(Received for publication, January 9, 1995; and in revised form, May 22, 1995)
From the
The recombinant porcine m2 muscarinic acetylcholine receptor
(rPm2R) from Chinese hamster ovary cells has been purified to
homogeneity. Two mg of purified rPm2R, with a specific activity of 12
nmol of R-(-)-quinuclidinyl benzilate/mg of protein,
were obtained from 30 ml of packed Chinese hamster ovary cells. The
apparent molecular mass (78.5 kDa) and specific activity for the rPm2R
preparation were the same as that for the Pm2R purified from atrial
tissue, but the yield was 100 times greater. Purified rPm2R bound
agonist and antagonist with the same affinities and coupled to the
inhibitory guanine nucleotide-binding protein with the same efficiency
as the purified native atrial Pm2R. Ligand binding studies were
consistant with a single class of antagonist binding sites but two
subclasses of agonist binding sites. The fraction of rPm2R having high
affinity for agonists was increased by mM
Mg mAcChRs The mAcChR was purified from
heart tissue with a yield of 30 µg of protein (5) and from
brain tissue at yields of 6-18 µg(6, 7) .
These yields are similar to those for other G protein-coupled receptors
from natural
sources(8, 9, 10, 11, 12, 13) .
Natural sources may also contain multiple subtypes that may be
difficult to resolve. Recombinant human In
this paper, we report the purification of fully active recombinant Pm2R
(12 nmol/mg) from a line of overexpressing CHO cells (15) with
yields in the mg range, up to 100-fold greater than that achieved for
the atrial Pm2R(5) . Sufficient material was obtained to
complete ultraviolet circular dichroism studies analyzing protein
secondary structure in the presence and absence of ligands. Agonists
interact with the purified atrial mAcChR(5) , purified brain
mAcChRs (16, 17) and purified brain A
Membranes
were adjusted to 4.5 mg of protein/ml with EI buffer, and the rPm2R was
solubilized with digitonin/cholate (24) by adding ¼
volume of 10 Combined detergent
extracts were diluted with an equal volume of EI buffer, brought to 5
mM MgCl The WGA eluate (120 ml) was
adsorbed onto hydroxylapatite (5.5 g of Bio-Gel HTP (Bio-Rad)
equilibrated with D/C-EB buffer) in a 3.1 cm 25
ml of ABT agarose was recycled with 125-175 ml of 1 M NaOH, 125-175 ml of 1 M NaCl, and 750-1000 ml of
H
RQ/R The receptor was diluted into either D/C-EP (1
mM EDTA, 10 mM potassium phosphate, pH 7.4) buffer or
Figure 1:
Silver-stained SDS-polyacrylamide gel
of the fractions obtained during purification of rPm2R from CHO cells.
The gel was an 8-18% polyacrylamide gradient in a minigel format
using the discontinuous buffer system of Laemmli(33) . Lane1, molecular mass standards (phosphorylase a, M
The CHO cell receptor
preparation was very susceptible to proteolysis, which could not be
eliminated even by judicious use of a wide spectrum of protease
inhibitors. Speed of processing during the early stages of the
preparation was essential to successful isolation of intact receptor.
Despite the high specific activity of the membranes that could be
isolated from the overexpressing CHO cells, we were unable to achieve
purification of the rPm2R without the WGA and hydroxylapatite
chromatography steps. Membranes from CHO cells banded at a higher
sucrose density than those from atrial tissue, and unlike the atrial
Pm2R (25) no purification was achievable during solubilization.
The CHO cell rPm2R bound to WGA agarose more tightly than did the
atrial receptor and required the use of chitotriose for elution. The
hydroxylapatite and ABT affinity purification steps gave results
similar to those found for the atrial preparation.
