Departments of
Receptor Biochemistry (G.C., C.W., P.I., T.R.,
T.K.),
Molecular Biology (W.-J.C., S.A., J.W., S.K.), and
Medicinal
Chemistry (J.C.), Glaxo Wellcome, Research Triangle Park, North
Carolina 27709, and
Amylin Pharmaceuticals, San Diego, California 92121 (K.B.)
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Introduction |
Amylin
is a peptide hormone that is synthesized and secreted from pancreatic
cells. There is evidence that an increase in the cosecretion ratio
of amylin to insulin exacerbates insulin tolerance, and in general,
there are data to suggest that this peptide may be important in the
pathology of type II diabetes (1-5). High affinity binding for
125I-rAmylin has been reported in the rat nucleus
accumbens (6-8), thus defining an operational and experimentally
accessible amylin receptor. Similar high affinity binding of both
125I-rAmylin and an sCAL antagonist analogue
radiolabel 125I-AC512 has been described (9) in human MCF-7
cells. These data raise the possibility that these cells contain a
human amylin receptor, and this information would be of value in the
study of the role of amylin in human type II diabetes.
We describe the expression cloning of the
125I-AC512 binding site from an MCF-7 cell cDNA
library and the subsequent identification of the gene products as
previously classified CTRs (10). The receptor pharmacology of these
gene products in various host cells is described, as are data to
suggest a relationship between the hCTR and the responses of human
systems to amylin.
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Materials and Methods |
Cell culture.
MCF-7 human breast adenocarcinoma cells from
pleural effusion (HTB 22; American Type Culture Collection, Rockville,
MD) were cultured in Eagle's minimal essential medium with
nonessential amino acids, sodium pyruvate [1 mM (90%)],
and fetal bovine serum (10%). Cells were grown as a monolayer, fed
fresh media every 3 days, and split 1:2 weekly. Transfected COS-7 or
HEK 293 cells were also cultured in Eagle's minimal essential medium
with nonessential amino acids, sodium pyruvate [1 mM
(90%)], and fetal bovine serum (10%). The cells were grown as a
monolayer, fed fresh media every 3 days, and split 1:2 weekly.
Membrane preparation.
The nucleus accumbens was dissected
from the brains of Sprague-Dawley rats (200-250 g) and homogenized
(three 15-second bursts) in ice-cold HEPES buffer (20 mM
HEPES, pH adjusted to 7.4 with NaOH at 23°). The homogenate was
centrifuged at 48,000 × g for 15 min and washed twice
by resuspension in fresh buffer. The membrane pellet from the third
centrifugation was resuspended in fresh buffer with 0.2 mM
PMSF, aliquoted, and stored at 
70°. The MCF-7 cells were harvested
at confluency by manual scraping of the tissue culture flasks. The
cells then were pelleted by centrifugation at 2000 rpm for 15 min and
homogenized as described above.
Cultured cells were harvested at confluency by manual scraping of the
tissue culture flasks, then pelleted by centrifugation at 2000 rpm for
15 min, and homogenized (three 15-second bursts) in ice-cold HEPES
buffer (20 mM HEPES, pH adjusted to 7.4 with NaOH at
23°). The homogenate was centrifuged at 48,000 × g
for 15 min and washed twice through resuspension in fresh buffer. The
membrane pellet from the third centrifugation was resuspended in fresh
buffer with 0.2 mM PMSF, aliquoted, and stored at 70°.
Synthesis of AC512.
To a solution of the peptide
(Ac)LGKLSQELHRLQTY-PRTNTGSNTY(NH2) (128.5 mg,
80% peptide content, 36.4 µmol; synthesized using normal solid-phase
techniques from Fmoc-protected amino acids and a Rink resin) in 25 mM aqueous NaHCO3 (30 ml, pH ~ 8) maintained at 0° we added dropwise a solution of 14.2 mg of cold
Bolton-Hunter reagent [3-(3-iodo-4-hydroxyphenyl)-propanoic acid
N-hydroxysuccinimide ester] in 8 ml of acetonitrile. The
resulting solution was stirred for 2 hr at 0°. An additional 10 mg of
the Bolton-Hunter reagent in 5 ml of acetonitrile was added at this
point, and the solution was stirred for 1 additional hr at 0°. The
reaction was quenched by the addition of sufficient 10% aqueous
trifluoroacetic acid to give pH 1.5 and warmed to room temperature.
This solution was filtered to remove a small amount of precipitated
material. The filtrate was injected onto a Waters (Milford, MA)
Delta-Prep HPLC equipped with a radial compression C-18 cartridge
(conditions: flow rate, 100 ml/min; solvent A = CH3CN, solvent B = 0.1% aqueous trifluoroacetic acid; initial conditions were 73% B; 4 min after the
injection a linear gradient was begun, decreasing the percentage of B
to 53% over 40 min). The desired product, AC512, eluted at 22.5 min.
Lyophilization gave 89.2 mg of AC512 as a white powder. Peptide content
was found to be 64.5%. High resolution mass spectrum (electrospray):
expected monoisotopic MH+ 3092.4091, found: 3092.4423. The most likely
spot for derivatization of the starting peptide is on the lysine, and
AC512 is assigned the structure
(Ac)LG(KBH)LSQELHRLQTYPRTNTGSNTY(NH2) with (KBH)
being lysine labeled on the 
-amino group with the Bolton-Hunter
reagent. The 125I-labeled material was
synthesized at Amersham (Arlington Heights, IL) using the same starting
peptide and radioactive Bolton-Hunter reagent. The Amersham
125I-labeled peptide coeluted with the
nonradioactive peptide synthesized as described above.
Receptor binding.
