(Received for publication, October 20, 1994; and in revised form, June 29, 1995)
From the
Muscarinic receptors were solubilized from porcine atria in
digitonin-cholate and were purified by chromatography on DEAE-Sepharose
and 3-(2`-aminobenzhydryloxy)tropane-Sepharose. The product identified
on Western blots migrated with an apparent molecular mass of
60-75 kDa, with additional bands indicative of homotrimers (190
kDa) and homotetramers (240 kDa). Receptor eluted from the affinity
column was accompanied by a mixture of guanyl nucleotide-binding
proteins (G-proteins) identified on Western blots as G,
G
, G
, and G
(preparation M2G);
the G-proteins were largely removed by further processing on
hydroxyapatite (preparation M2). Solubilized purified receptors bound
muscarinic ligands in an apparently cooperative manner. In studies at
equilibrium, the antagonists [
H]AF-DX 384, N-[
H]methylscopolamine (NMS), and
quinuclidinylbenzilate (QNB) revealed Hill
coefficients between about 0.8 and 1.2. Also, the apparent capacity for
[
H]QNB exceeded that for
[
H]AF-DX 384 and [
H]NMS by
about 1.5-fold in M2 and by 2-fold in M2G. Binding to M2G at high
concentrations of [
H]QNB was fully inhibited by
unlabeled NMS, which therefore affected sites not labeled at similar
concentrations of [
H]NMS. Oxotremorine-M
displayed a biphasic inhibitory effect on the binding of
[
H]AF-DX 384 in M2 and M2G, suggesting that
multiple states of affinity are intrinsic to the receptor;
5`-guanylylimidodiphosphate was without appreciable effect in M2 but
resulted in a bell-shaped binding profile for the agonist in M2G. All
of the data can be described in terms of cooperative interactions
within a receptor that is at least tetravalent and presumably an
oligomer. In the context of the model, copurifying G-proteins and
guanyl nucleotides serve to regulate the degree of cooperativity
between successive equivalents of muscarinic ligands.
G-protein-mediated signaling is widely held to occur via a transient complex between the G-protein and the receptor; similarly, a mixture of uncoupled and G-protein-coupled receptors generally is assumed to account for heterogeneity seen in the binding of agonists (Gilman, 1987; Birnbaumer et al., 1990; Conklin and Bourne, 1993). The binding patterns are analyzed almost universally in terms of distinct, noninteracting, and noninterconverting sites (e.g. Munson and Rodbard(1980)), but that approach embodies a mechanistic paradox: in particular, the receptors appear to interconvert between the different states. Guanyl nucleotides act via the G-protein to effect an interconversion from higher to lower affinity for the agonist; there is a similar effect of sodium in some systems, whereas an interconversion from lower to higher affinity is promoted by magnesium (Birnbaumer et al., 1990). Also, the relative number of receptors ostensibly in one state or another differs among different agonists (e.g. Kent et al.(1980), Wong et al.(1986), Sinkins et al.(1993)).
Variants of the ``mobile receptor'' or ``ternary complex'' model (De Lean et al., 1980) accommodate the apparent interconversion and complement biochemical evidence that a complex between receptors and G-proteins is stabilized by agonists and destabilized by guanyl nucleotides (Gilman, 1987; Birnbaumer et al., 1990). In such schemes, the allosteric interactions between agonists and guanyl nucleotides are described explicitly in terms of ligand-regulated equilibria between free receptors (R) and free G-proteins (G), or subunits thereof, on the one hand and one or more complexes on the other (e.g. De Lean et al. (1980), Onaran et al.(1993), Samama et al. (1993)). Agonists bind with higher affinity to RG than to R, and therefore promote coupling, whereas antagonists are indifferent or exhibit the opposite preference; guanyl nucleotides bind with higher affinity to G than to RG and therefore promote uncoupling. Schemes based on the mobile receptor hypothesis have been used to describe the binding properties of several receptors (e.g. Wreggett and De Lean(1984), Ehlert(1985), Neubig et al.(1988), Leung et al.(1990)), but a model that is fully consistent with the data has proved to be elusive (Lee et al., 1986; Wong et al., 1986; Wreggett, 1987; Ehlert and Rathbun, 1990).
In the absence of a viable mechanistic scheme, there is no conceptual basis for interpreting the consequences of disease, mutagenesis, or other perturbation that affects the binding of agonists. Since the multiple states of affinity and their sensitivity to guanyl nucleotides are a measure of efficacy and intrinsic activity (e.g. Kent et al.(1980), Ehlert(1985)), an inability to account for the binding properties implies uncertainty over the process whereby a receptor elicits a response. Recent studies in our laboratory have focussed on an alternative to the notion of a ligand-regulated distribution of receptors between free and G-protein-coupled states. Several lines of evidence suggest that the binding properties of membrane-bound receptors and their attendant G-proteins are a manifestation of cooperative effects between successive equivalents of the ligand bound at interacting sites; moreover, the reciprocal allosteric interactions between agonists and guanyl nucleotides emerge as changes effected by one ligand in the degree of cooperativity associated with the other (Chidiac and Wells, 1992; Sinkins and Wells, 1993).