Figure 2:
Binding of [
Figure 3:
Effect of [Mg
Detergent effects on carbachol binding to the purified rPm2R
are shown in Fig. 4A. The competition curves were
slightly left-shifted when the detergent concentration was lowered to
0.075% digitonin alone. Under conditions of reduced detergent, K
Figure 4:
Effect of detergent concentration and
temperature on the carbachol titrations of purified rPm2R. Total
binding site concentrations were about 400 pM, and total
[
Fig. 4B shows the
effect of temperature on the carbachol displacement curves for the
purified rPm2R in 18.75 mM MgCl The
effects of temperature on carbachol/[
Figure 5:
CD spectra of the purified rPm2R. Aliquots
of one preparation of purified rPm2R preparation (14.4 nmol of
[
Purification of the rPm2R from a clonal line of CHO cells
overexpressing this receptor resulted in a preparation of 2 mg of
homogeneous receptor protein at theoretical specific activity. The
yield is nearly 100-fold greater than the yield of purified atrial Pm2
receptor(5) . The purification of rPm2R also compares favorably
with the rHm2R preparations from the baculovirus-infected insect Sf9
cell system(14) . These preparations of recombinant m2R will
permit more extensive characterization of the purified receptor and
provide a model system to advance our understanding of the molecular
structure and function of G protein-coupled receptors in general. In
most respects the purified rPm2R was indistinguishable from the
purified atrial Pm2R. Ligands bound to both preparations with the same
affinities. Both preparations coupled to purified G proteins with the
same efficiency in reconstitution experiments.
Carbachol/[ Agonists compete with
[ Mg The leftward shift of the carbachol competition
curves at lower temperatures was due to a large increase in the
proportion of the receptor in the high affinity agonist state (F K The CD spectra demonstrated that little
change in secondary structure was associated with agonist occupancy of
the purified rPm2R at room temperatures, but small changes were
observed with antagonist binding. Ligand-receptor interactions may not
require large changes in secondary structure, but the results observed
with QNB were reproducible. At this time, it is not clear how the two
agonist affinity states relate to the CD measurements, but if there are
two distinct agonist-receptor conformations, the CD spectrum should be
a weighted average of the two. The overall secondary structure analysis
of the CD spectra are consistent with the putative
seven-transmembrane-spanning structure for the mAcChR. These domains
account for about 36% of the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, low detergent concentration, and low temperature.
Circular dichroism spectra obtained for the purified rPm2R with and
without agonists were indistinguishable, but spectra for the
antagonist-occupied receptor showed reproducibly deeper characteristic
negative deflections at 208 and 220 nm. Secondary structure analysis of
the CD spectra predicted 53%
-helix for the free receptor and 49%
-helix for the R-(-)-quinuclidinyl
benzilate-receptor complex.
(
)belong to the family of
seven-transmembrane domain receptors that transduce extracellular
neurotransmitter signals by activating G proteins within the cell.
Effector systems activated by muscarinic agonists include inhibition of
adenylyl cyclase and stimulation of phosphatidylinositol hydrolysis (1) . Five mAcChR subtypes have been identified, and most
tissues that have muscarinic activity express multiple
subtypes(2, 3, 4) . Studies of purified
receptor alone and in reconstitution with other purified signaling
components permit analysis under more defined and tractable conditions.
However, the supply of purified mAcChR like that of other G
protein-coupled receptors is limited.
-adrenergic receptor and
Hm1 and Hm2 muscarinic receptors were purified from
baculovirus-infected insect Sf9 cells(14) , with yields for the
rHm2R estimated at 6 mg of protein/liter of culture with a specific
activity of 2 nmol/mg (10-20% active), and at 40 µg of
protein/liter of culture at a specific activity of 8 nmol/mg.
adenosine receptors (13) at two subclasses of sites. The
assay conditions affecting the equilibrium binding parameters and the
distribution between high and low affinity states were examined for the
interaction of carbachol with the purified rPm2R.