Membranes were incubated with
125I-rAmylin (Bolton Hunter labeled at the
amino-terminal lysine; Amersham) or 125I-AC512
(Bolton Hunter-labeled [Arg18,Asn30,Tyr32]9-32 sCAL, 2000 Ci/mmol;
Amersham) in 20 mM HEPES buffer, containing 0.5 mg/ml bacitracin, 0.5 mg/ml bovine serum albumin, and 0.2 mM PMSF
(all from Sigma Chemical, St. Louis, MO) plus test ligand or ligands, for 60 min at 23° (samples mixed on a Titer Plate Shaker; Lab-Line Instruments). Nonspecific binding was defined as the radioactivity remaining in the presence of 100 nM sCAL. Incubations were
carried out in triplicate tubes and were started by the addition of
membrane. Binding was terminated by filtration through glass-fiber
filters (presoaked 30 min in 0.5% polyethyleneimine), using the
Skatron semiautomatic cell harvester. Filters were placed in Sarsted
68.752 51 × 12 mm polypropylene tubes and counted for 1 min in a
-counter.
Binding data were analyzed with the Glaxo Wellcome statistical fitting
package RADLIG (Glaxo Wellcome Scientific Computing) to simultaneous
equations describing total binding (saturable binding according to a
logistic equation plus a linear nonspecific binding curve) and a linear
nonspecific binding curve. Statistical analyses were used to determine
whether data could be fit to a single or double population of binding
sites. Saturation analysis yielded a nonlinear least-squares fit to the
logistic equation with a half-maximal fitting parameter (the
equilibrium dissociation constant of the ligand/receptor complex under
ideal conditions, denoted Kd) and a
maximal asymptote (denoted Bmax, providing
an estimate of the maximal number of binding sites in fmol/mg of protein). Displacement analysis (radioligand concentrations = 0.3 × Kd; incubations for 90 min) yielded concentrations of nonradioactive ligand that
half-maximally displaced a given concentration of radioligand (denoted
IC50). This value was used to calculate an estimate of the equilibrium dissociation constant of the nonradioactive ligand/receptor complex (denoted Ki)
by correction for the amount of radioactive ligand and the
Kd value (11). Complex displacement curves were fit to a two-population model, yielding two apparent affinities and the relative quantities of two apparent sites (or receptor states).
Molecular biology.
Standard molecular biology techniques
were used (12). Poly(A)+ RNA was isolated using a
FastTrack RNA isolation kit (InVitrogen, San Diego, CA). Both strands
of the CTR cDNA were sequenced with an ABI394 automatic sequencer with
use of the Analysis (Applied Biosystems, Foster City, CA) and Assembly
LIGN (Kodak IBI, New Haven, CT) software programs.
Construction of cDNA library.
Five micrograms of
poly(A)+ RNA, isolated from MCF-7 cells, was used
to construct a size-selected cDNA library according to the
manufacturer's protocol (InVitrogen). Double-stranded,
oligo(dT)-primed cDNA was synthesized and ligated to
NotI/EcoRI adaptors. Fragments of cDNA of >1.6
kb were isolated and subsequently ligated into the EcoRI
site of expression vector pMT4 (13). Aliquots of the library were
titered by electroporation into Top10 competent cells (Stratagene, La
Jolla, CA). Pools of colonies, representing 
1000 independent
cDNAs/pool, were scraped from plates and grown for 4-6 hr in 25 ml of
LB-ampicillin. Plasmid DNA from each pool was isolated using the Wizard
Midiprep DNA purification system (Promega, Madison, WI).
Expression cloning.
Pools of cDNA were transfected into
COS-7 cells and analyzed for their ability to bind
125I-sCAL or 125I-AC512 in
transient transfection assays. Initial experiments were done with
125I-sCAL; however, in view of the nearly
irreversible kinetics of this radioligand, later experiments were done
with 125I-AC512. On day 0, 3-4 × 105 COS-7 cells/well were plated onto six-well
dishes. On day 1, cells were transfected with 1 µg of DNA from each
pool per well using lipofectamine (GIBCO BRL, Gaithersburg, MD)
according to the manufacturer's instructions. Cells were assayed 48 hr
after transfection by binding analysis (see below). After screening 0.6 × 106 clones, four pools that bound
125I-ligand were identified. One pool was
progressively subdivided into smaller pools until a single positive
clone was obtained.
Generation of stable cell lines.
On day 0, HEK 293 cells
were plated at a concentration of 106
cells/100-mm dish. On day 1, cells were cotransfected with clone 77/pMT4 or clone 134/pMTR with pRSV/neo at a 10:1 ratio, respectively, according to the calcium phosphate method (Promega). On day 3, transfected cells were selected using G418-supplemented media at a
concentration of 600 µg/ml. After a 2-week selection period, several
colonies from each transfection were selected and expanded. Stable
lines were checked for expression by binding of
125I-sCAL or 125I-AC512.
Baculovirus Ti ni cells.
The
BglII/NotI fragment of CTR cDNA was subcloned
into pFASTBACI vector (GIBCO BRL). Recombinant baculovirus was
generated according to Life Technologies Bac-To-Bac Baculovirus
Expression System Manual (14) efficient generation of infectious
recombinant baculoviruses by site-specific transposon-mediated
Expression System Manual. Spodoptera frugiperda
(Sf9) cells (American Type Culture Collection) were used for
transfection, virus amplification, and titering and were grown in
supplemented Grace's insect culture medium (GIBCO BRL) with 10% fetal
bovine serum (Hyclone Laboratories, Logan, UT), 1% pluronic F-68
(GIBCO BRL), and 50 mg/ml gentamycin (GIBCO BRL). Ti ni
cells (gift from JRH Biosciences, Lenexa, KS) were used for recombinant
protein generation and were grown in Ex cell 405 insect medium (JRH
Biosciences) with 50 mg/ml gentamycin. Using the initial transfection
mix harvested 48 hr after infection, recombinant viruses were amplified
in Sf9 cells and titered. Ti ni cells at 1.2 × 106 cells/ml were infected at a multiplicity of
infection of 2 plaque-forming units/cell and were harvested 48 hr after
infection. Cells pellets were washed once with phosphate-buffered
saline and then frozen until assayed.
