikely to be involved in ligand recognition. We are currently performing mutational analysis of the potential N-linked glycosylation sites to further dissect the role of the carbohydrate in the function of the receptor, particularly that which is inaccessible under nondenaturing conditions.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Biology, Wollongong University,
Wollongong 2522, N.S.W., Australia.
3 Research Fellow of the Australian Research Council.
Received July 15, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 F. Naro, M. Perez, S. Migliaccio, D. L. Galson, P. Orcel, A. Teti, and S. R. Goldring Phospholipase D- and Protein Kinase C Isoenzyme-Dependent Signal Transduction Pathways Activated by the Calcitonin Receptor Endocrinology, July 1, 1998; 139(7): 3241 - 3248. [Abstract] [Full Text] [PDF]
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 L. J. Suva, M. S. Flannery, M. P. Caulfield, D. M. Findlay, H. Jüppner, S. R. Goldring, M. Rosenblatt, and M. Chorev Design, Synthesis and Utility of Novel Benzophenone-Containing Calcitonin Analogs for Photoaffinity Labeling the Calcitonin Receptor J. Pharmacol. Exp. Ther., November 1, 1997; 283(2): 876 - 884. [Abstract] [Full Text]
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K. J. Perry, M. Quiza, D. E. Myers, M. Morfis, G. Christopoulos, and P. M. Sexton Characterization of Amylin and Calcitonin Receptor Binding in the Mouse {alpha}-Thyroid-Stimulating Hormone Thyrotroph Cell Line Endocrinology, August 1, 1997; 138(8): 3486 - 3496. [Abstract] [Full Text] [PDF]
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), rCT (
), or rat amylin (
).
Points are mean of duplicate determinations in a representative
experiment (n = 3).