Materials
WGA was purified according to Kahane et al.(18) and coupled to Affi-Gel 10 (Bio-Rad) at 20
mg of WGA/ml of resin. ABT prepared according to Haga and Haga (19) was coupled to agarose at 1 µmol/ml resin. Chitotriose
was prepared according to Rupley(20) . Cholate, recrystallized
three times as the free acid, R-(-)-hyoscyamine,
acetylcholine, and gallamine were from Sigma. Carbachol and
(+)-pilocarpine were from Aldrich. QNB and oxotremorine M were
from Research Biochemicals International. [H]QNB
(30-46 Ci/mmol), [
S]GTP
S
(1000-1500 Ci/mmol), and [
-
P]GTP
(30-44 Ci/mmol) were from DuPont NEN. Protease inhibitors,
purchased from either Sigma or Boehinger Mannheim, were prepared as
1000-fold concentrated stocks: 1 mg/ml pepstatin A and 17 mg/ml
phenylmethylsulfonyl fluoride in dry methanol; 1 M benzamidine, 10 mg/ml bacitracin in 95% ethanol; 2.5 mg/ml
leupeptin, 2.5 mg/ml aprotinin, 1 mg/ml E-64 in H
O. Cell
growth media was FD (1:1 mix of Ham's F-12
medium:Dulbecco's modified Eagle's medium) plus 10% calf
serum.
Cell Culture
A transfected clone of
dhfr CHO cells expressing high levels of rPm2R (15) was adapted to spinner culture and grown in 4-liter
spinner flasks containing
1.5 liters of growth media. Expression
levels were increased 5-9-fold by treatment with 5 mM sodium butyrate for 15-18 h prior to harvesting. Butyrate
treatment can increase gene transcription in CHO cells(21) ,
which may be due to hyperacylation of histones(22) . Cells were
harvested by centrifugation at 4000 rpm for 5 min at 4 °C. Cells
from each spinner flask were washed with 45 ml of CHM (250 mM sucrose, 50 mM EDTA, 1 mM EGTA, 25 mM imidazole, pH 7.4) plus protease inhibitors by centrifugation at
1500
g for 2 min in a clinical centrifuge, resuspended
in 10 ml of CHM plus protease inhibitors and frozen at -80
°C.
Receptor Purification
Atrial Pm2R was purified as
described previously(5) . Purification of the recombinant Pm2R
was significantly modified from that for the atrial preparation.
Protease inhibitors were added to frozen CHO cells during thawing and
to all solutions before use. 30-35-ml batches of packed cells
expressing 1-2 10
surface receptors/cell were
homogenized under argon for 2
20 s with a PTA 10S polytron
probe operated at 60% of maximum speed. The homogenate was centrifuged
at 1500
g for 1 min in a clinical centrifuge, brought
to 120 ml with CHM, and layered over sucrose gradients prepared in
Beckman SW28 rotor tubes. Each tube contained 9 ml of 42.5% sucrose and
9 ml of 20% sucrose in 5EI buffer (5 mM EDTA, 1 mM EGTA, 25 mM imidazole, pH 7.4) and 20 ml of homogenate.
After centrifugation at 27,000 rpm for 1 h at 4 °C, the membrane
fraction was collected from the 20-42.5% sucrose interface.
Homogenization and sucrose gradient steps were repeated on the pellet,
and the combined membrane fractions were diluted with EI buffer (1
mM EDTA, 25 mM imidazole, pH 7.4) and pelleted by
centrifugation at 70,000
g for 1 h at 4 °C. The
pellet was resuspended to 20 ml in EI buffer and assayed for total
protein by the Lowry Folin phenol method(23) .
D/C-EI buffer. After 10 min. at room temperature,
the mixture was centrifuged at 70,000
g for 1 h at 4
°C. The supernatant was saved on ice, and the pellet was
resuspended in EI buffer with a glass Teflon homogenizer to a total
volume equal to one-half of the initial volume and extracted again with
¼ volume of 10
D/C-EI buffer.