Microphysiometry.
This technique is based on the principle
that the metabolism of cells is tightly linked to hydrogen ion output.
A thin disk of cells is cultured over a pH detector and perfused with
medium. Although perfusion takes place, the pH registered by the
detector is constant. At regular intervals, the perfusion is stopped
and the hydrogen ion allowed to accumulate in the chamber. The
resulting decrease in pH with time is measured as a rate; this rate is
proportional to the metabolic state of the cell. The overall cellular
metabolism is measured as a succession of rates of secretion of
hydrogen ion.
At 16 hr before the experiment, cells were seeded (300,000 cells/chamber) at 75-85% confluency in microphysiometer capsule cups.
Capsules were then kept in a CO2 incubator at
37°. For experimental procedures, the microphysiometer was primed
with low buffer media (modified RPMI 1640 medium; Molecular Devices,
Menlo Park, CA) for 10 min, sensor chambers were put in place, and cell
capsules were placed in the sensor chambers. After calibration of the
microphysiometer, cells were allowed to equilibrate in a constant flow
of media for 30-60 min to attain a steady base-line of hydrogen ion
output. Perfusion was then changed to media containing the test drugs and effects recorded by computer .
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Results |
Rat nucleus accumbens binding.
High affinity reversible
binding was observed with the radiolabel sCAL analogue
125I-AC512 and
125I-rAmylin. Binding reached steady state by
60-120 min and was displaceable with nonradioactive sCAL and amylin.
125I-AC512 showed considerably less filter
binding and nonspecific binding than
125I-rAmylin. Over a range of concentrations,
125I-AC512 yielded much higher specific binding
in the rat nucleus accumbens than 125I-rAmylin.
In addition to these improved binding characteristics and in contrast
to 125I-rAmylin, 125I-AC512
is an antagonist with no observable agonist activity in amylin
functional tissue systems, thereby reducing the possible complication
of binding effects by G protein coupling.
125I-AC512 bound with high affinity to membranes
prepared from rat nucleus accumbens in a saturable manner (see Table
1
for saturation binding data). Bound 125I-rAmylin
could be displaced with agonists and antagonists. Table 2 shows the chemical structures of the
antagonists (AC512, AC66, AC413, hCGRP8-37) and
agonists (rAmylin; human, rat, human, and eel CAL; and rCGRP). The
equilibrium dissociation constants of the nonradioactive displacing
ligand/receptor complex (Ki) for the
displacement of 125I-amylin are given as negative
log values (pKi) (see Table
3). In general, all displacement curves were monophasic with Hill coefficients not significantly different from unity.
Bound 125I-AC512 also could be displaced with
nonradioactive ligands. The data describing the displacement by these
ligands are given in Table
4. In
contrast to the simple curves obtained with the antagonists,
displacement of 125I-AC512 with agonists produced
complex biphasic curves. The data could be fit with a two-population
model; the apparent affinities for the two populations and their
relative abundance are given in Table 4. As seen from these data, the
affinity of the agonists varied with the radioligand used. When
125I-rAmylin was displaced, the agonists produced
monophasic curves with high affinities. In contrast, during
displacement of 125I-AC512, biphasic curves with
two apparent affinities were obtained.
Human MCF-7 cells.
The human breast carcinoma cell line MCF-7
binds 125I-rAmylin with high affinity; this was
confirmed in the current study with membrane from MCF-7 cells. The
quantitative data describing this binding activity are given in Table
5. 125I-rAmylin bound with affinity similar to
that found in the rat nucleus accumbens, but the estimate for the
maximal number of 125I-rAmylin binding sites in
MCF7-7 cells was considerably larger (155.2 fmol/mg of protein; 95%
CI, 64-246 fmol/mg of protein). As was seen in the rat nucleus
accumbens, 125I-AC512 bound with high affinity
(77.6 pM; 95% CI, 50-123 pM) and a greater
Bmax value (337 fmol/mg of protein; 95%
CI, 180-493 fmol/mg of protein) than that found for
125I-rAmylin.
A range of peptide antagonists and agonists displaced
125I-rAmylin from the binding sites, all with
monophasic displacement curves with Hill coefficients not significantly
different from unity (Fig. 1, A and B). Table 6
shows pKi estimates from displacement studies. The affinity of AC512, AC66, and AC413 was comparable to that
found for the rat nucleus accumbens. Of note was the 30-fold loss in
potency in the MCF-7 cell over the rat nucleus accumbens for
hCGRP8-37.
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Fig. 1.
Displacement of bound 125I-AC512
with nonradioactive antagonist (A and B) and agonist (C and D) ligands
in MCF-7 cells. [Bound], bound radioactivity.
Log[Amylin], logarithms of molar concentrations of
nonradioactive ligand. Displacement by AC413 (A), AC66 (B), rAmylin
(C), and hCAL (D).
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As with rat nucleus accumbens, differences in potencies of displacing
ligands were observed with the displacement of bound 125I-AC512. Although monophasic single
displacement curves were obtained for the antagonists, complex
displacement curves were obtained for agonists (Fig. 1, C
and D). These curves yielded two apparent affinities and two
apparent binding site populations (Table 7).
The pKi values in membranes from
MCF-7 cells for agonists differed for displacement of
125I-rAmylin and
125I-AC512, as observed in the rat nucleus
accumbens. A composite experiment of the differences in the
displacement curves with rAmylin against
125I-rAmylin and 125I-AC512
is shown in Fig. 2. The
levels of initial radioligand binding were comparable, but the location
parameters for the displacement curves differed by a factor of nearly
100.
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Fig. 2.
Comparison of displacement of
125I-rAmylin ( ) and 125I-AC512 ( ) with
nonradioactive rAmylin in MCF-7 membranes. [Bound],
bound radioactivity. Log[Amylin], logarithms of molar
concentrations of nonradioactive ligand.
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Expression cloning.
Transiently transfected COS-7 cells
cultured in six-well plates were exposed to
125I-sCAL or 125I-AC512
(20,000 cpm/well). Both radiolabels were used in separate experiments to maximize the possibility of obtaining a specific amylin-binding protein. A total of 171 pools screened yielded 4 pools
exhibiting 125I-sCAL and
125I-AC512 binding. From positive pool 77, an
additional 20 pools of 100 clones each were prepared, and 2 subpools
were found to be positive on further assay. The positive subpool was
subfractionated until a single clone (clone 77) was isolated. A search
of the sequence database revealed that the DNA and the deduced amino acid sequence for clone 77 were identical to those for hCTR1 (15). The
only difference between clone 77 and the published sequences was a
78-bp segment missing at the 5
untranslated region of clone 77. Insertion of the 78-bp segment created an in-frame ATG by the addition
of 26 extra amino acids at the amino terminus of the CTR.
Using the polymerase chain reaction with CTR-specific primers, the
three remaining pools were examined and determined to contain the CTR
cDNA. Clone 77 cDNA then was used to screen the three positive pools by
colony hybridization to isolate additional clones. Sequences of these
three clones (clones 40, 134, and 167) revealed different lengths for
the 5- and 3-untranslated regions but with no apparent difference in
the coding region. Deduced protein sequences were identical to those of
hCTR2 isolated from T47D cells (10). Clone 134 was the longest cDNA
among the three and was chosen for further characterization. Comparison
of clones 77 and 134 revealed that clone 77 contained a 16-amino acid
insert in the first intracellular loop that was absent in clone 134. The differences between the two cDNAs are shown in Fig.
3.
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Fig. 3.
cDNA for clones 77 and 134 shows the similarity to
the CTR cloned by Gorn et al.. (15) from BIN-67 cells.
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Receptor binding: clone 77 (hCTR1).
hCTR1 transfected into
COS-7 cells produced saturable binding with
125I-AC512 and 125I-sCAL.
For 125I-AC512, the equilibrium dissociation
constant (Kd) was 71 pM (95% CI, 30-158 pM)
with a Bmax estimate of 597 ± 66 fmol/mg of protein (95% CI, 470-722 fmol/mg of protein; four
experiments). This maximal number of binding sites was confirmed with
saturation binding with 125I-sCAL (680 fmol/mg of
protein; 95% CI, 540-816; three experiments). The
Kd value for
125I-sCAL was 2.8 pM (95%
CI, 0.7-10.5 pM). Displacement experiments indicated that 125I-AC512 could be displaced by
sCAL (pKi = 11.16), AC512
(pKi = 10.4), and
hCGRP8-37 (pKi = 10.4). In view of these data, clone 77 was transfected into HEK 293 cells, and a stable cell line expressing hCTR1 was made.
As shown in Table 8, hCTR1 expressed in HEK 293 cells furnished
membranes that saturably bound 125I-AC512 with a
Kd value of 200 pM (95% CI, 125-316 pM)
and a Bmax value of 1493 ± 276 fmol/mg of protein (95% CI, 970-2017). These membranes also saturably
bound 125I-sCAL with a
Kd value of 3.5 pM (95% CI, 2.7-4.6 pM).
The Bmax value of 1260 ± 360 fmol/mg
of protein (95% CI, 576-1944) was not significantly different from
that found for the binding of 125I-AC512. No
appreciable binding of 125I-hCAL or
125I-rAmylin was observed. The potency of
nonradioactive antagonists and agonists in displacing
125I-AC512 is shown in Table 9. No high affinity
binding was observed with agonists in these membranes.
Receptor binding clone 134 (hCTR2): COS-7 cell membranes.
125I-AC512 bound with high affinity to membranes
prepared from COS-7 cells transiently transfected with hCTR2 cDNA. The
equilibrium dissociation constant of the AC512/receptor complex was 245 pM (95% CI, 165-370 pM; see Table
10). The maximal number of binding sites was 4423 fmol/mg of protein (95% CI, 3603-5242). Binding of
somewhat higher affinity was observed with
125I-sCAL. The
Kd value was 28.2 pM (95% CI, 7-110 pM).
The Bmax value was 5225 fmol/mg of protein
(95% CI, 4235-6214), a value not significantly different from that
found for 125I-AC512 (Table 10).
The difference between hCTR1 and hCTR2 was that the latter, when
transiently transfected into COS-7 cells, also bound
125I-hCAL in a saturable manner
(Kd = 417 pM;
95% CI, 269-655 pM). In contrast to the data
with 125I-AC512 and
125I-sCAL, the Bmax
value for 125I-hCAL binding was significantly
lower (3025 fmol/mg of protein; 95% CI, 1898-4150).
Bound 125I-AC512 and
125I-hCAL could be displaced with agonists and
antagonists for CTRs. Although the
pKi estimates for the displacement of
both radioligands with antagonists were uniform (Table 11), hCAL had a
higher estimated affinity for the displacement of
125I-hCAL (as opposed to
125I-AC512). Fig. 4
shows the displacement of 125I-AC512 and
125I-hCAL by hCAL. It can be seen from this
figure that the curve for displacement of
125I-hCAL is monophasic, whereas that for
displacement of 125I-AC512 is biphasic and
shifted to the right.
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Fig. 4.