, combined with 40-50 ml of freshly
recycled WGA-agarose, and mixed overnight at 4 °C. WGA agarose,
stored in 0.2 M N-acetylglucosamine, was recycled with 130 ml
of 0.1 M acetic acid, 250 ml of EI buffer, and finally 50 ml
of 5 mM MgCl
in D/C-EI buffer. The next day the
mixture was transferred to a 3.8
16-cm acrylic column, and the
flow-though was collected at 3 ml/min. The WGA resin was washed with
two 50-ml batches of 5 mM MgCl
in D/C-EI buffer,
one 50-ml batch of D/C-EB (1 mM EDTA, 25 mM bicine,
pH 9) buffer, and eluted with two 50-ml and one 20-ml batch of 4 mM chitotriose in D/C-EB buffer.
16-cm acrylic
column. The WGA eluate was applied at 1 ml/min, washed with 5 ml of
D/C-EB buffer, and eluted at 1 ml/min by sequential addition of 30 ml
of 0.12 M potassium phosphate, 1 mM EDTA, D/C, pH 9;
30 ml of 0.05 M potassium phosphate, 1 mM EDTA, D/C,
pH 7.4; and 60 ml of 0.5 M potassium phosphate, 1 mM EDTA, D/C, pH 7.4. The eluate contained 15-20% pure rPm2R
and was collected in a siliconized 120-ml polypropylene bottle.
O. The resin was gently mixed with the HTP eluate for
18-36 h at 4 °C; transferred to a siliconized 250-ml sintered
glass funnel and washed under vacuum with three 25-ml batches of 0.25 M NaCl in D/C-EI buffer; and transferred to a siliconized
50-ml conical centrifuge tube and eluted three times by rotation for
12-18 h with 25 ml of D/C-EI buffer containing 0.5-1 mM hyoscyamine, 0.25 M NaCl, pepstatin A, and
phenylmethylsulfonyl fluoride followed by centrifugation for 20 s for
eluate recovery. Combined ABT eluates were dialyzed against two changes
of 1 liter of EI buffer plus 25 ml of 10
D/C to reduce the NaCl
concentration to below 25 mM. The dialysate was transferred to
a siliconized 120-ml polypropylene bottle and adsorbed to 1 ml of
TSK-Gel Toyopearl DEAE-650M per 4000 pmol of HTP fraction receptor
sites by mixing for 2 h at 4 °C. The resin was transferred to a 3.1
16-cm acrylic column, washed with 200-300 ml of digitonin
or digitonin/cholate buffer containing <20 mM salts to
remove the remaining hyoscyamine, and the purified rPm2R was eluted
with detergent buffer containing 0.25 M NaCl or potassium
phosphate, pH 7.4. Sephadex G25F spin columns were used for desalting,
with 80-90% recoveries. For long term storage the purified
receptor was frozen at -80 °C in siliconized microfuge tubes.
About 5% of the receptor activity was lost with one freeze-thaw cycle.
Ligand Binding
[H]QNB
binding activity was analyzed using the DEAE filter disc assay (25) with nonspecific binding determined in the presence of
10-100 µM hyoscyamine. The dissociation constant for
[
H]QNB binding to the receptor, K
, was determined by
Scatchard analysis(26) . Binding parameters for nonradioactive
ligands were determined from competition experiments against
[
H]QNB. The competition binding data were fit to
a model consisting of either one (antagonist) or two (agonist)
subclasses of noninteracting binding sites as
described(25, 27) .
is the fractional saturation of
receptor with [
H]QNB, and
[Q
] and
[I
] are the concentrations of
free [
H]QNB and inhibitor, respectively.
[I
] was assumed to be equal to
[I
] since no displacement was
observed until [I
] >
[R
], and the data were normalized to the
fractional saturation in the absence of inhibitor. K
and K
are the dissociation
constants for the higher and lower affinity agonist sites,
respectively, with F
and F
= 1 - F
their corresponding
fractional amounts.
D/C, MgBB (5 mM MgCl
, 10 mM
HEPES, 1 mM EDTA, 1 mM EGTA, pH 7.4) for ligand
binding studies. Variations on these conditions are noted in the table
and figure legends. In competition experiments final buffer
concentrations were reduced by 25%, and calculated
[Mg
]
(28) was
reduced to about 3 mM.
Reconstitution
Purified rPm2R or atrial Pm2R was
reconstituted with purified atrial G as
described(27) . Coupling was assessed by measuring the
muscarinic receptor stimulated GTPase activity of G
,
determined by the difference in GTPase activity between 20 mM
carbachol and 10 µM hyoscyamine at 32 °C (27) . Purified rPm2R was also reconstituted into lipids by the
same procedure but without added G
to assess ligand binding
parameters between detergent and lipid environments.
Circular Dichroism
Purified rPm2R was prepared for
CD analysis by elution of the DEAE resin with 0.25 M sodium
phosphate, pH 7.4, in CD buffer (0.1% digitonin, 0.02% cholate, 0.2
mM EDTA, 10 mM potassium phosphate, pH 7.4), and the
excess phosphate was removed by gel filtration. The receptor
concentration was 1.4 µM in [H]QNB
sites with a specific activity of 14.4 nmol/mg of protein. Protein
content was measured by analysis for total amino acids (23) in
order to directly determine the total concentration of amide bonds.
Buffer controls were prepared in parallel with the receptor
preparation. rPm2R was incubated for 1 h at room temperature with 1
mM acetylcholine, 1 mM carbachol, 20 µM oxotremorine M, 100 µM (+)-pilocarpine, 10
µM(-)-hyoscyamine, or 10 µM QNB and
stored for 1-2 days at 4 °C before CD analysis. The CD of
each sample was measured in 100-micron path length cells from 260 to
178 nm with total absorbance less than 1.0 to ensure sufficient light
transmission. Spectra were measured at 20 °C using a model J-720
Jasco spectrometer calibrated with (+)-10-camphorsulfonic acid at
two points to ensure reliability(29) . The data were collected
at 1-nm intervals using an on-line PC computer, and at least 6 spectra
for each sample were averaged, corrected by subtraction of buffer
spectra (usually negligible), and then smoothed. Secondary structure
was analyzed for
-helix, antiparallel
-sheet, parallel
-sheet,
-turn, and random coil by the variable selection
method(30) .
Purification
Table 1shows a
representative purification. Final receptor yield was 16% of the total
homogenate activity and over 30% of that for isolated membranes. The
final specific activity was 11.9 (± 2.0) nmol/mg of protein
(average of four preparations), the same specific activity obtained for
the purified atrial Pm2R(5) . The yield of purified CHO cell
rPm2R protein was 100-fold greater than for the purified atrial Pm2R. A
specific activity of 12.5 nmol/mg corresponds to a mass of 80 kDa for
the receptor, assuming one ligand binding site per receptor molecule.
The rPm2R preparation consisted of a single diffuse silver-stained band
on SDS-polyacrylamide gel electrophoresis, similar to that of the
atrial Pm2R (Fig. 1, and (5) ), with a mass of 78.5 kDa
(range 60-100 kDa) for the rPm2R and 80 kDa (range 60-112
kDa) for the atrial Pm2R when analyzed together(32) . Thus, the
rPm2R and atrial Pm2R preparations were purified to homogeneity with
full retention of ligand binding capacity. Atrial and recombinant
preparations were stable on ice for a month or more, and considerably
longer at -80 °C, with only small losses (5%) in binding
activity associated with a freeze-thaw cycle.
97114; bovine serum albumin, M
66296; ovalbumin, M
42807; aldolase, M
39210; concanavalin A, M
25271; soybean trypsin inhibitor, M
20095;
lysozyme, M
14314); lane2, 1
µg of homogenate; lane3, 1 µg of membranes; lane4, 1 µg of extract; lane5, 1 µg of WGA eluate; lane6, 0.5
µg of HTP eluate; lane7, 0.1 µg of ABT
eluate. The gel was silver-stained according to the method of Wray et al.(34) .