Displacement of 125I-AC512 () and
125I-hCAL ( ) by hCAL in membranes from COS cells. A,
[BO], bound
radioactivity. Log[hCal], logarithms of molar
concentrations of nonradioactive ligand. B, Data in A with ordinate
values recalculated as a percentage of the initial BO
binding of radioligand.
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Membranes from COS-7 cells transiently transfected with cDNA for hCTR2
also saturably bound 125I-rAmylin
(Kd = 275 pM;
95% CI, 30-2000 pM). The
Bmax value for 125I-rAmylin binding was much lower (65 fmol/mg
of protein; 95% CI, 36-94) than that found for
125I-AC512, 125I-hCAL, or
125I-sCAL. A limited displacement study in COS-7
cell membranes indicated that the saturable
125I-rAmylin binding could be displaced by CAL
and amylin antagonists and agonists (Table 11). Fig.
5 shows the effects of amylin
displacement of 125I-rAmylin and
125I-AC512. The low level of
125I-rAmylin binding made rigorous quantification
of this effect in COS-7 cells difficult.
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Fig. 5.
Displacement of 125I-AC512 () and
125I-rAmylin () by rAmylin in membranes from COS cells.
[Bo], bound
radioactivity. Log[Amylin], logarithms of molar
concentrations of nonradioactive ligand. B, Data in A with ordinate
values recalculated as a percentage of the initial BO
binding of radioligand.
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Receptor binding: HEK 293 cell membranes.
Saturable binding of
the radioligands was observed in membranes from HEK 293 cells stably
transfected with cDNA for hCTR2. The equilibrium dissociation constant
of the 125I-AC512/receptor complex was 209 pM (95% CI, 133-331 pM; see Table 12). The maximal number of binding
sites (Bmax) was considerably larger than
that found for COS-7 cell membranes (30,047 fmol/mg of protein; 95%
CI, 24,130-35,964). Similar binding was observed with
125I-sCAL (Kd = 195 pM; 95% CI, 56-692
pM) with a Bmax value
not significantly different from that found with
125I-AC512 (Bmax = 34,400 fmol/mg of protein; 95% CI, 24,063-44,737). Saturable
125I-hCAL binding also was observed in these
membranes (Kd = 219 pM; 95% CI, 151-316 pM).
As was seen in COS-7 cell membranes, the
Bmax value for
125I-hCAL binding was significantly lower (6511 fmol/mg of protein; 95% CI, 3692-9329) than that seen with
125I-AC512 and 125I-sCAL.
Membranes from HEK 293 cells also saturably bound
125I-rAmylin
(Kd = 49 pM;
95% CI, 28-89 pM). The
Bmax value for
125I-rAmylin binding was again very much lower
(2182 fmol/mg of protein; 95% CI, 1284-3080) than that found for
125I-AC512 and 125I-sCAL
and, notably, also that found for 125I-hCAL. The
different Bmax values were studied further
in HEK 293 cell membranes. Specifically, the saturation binding data for 125I-hCAL and
125I-rAmylin were fit to a two-site (or
two-state) model. A prerequisite to this procedure was a reliable
estimate of the total number of binding sites. The saturation binding
for 125I-sCAL was used since it clearly
demonstrated a maximal asymptote for the saturation binding curve (Fig.
6). It should be noted that although the
dissociation kinetics of AC512 and hCAL were reversible,
125I-sCAL gave essentially irreversible binding.
Therefore, displacement of 125I-sCAL was an
unsuitable method for receptor and/or ligand characterization. Specifically, the potency of displacing ligands and the magnitude of
nonspecific binding of the pseudoirreversible ligand
125I-sCAL was dependent on the binding protocol.
Accordingly, if nonradioactive sCAL was added 30 min before the
addition of 125I-sCAL, the observed
IC50 was significantly lower than if the two
ligands were added concomitantly to start the reaction. If the
radioligand was added 30 min before the nonradioactive ligand, far less
displacement was observed. Similar effects were observed with other
nonradioactive ligands (i.e., hCAL). For these reasons, the use of
125I-sCAL for receptor characterization was not
pursued in these studies. However, the quantification of the maximal
number of receptors with 125I-sCAL binding is
still useful and, this was used to model the saturation binding curves
for the agonist radioligands.
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Fig. 6.
Saturation binding curve for 125I-sCAL
to membranes from HEK 293 stably transfected with hCTR2 (clone 134).
[Bound], Specific binding of 125I-sCAL.
Log (125I-cCal),
logarithms of free molar concentrations of 125I-sCAL.
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Fig. 7A (inset) shows the
saturation curve for 125I-hCAL in membranes from
stably transfected HEK 293 cells. The linear abscissal scale was
transformed to a logarithmic scale and a fit to a model containing two
affinity states for the maximal number of
125I-sCAL binding sites
(125I-sCAL saturation curve [dotted line]). For
this particular experiment, the 125I-hCAL data
indicated a 24.8% high affinity state. A similar procedure for the
saturable binding for 125I-rAmylin (Fig. 7B)
showed a significantly smaller number of high affinity sites (8.8%
high affinity sites).
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Fig. 7.
Saturation binding curves for
125I-hCAL (A) and 125I-rAmylin (B) for hCTR2
(clone 134) expressed in HEK 293 cells. A, Inset, total (), nonspecific ( ), and specific ( ) binding curves
(inset is represented on larger axes as the
shaded area). Log
(125I-hCal), logarithms
of molar concentrations of 125I-hCAL. Dotted
line, saturation binding curve for 125I-sCAL (see
Fig. 6). The maximal asymptote of this latter curve was used to fit the
binding data for 125I-hCAL to a two-site model. Under these
conditions, the data for the specific binding of 125I-hCAL
could be fit to a two-site model in which 24.8% of the sites had a
high affinity for 125I-hCAL. B, Same as for A with
125I-rAmylin as the radiolabel. The two-site model could be
fit for an 8.8% population of high affinity sites for
125I-rAmylin.
|
|
Bound radioligand could be displaced with agonists and antagonists for
CTRs. The equilibrium dissociation constants of the nonradioactive
displacing ligand/receptor complex (denoted as the
Ki) for the displacement of the
radioligands are given as negative log values
(pKi) in Table
13. The
pKi estimates for the displacement of
all three radioligands with antagonists was uniform (Table 13);
agonists had a higher estimated affinity for the displacement of
125I-hCAL and 125I-rAmylin
(as opposed to 125I-AC512).