Equilibrium Ligand Binding
Representative
Scatchard plots for the binding of [H]QNB to the
purified rPm2R and atrial Pm2R are shown in Fig. 2. For three
different preparations incubated for 24 h at room temperature
(18-22 °C) in D/C-EP buffer the apparent K
for
[
H]QNB binding was 98 ± 26 pM for
the recombinant receptor and 76 ± 8 pM for the atrial
receptor. Agonist interactions with the purified receptor were best fit
to a model predicting two subclasses of sites, and conditions affecting
agonist binding were examined in the carbachol competition experiments
described below. Under similar conditions the carbachol binding
parameters were essentially the same for both the purified rPm2R and
atrial Pm2R.
H]QNB to
purified Pm2R. Purified recombinant Pm2R and atrial Pm2R were diluted
into D/C-EP buffer and incubated with various concentrations of
[
H]QNB for 24 h at room temperature (18-22
°C). Nonspecific binding was determined by preincubation with 10
µM hyoscyamine for 20 min prior to addition of
[
H]QNB. The binding data were analyzed by the
method of Scatchard (26) (inset) and fitted by linear
regression. The lines represent the fitted curves, which gave K
values of 82 ± 10 pM and 83
± 5 pM for the recombinant and atrial receptors,
respectively.
Reconstitution of Purified Pm2R and Purified
G
The ability of purified rPm2R to couple to G in a reconstituted system was compared with that for the purified
atrial Pm2R(27) . The results of these studies are shown in Table 2. Similar levels of incorporation were obtained for both
receptor preparations (
30%), and there were no significant
differences in their ability to couple to the purified atrial G
proteins. Under parallel conditions, both receptor preparations
showed similar levels of agonist-stimulated GTPase activity.
Conditions Affecting Ligand Binding
Fig. 3shows the effects of Mg on carbachol
binding. At 20 °C and 0.075% digitonin, increasing Mg
had the effect of shifting the carbachol competition curves
leftward (panelA), due in large part to an increase
in F
from zero in the absence of Mg
to 30% in the presence of 18.75 mM MgCl
(Table 3). At 4 °C and higher detergent concentration,
increasing Mg
also caused a leftward shift of the
titration curve, but in this case changes in F
were small (panelB). Instead, K
was reduced about 6-fold
when Mg
was present. K
was reduced about 3-fold in
the presence of Mg
under both experimental
temperature/detergent conditions. There was no apparent affect of
Mg
on K
. Effects of
Mg
on the purified atrial Pm2R were similar (data not
shown).
]
on the carbachol titrations of purified rPm2R. Total binding site
concentrations were about 400 pM, and total
[
H]QNB was about 750 pM. PanelA experiments were done with receptor diluted into BB
containing 0.075% digitonin (final) and the indicated final
concentrations of MgCl
. Incubations were for 18-20 h
at room temperature (18-22 °C). PanelB experiments were done in the presence of BB and 0.75
D/C
(final) and the indicated final concentrations of MgCl
.
Incubations were for 70 h at 4 °C. The fitted binding parameters
are given in Table 3.
and K
were reduced, and F
was increased (Table 4). These differences
were greater at 20 °C than that at 4 °C. With the purified
atrial Pm2R, lower detergent concentrations also reduced K
and K
, but effects on F
were more dramatic. The K
site of the atrial receptor
was undetectable in experiments carried out with 0.3% digitonin, 0.15
D/C, or 0.75
D/C in MgBB for 21 h at 25 °C but was
clearly observable with 0.075% digitonin (F
= 26 ± 6%).
H]QNB was about 750 pM. PanelA depicts the effects of detergent under two different
conditions: 18.75 mM MgCl
at 20 °C (circles) and 3.75 mM MgCl
at 4 °C (squares). PanelB depicts the effect of
temperature with the receptor in 18.75 mM MgCl
in
0.75
D/C, BB. The fitted binding parameters for these
experiments are given in Table 4.