As was observed in COS-7 membranes, the curve for displacement of
125I-hCAL was monophasic, whereas that for
displacement of 125I-AC512 was biphasic and
shifted to the right. In contrast, there was no significant difference
in the potency of AC512 in displacement of these two radioligands. A
similar effect, but more pronounced, was seen for amylin displacement
of 125I-rAmylin and
125I-AC512. Fig. 8
shows the difference in potency demonstrated for amylin in displacement
of these two radioligands.
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|
Fig. 8.
Displacement of 125I-AC512 () and
125I-rAmylin () by rAmylin in membranes from HEK 293 cells. A, BO, bound
radioactivity. Log [hCal], logarithms of molar
concentrations of nonradioactive ligand. B, Data recalculated as a
percentage of the initial BO binding of radioligand.
|
|
Receptor binding: baculovirus expression.
Membranes from
Ti ni cells infected with baculovirus containing cDNA for
hCTR2 demonstrated saturable binding of
125I-AC512 with a
Kd value of 575 pM (95% CI, 550-602 pM)
and a Bmax value of 8340 fmol/mg of
protein; 95% CI, 6,184-10,496). A comparable but somewhat larger
number of sites was observed with 125I-sCAL
(Bmax = 10,620 fmol/mg of protein; 95% CI,
9,208-12,031; Kd = 59 pM; 95% CI, 38-925 pM).
In contrast, a significantly lower number of binding sites was observed
for 125I-hCAL
(Kd = 417 pM;
95% CI, 269-655 pM) with a
Bmax value of 5460 fmol/mg of protein; 95%
CI, 4,742-6,177). The data are summarized in Table
14. No significant
125I-rAmylin binding could be obtained in these
membranes. Both agonist and antagonist ligands displaced
125I-AC512 (see Table
15 for
pKi values).
Functional responses with hCTR2: microphysiometry.
In view of
the complex displacement curves seen with some of these ligands and the
submaximal saturation kinetics, it was useful to determine which of
these ligands produced cellular responses (and thus could be classified
as having efficacy with concomitant complex binding behavior). HEK 293 cells transfected with hCTR2 were tested in the cytosensor
microphysiometer for the study of functional responses. A HEK 293 cell
line expressing a high density of hCTR2
(Bmax = 28,000 fmol/mg of protein) yielded
responses with complex wave forms that changed with hCAL concentration
(Fig. 9A). Due to the rapidly declining
phase of the response, dose-response curves could not be obtained in a
cumulative manner (i.e., increases in concentration resulted in
capricious secondary responses). Interestingly, a clone with a much
lower receptor expression level (Bmax = 65 fmol/mg of protein; 95% CI, 40-96 fmol/mg of protein; Kd = 316 pM;
95% CI, 165-650 pM) provided an excellent
functional response. In these cells, responses to hCAL were sustained
(Fig. 9B) and yielded cumulative concentration-response curves (Fig. 9C). AC512 produced concentration-dependent dextral displacement of the
hCAL dose-response curve (Fig. 9D). Schild analysis with the antagonist
AC512 provided a regression with a slope not significantly different
from unity and a pKB value of 9.1 (95% CI, 9.48-8.7). This was not significantly different from the
pKi or
pKd value obtained in binding studies
(see Tables 12 and 13).
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Fig. 9.
Microphysiometry responses obtained from HEK 293 cells transfected with hCTR2. A. Response of a high receptor expression
clone (clone C134-2-23; Bmax = 34, 400 fmol/mg of protein) to 10 nM hCAL. Percent,
increase in hydrogen ion output as a percentage of the basal. B,
Response of a low receptor expression clone (clone C134-4-7;
Bmax = 67 fmol/mg of protein) to 10 nM hCAL. Percent, increase in hydrogen ion
output as a percentage of the basal. C, Cumulative dose-response curve
to hCAL. hCAL added at designated points a (10 pM), b (100 pM),
c (1 nM), d (10 nM), and e (100 nM).
Percent, increase in hydrogen ion output as a percentage of the basal. D, Dose-response curves to hCAL in the absence (, six
experiments) and presence of AC512 10 nM (, three
experiments), 100 nM (, three experiments), and 300 nM ( , three experiments). Percent,
responses as a percentage of the maximal response to 100 nM
sCAL. Log [hCal], logarithms of molar concentrations
of hCAL.
|
|
Receptor-transfected HEK 293 cells responded to a variety of agonists
for CTRs and amylin receptors. Fig. 10A
shows concentration-response curves to eel, porcine, and rat CAL; hCAL;
rCGRP; and rAmylin. No responses to the antagonists AC66, AC413,
hCGRP8-37, and AC512 were observed (data not
shown). These data are in agreement with those obtained with binding,
which showed that the observed affinity of the antagonists did not vary
when displacing either the radioligand agonists
(125I-hCAL or 125I-rAmylin)
or antagonist (125I-AC512), whereas that of the
agonists did. The dose-response curve to rAmylin was shifted to the
right by AC512 (Fig. 10B). The resulting
pA2 value of 9.1 was not significantly
different from the pKB estimated by
Schild analysis for blockade of responses to hCAL.
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Fig. 10.