, D/C, BB (10
mM HEPES, 1 mM EDTA, 1 mM EGTA, pH 7.4). The
carbachol curves were left-shifted 1 order of magnitude from 37 to 20
°C and again from 20 to 4 °C. As shown by the fitted binding
parameters for these experiments (Table 4), this leftward shift
resulted from a decrease in K
and K
and an increase
in F
. These same trends were observed under the
conditions of low detergent and 3.75 mM Mg
for both the recombinant and atrial Pm2R (data not shown). The
carbachol competition curves done at 4 °C in either 18.75 mM MgCl
, 0.75
D/C, BB or 3.75 mM MgCl
, 0.075% digitonin, BB were superimposible.
H]QNB
competition experiments on purified rPm2R following detergent removal
by reconstitution into phospholipid vesicles were similar to that for
the receptor in detergent. The displacement curves were left-shifted
with decreasing temperatures and appeared much like those of Fig. 4B, except that the corresponding shifts were
slightly smaller. The fitted binding parameters for the reconstituted
preparations are given in Table 4and are similar to the receptor
in detergent except that K
was less temperature-sensitive. The overall effects of decreasing
temperature, namely, decreasing K
and K
, increasing F
, and no detectable K
at 37 °C, was observed
for purified receptor in both detergent and liposomes.
Pm2R Secondary Structure from Circular
Dichroism
Representative CD spectra for the purified rPm2R are
shown in Fig. 5. These spectra showed strong negative
deflections with minima at 220 and 208 nm and a strong maximum positive
deflection at 190 nm indicative of the -helical structure.
Analysis of the spectrum for the nonliganded receptor indicated 53%
-helix (Table 5). Addition of agonists to the receptor (Fig. 5, A and B) produced little change in
the CD spectra, whereas antagonists (Fig. 5, C and D) showed more negative deflections at the 220 and 208 nm
minima. In all CD experiments, the QNB-receptor complex always produced
deeper negative deflections at 220 and 208 nm than either the
nonliganded or agonist liganded receptor. Secondary structural analysis
of the QNB-liganded receptor spectrum showed a marginally significant
4% decrease in
-helix and 4% increase in parallel
-sheet
structure compared with the free receptor (Table 5).
H]QNB sites/mg of protein) were incubated with
ligands, and the CD spectra were collected as described under
``Experimental Procedures.'' The spectra for the agonist
oxotremorine M and the partial agonist pilocarpine are not shown but
were identical to the acetylcholine and carbachol spectra. In each panel the solidline represents the spectrum
of the free rPm2R and the dashedline the spectrum
for the receptor plus ligand. Spectral data were analyzed on a per
amide basis calculated from protein content and sequence (
is
in units of M
cm
).
Analyses of secondary structures are given in Table 5for the
free receptor and QNB-receptor complex (panelD).
H]QNB competition assays were
similarly affected by Mg
, detergent, and temperature
in both preparations. There were only slight differences in
SDS-polyacrylamide gel electrophoresis migration between the
recombinant and atrial receptors, suggesting that their overall extent
of glycosylation was similar. The recombinant receptor did bind more
tightly to WGA than the atrial receptor, indicating that it differs in
the amount of surface-exposed sialic acid residues. The mass of the
rHm2R prepared from the insect cell system was 55 kDa on
SDS-polyacrylamide gels(14) , which is considerably smaller
than the 80-kDa mass found for the Pm2R preparations and indicates that
glycosylation of recombinant m2R in the insect cell system is different
from the atrial or CHO cell system.
H]QNB for more than one subclass of sites in the
purified mAcChR (5, 16, 17) and adenosine
receptor (13) , but the significance of two agonist affinity
states in the purified receptor has not been addressed. Agonist
competition curves for the membrane-bound rPm2R can be analyzed in
terms of three agonist affinity states(36) , similar to natural
tissue sources(37, 38, 39) . The highest
affinity site (K
about 3 nM for
carbachol) is sensitive to guanine nucleotides, indicating it is the
site associated with activation of G proteins. The two remaining
membrane agonist affinity states bind carbachol with an apparent K
of 1.6 and 30 µM.
(
)These K
values are
within range of the two carbachol affinity states of the purified rPm2R
and atrial Pm2R in detergent as well as in phospholipids after
reconstitution and may represent the same receptor states. Moreover,
the common characteristics found for the two G protein-independent
agonist affinity states, K
and K
, among
membrane-bound, detergent-solubilized, and phospholipid-reconstituted
pure receptor demonstrate that the purified mAcChR in detergent is a
good system for further probing these states.
in the 1-10 mM range is needed for hormone
activation of G proteins and differs from the high affinity
(nM) Mg
site associated with G protein
GTPase activity(40) . The low affinity Mg
site(s) is required for formation of nucleotide-sensitive high
affinity agonist binding and for dissociation of activated G protein
into its
and
subunits(41, 42, 43) . Formation of this site
appears to be primarily at the expense of depletion of the intermediate
affinity agonist site(36, 38, 42) .
Mg
effects on the purified Pm2R ( Fig. 3and Table 3) demonstrate that mM Mg
also
promotes formation of the intermediate affinity agonist state. Thus
mM Mg
may play a regulatory role in
stabilizing the intermediate affinity agonist conformation prior to G
protein interaction.
) and to a decrease in K
. Low temperature-induced
increases in agonist affinity were reported in studies of the
membrane-bound mAcChR(44, 45, 46) ,
and
adrenergic
receptors(47, 48, 49, 50) , dopamine
receptors(51) , and in the detergent solubilized preparations
of these receptors (47, 50) as well as in intact cells (50, 52) and membranes in which G proteins were
removed by alkalinization or proteolysis(53, 54) . In
two studies(49, 50) , the major low temperature
effects were attributed to an increase in F
while K
remained unchanged. The
above results show that this property also persists in purified
receptor preparations either in detergent or in liposomal environments,
demonstrating that it is an intrinsic characteristic of the receptor.
and K
may be characterized by
similar lipid-receptor interactions, which are not shared with K
. Low concentrations of
detergents and low temperatures favored decreases in both K
and K
, while K
was largely unaffected.
Detergent effects were more prominent at 20 than at 4 °C, and
differences were observed between the recombinant and atrial
preparations, perhaps because of different boundary lipid compositions. K
and K
for the purified rPm2R in
both detergent and phospholipid were greater than their corresponding
membrane-bound values, but K
was essentially identical under all such conditions. The large
temperature effects on K
and K
indicate that ligand
binding energies for these interactions involve large enthalpy changes,
but K
was unaffected by
temperature (
H
0), and the favorable
standard free binding energy
G
=
-8.2 kcal/mol (for K
≅ 1 µM) must arise from mostly entropic
contributions (
S
= +27.4 cal/deg
mol), a feature characteristic of hydrophobic
interactions(55) .
-helical content. Thus the remaining
17% suggests that the intra- and extracellular domains must also
contain a significant amount of
-helix. The secondary structure
analysis for the rPm2R was found to be very similar to that reported
for the related G protein-coupled membrane protein rhodopsin analyzed
by the same method (35) and by Fourier transform infrared
spectroscopy(56) , which gave an estimated 51%
-helical
content.
CHO cells, CHO cells deficient in dihydrofolate
reductase; QNB, R-(-)-quinuclidinyl benzilate; WGA,
wheat germ agglutinin; ABT, 3-(2`-aminobenzhydryloxy)tropane; HTP,
hydroxylapatite; CD, circular dichroism; D/C, 0.4% digitonin, 0.08%
cholate; BB, binding buffer; CHM, cell-homogenizing medium; GTP
S,
guanosine 5`-O-(thiotriphosphate).
We acknowledge Dr. Daniel Capon of Genentech, Inc. for
providing the pSVE expression system and the clone of the Pm2R gene,
and we thank Bipei Chen for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.