Microphysiometry responses obtained from HEK 293 cells transfected with hCTR2 to agonists and antagonists for CTRs and
amylin receptors. Max., increases in hydrogen ion output
expressed as a percentage of the maximal output produced by 100 nM sCAL. Log, logarithms of molar
concentrations of agonists. A, Responses to eel CAL (, five
experiments), hCAL (, five experiments), rat CAL (, six
experiments), porcine CAL (, five experiments), rAmylin ( , six
experiments), and rCGRP ( , six experiments). B, Blockade of amylin
dose-response curve by AC512. Responses obtained in the absence (,
three experiments) and presence (, three experiments) of AC512 (10 nM). Estimated pA2 = 9.1.
|
|
| |
Discussion |
These data confirm the results reported previously by Beaumont
et al. (6) that described the presence of a high affinity binding site for 125I-rAmylin in membranes
prepared from rat nucleus accumbens. High affinity binding for the
radiolabeled amylin receptor antagonist 125I-AC512 also was confirmed, as reported
previously by Watson et al. (9). The data for MCF-7 cell
binding raise the question of the classification of the MCF-7 site as
an operational human amylin receptor. This can be done by comparing the
binding of ligands to the gene products expressed in the recombinant
systems with the characteristics of amylin receptors in the natural
systems, namely, the rat nucleus accumbens and MCF-7 cells. There are
four unique characteristics of ligand binding to amylin receptors: (i)
the high affinity binding for both 125I-rAmylin
and 125I-AC512, (ii) the complex displacement
kinetics between nonradioactive agonist amylin and antagonist
radioligand 125I-AC512, (iii) the
characteristically high affinity of rCGRP, and (iv) the similar
affinities of the antagonists AC413, AC512, and AC66 in the rat nucleus
accumbens and MCF-7 cells. In general, the binding with
125I-AC512 and 125I-amylin
showed these characteristics and, thus, support the notion that this
site can be considered as an amylin receptor. However, the fact that
hCGRP8-37 clearly distinguishes the rat and human receptor underscores the importance of species differences between the two receptors.
The original intent of this study was to define the human high affinity
receptor for amylin in MCF-7 cells. Expression cloning from a library
prepared from the human breast carcinoma MCF-7 cell line with a
radiolabeled form of the amylin and CAL peptide antagonist AC512
yielded two hCTR isoforms, hCTR1 and hCTR2 (10, 15-17). The presence
of these receptor isoforms in this cell line has been demonstrated by
Albrandt et al. (18) through reverse transcription-polymerase chain reaction amplification from reported sequences. The hCTR1, first described in BIN 67 cells (15), saturably
bound 125I-AC512 and
125I-sCAL. It is interesting to note that the
affinity of 125I-sCAL is considerably higher in
the current membrane study than that reported for whole-cell binding
[estimated pKi = 10 (16) to 8.0 (19)]. The higher affinity reported in this study may reflect receptor
isomerization due to formation of G protein complexes, a common
difference between membrane and whole-cell binding studies; however, it
is difficult to interpret estimated affinities with this ligand because
the high affinity for the receptor also may reflect the
pseudoirreversible kinetics of onset. The unsuitability of
125I-sCAL in receptor characterization (other
than for quantification of receptor number) provided the impetus to
characterize the affinities to the agonist and antagonist ligands with
125I-AC512. The lack of high affinity
displacement by rAmylin and the lack of saturable binding for
125I-rAmylin precluded further exploration of a
possible relationship between hCTR1 and amylin in this study; in
contrast, the high affinity binding of
125I-rAmylin to expressed hCTR2 provided a basis
for further study. As a prerequisite to a discussion of the data with
hCTR2, it is useful to delineate the data in terms of those obtained
with agonists and those obtained with antagonists. Ligands with no
efficacy are very useful in the process of expression cloning procedure and subsequent identification of products. Unlike those of agonists, the observed binding affinities of antagonists do not vary because of
receptor/G protein complexation. The studies with the microphysiometer were helpful in classifying the ligands used in this study; the microphysiometry data with these ligands indicated AC512, AC66, AC413,
and hCGRP8-37 produced no visible response and,
thus, ostensibly qualified as antagonists. By implication,
125I-AC512 also was considered to be a
nonefficacious antagonist. The affinity of this radioligand from
saturation binding varied <2.5-fold among the various expression
systems, indicating that the environment of the gene product did not
appreciably affect the binding of this ligand. Similarly, the affinity
of the antagonist AC413 in displacing the radioligand antagonist
125I-AC512 was constant over the three expression
systems (COS-7, HEK 293, and Ti ni cells). However, some
variation for AC66 (Ti ni cells) and
hCGRP8-37 was observed. In general, the data suggest that the hCTR2 antagonist binding was fairly consistent in the
various expression systems used in this study.
A recurrent finding in this work was that agonists were more potent at
displacing radiolabeled agonists (125I-hCAL,
125I-rAmylin) than they were at displacing the
radiolabeled antagonist 125I-AC512. This is
consistent with the idea that the agonist radioligands select the high
affinity species in a G protein-deprived environment, whereas the
antagonist labels a random sampling of bare receptors. The concept of G
protein deprivation does not necessarily refer to a stoichiometric
deficiency in the ratio of expressed receptors to G proteins but rather
to an inability of the expressed receptors to adequately access the
existing G proteins due to constraints in the membrane architecture
(20). On displacement with nonradioactive agonist, insufficient G
protein exists for complete formation of the high affinity ternary
complex; thus, a lower affinity (agonist binding to the receptor not
complexed with G protein) is observed. This effect of high affinity
selection is made manifest in the significantly different
pKi values for agonists when
displacing agonist and antagonist radioligands. The idea that there is
a G protein insufficiency in some of these systems is supported by the
significantly lower Bmax values for
radioactive agonists versus the antagonist
125I-AC512. The data for
125I-hCAL indicate that COS-7 cells and
baculovirus-expressed Ti ni cells have an equal capability
to form the high affinity ternary complex for hCAL (68% and 65% of
the receptors, respectively), whereas HEK 293 cells are limited (only
22%). In all cases, however, the maximal number of binding sites
measured by agonist saturation binding was much less than the number of
receptors as quantified by binding of the antagonist
125I-AC512. The observance of differences in
apparent receptor densities when measured with agonist and antagonist
radioligands is known (21). However, differences in the potencies of
agonist ligands when displacing agonist and antagonist radioligands
usually produce complex biphasic displacement curves for antagonist
displacement. The production of parallel displacement curves with Hill
coefficients near unity is more uncommon but also not unknown (22).
In general, the experimental results can be discussed in terms of two
possible hypotheses. The first is that amylin is simply a low efficacy
agonist for the CTR. Under these circumstances, the hCTR2 clone is not
associated with the amylin binding found in MCF-7 cells (i.e., the
amylin receptor gene product was not recovered from these studies). The
second is that the CTR functions as the amylin receptor when coupled to
certain G proteins in some membranes. These ideas are considered
separately.
The first hypothesis to consider is the proposition that amylin is
simply a lower efficacy agonist for hCTR2 in these systems and that the
amylin effect is not relevant to the amylin binding seen in native
MCF-7 cells. In terms of this idea, the two gene products isolated from
the MCF-7 cell library are not related to the high affinity amylin
binding found in the MCF-7 cells. The small quantity of high affinity
binding of 125I-rAmylin found in COS-7 and HEK
293 cells and the high affinity selection effects would then represent
a separate activity of rAmylin for the hCTR2. In terms of this
hypothesis, a human amylin receptor awaits discovery in the MCF-7 cell
library. This hypothesis is based on the interaction between receptors
and G proteins. The ternary complex models for seven-transmembrane
receptors incorporate an intrinsic affinity constant between the
activated receptor and G proteins (23-26). Under these circumstances,
there is a stoichiometric relationship between the receptor and G
protein that depends on this affinity constant and the relative amounts
of receptor and G protein. In G protein-deprived systems, the amount of
activated receptor dictates the amount of complex of receptor and G
protein. Thus, a low efficacy partial agonist may produce a lower
amount of activated receptor than a high efficacy agonist with a
subsequently lower quantity of high affinity agonist complex. These
effects have been observed directly with cholinergic agonists (27, 28) (see Ref. 29 for a discussion). The fact that hCAL produces 24.8% high
affinity complex in HEK 293 cells while amylin produces only 8% is
consistent with the idea that amylin produces less of the activated
state than hCAL (i.e., it simply is a lower efficacy agonist for the
CTR).
A second hypothesis describes a system in which hCTR2 couples to one G
protein for CAL function and another for amylin function. This latter G
protein may not be present in all cellular systems, thus conferring
cellular selectivity for amylin effect. There is a considerable body of
evidence to show that CTRs in general couple to a variety of G proteins
[i.e., Gs, Gi, and
Gq; see Horne et al. (30) for a
review]. In addition, the promiscuous coupling of this receptor has
been shown to alter with cell cycle as well (31). In the current series
of experiments, there are two lines of evidence consistent with a
hypotheses of separate G protein coupling for hCAL and rAmylin. The
first is the disparate formation of high affinity ternary complexes
formed by hCAL and rAmylin as measured by radioligand saturation
binding in all of these systems (i.e., 24.8% for
125I-hCAL versus 8% for
125I-rAmylin). In terms of this hypothesis, the
different Bmax values for the two agonists
may reflect different stoichiometries of the different G proteins.
However, this is not definitive because this also is consistent with
amylin simply being a lower efficacy agonist for hCTR2 (see above).
The main support for the second hypothesis is related to the extremely
selective affinity of rAmylin and rCGRP for displacement of
125I-rAmylin and the considerably lower potency
in displacing 125I-hCal (i.e., they are of low
activity at CTRs). This suggests that the CTR changes character when
bound to 125I-amylin. In view of the high degree
of high affinity selection for 125I-rAmylin
displacement over displacement of 125I-AC512
observed for amylin in MCF-7 cells (the source of the transfected
receptor hCTR2) and the fact that the same is not true for hCAL suggest
that the MCF-7 cell possesses a G protein or other factor to confer
high rAmylin binding in that system and that this factor is lost on
transfection into COS-7, HEK 293, and Ti ni cells. The
factor need not be a specific G protein; it could be an auxiliary
player in the receptor coupling process. For example, protein factors
that tightly couple adenosine receptors (32) and
2A/D-adrenergic receptors (33) have been
described recently. Removal of these from host membranes results in
loss of high affinity binding of agonists to receptors.
In conclusion, these data describe receptor binding characteristics of
two hCTR isoforms in various expression systems for CAL and amylin
ligands. The provocative association of the hCTR2 with high affinity
amylin binding requires further study and may have implications for the
understanding of selective cellular signaling of hormones and the use
of gene products to attain signaling diversity. In view of the similar
affinities of all of these agonist and antagonist radioligands for
amylin binding in MCF-7 cells and that found in these expression
studies with hCTR2, it could be inferred that these effects are
mediated either by one receptor coupling to various membrane components
or by separate but similar receptors.
rAmylin, rat amylin;
CTR, calcitonin
receptor;
hCTR, human calcitonin receptor;
HEK, human embryonic kidney;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
sCAL, salmon
calcitonin;
hCAL, human calcitonin;
Ti ni, Trichoplusia ni;
rCGRP, rat calcitonin gene-related
product;
hCGRP, human calcitonin gene-related product;
CI, confidence
interval;
sCAL, salmon calcitonin;
hCAL, human calcitonin.
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Summary
Introduction