If a multivalent
receptor retains its structural integrity over time in a system at
equilibrium, multiple states of affinity can arise from asymmetry alone
or from cooperativity. The potential complexity is much greater,
however, if the ligand itself promotes either the formation or the
dissociation of an oligomer (e.g. Hirschberg and
Schimerlik(1994)). Similarly, guanyl nucleotides may regulate the
oligomeric status of G-proteins (Jahangeer and Rodbell, 1993) in
addition to affecting the interaction between and
subunits within the G-protein heterotrimer itself (Gilman, 1987).
Finally, agonists, antagonists, and guanyl nucleotides may regulate the
interaction between receptors and G-proteins as described above. In
systems comprising ligand-regulated equilibria and transient complexes,
the binding properties depend upon the local concentrations of the
interacting proteins (Lee et al., 1986). In contrast,
cooperative systems in which oligomeric integrity is ligand-independent
might be expected to behave similarly under disparate conditions. To
investigate these possibilities under controlled conditions, M
muscarinic receptors have been purified from porcine atria and
studied for their binding properties in solution.
Fat, blood clots, and excess
tissue were removed, and the atria were rinsed in ice-cold PBS. ()The rinsed tissue was minced in a Waring blender (20 s at
full power) in 2.5 volumes (by original weight of tissue) of ice-cold
buffer A, and the mixture was homogenized on ice with three bursts of a
Polytron (20 s at setting 7, 20-s intervals). The homogenate then was
centrifuged for 15 min at 4 °C and 1000
g; this
step was repeated once following resuspension of the pellet in 1 volume
of buffer A in the Waring blender. The pooled supernatant was poured
through eight layers of cheesecloth and centrifuged for 40 min at 4
°C and 100,000
g (Beckman, 45Ti). The pellet then
was resuspended in a few milliliters of buffer A, briefly homogenized
(Polytron, 10 s, setting 7), and diluted with the same buffer to obtain
a convenient volume for the next step (0.2-0.6 ml/g of original
weight). The diluted homogenate (about 20 ml/tube) was centrifuged for
60 min at 2 °C and 100,000
g (Beckman, SW28)
through a stepwise gradient of 13% (w/v) sucrose (6 ml) and 28% (w/v)
sucrose (7 ml) in buffer A. Material at the sucrose interface was
recovered with a Pasteur pipette, diluted at least 3-fold with buffer
A, and centrifuged for 40 min at 4 °C and 100,000
g (Beckman, 45Ti). The final pellet was resuspended in a few
milliliters of buffer A, briefly homogenized (Polytron, 10 s, setting
7), and diluted to about 0.05 ml/g of original weight. Sarcolemmal
membranes obtained from this step usually were stored overnight at 2
°C in a polypropylene tube.
To solubilize the sarcolemmal
membranes, the suspension described above was diluted with buffer A to
obtain a protein concentration of 5.5 mg/ml. Nine volumes of the
diluted suspension then were mixed with 1 volume of a stock solution
containing 4% digitonin (Wako) and 0.8% sodium cholate in buffer A.
After gentle mixing by vertical rotation (30 rpm) for 10 min at room
temperature, the membranes were collected by centrifugation for 40 min
at 4 °C and either 100,000 g (Beckman, 45Ti) or
150,000
g (Beckman, 70Ti). The pellet was resuspended
in a few milliliters of buffer A, gently homogenized (Polytron, about
10 s, setting 4), and diluted with buffer A to 39% of the original
volume. This preparation contained about 10 mg of protein/ml, assuming
a 30% loss of protein from the first step (Peterson and Schimerlik,
1984). Four volumes of the suspension were mixed with 1 volume of the
buffered detergent stock, and the preparation was rotated for 10 min as
described above. An equal volume of ice-cold buffer A then was added,
the mixture was centrifuged as described above, and the supernatant was
stored at 2 °C in a polypropylene tube. The material from this step
was the crude solubilized preparation used in all subsequent
procedures.
The material recovered from
the affinity column was divided in two, and each half was processed at
room temperature according to one of two chromatographic methods.
Originally introduced to remove carbachol, each method subsequently was
found to yield a distinct preparation of the receptor (see below). The
material was processed by hand-held disposable plastic syringes, and
the two procedures were carried out in parallel. In the first
procedure, the receptor-containing sample was supplemented with 1
mM EDTA from a solution previously adjusted to pH 7.0 with
NaOH. That sample then was concentrated to less than 0.2 ml (Amicon,
Centricon-10), adjusted to a volume of 0.2 ml with buffer C, and
applied to an Econo-Pac P6 cartridge (5 ml, size-exclusion column)
previously equilibrated with buffer C. Elution was carried out with
buffer C at a rate of about 4 ml/min. The receptor was collected in
1.7-4.0 ml of eluate, and that fraction is designated here as
preparation M2G. In the second procedure, the material eluted from the
affinity column was applied to an Econo-Pac HPT cartridge (1 ml,
ceramic hydroxyapatite column) at a rate of about 0.5 ml/min. The resin
was washed first with 3 ml of EDTA-free buffer C and then with 15 ml of
a phosphate buffer (0.12 M KHPO
)
containing 0.1% digitonin (Wako), 0.02% sodium cholate, and 0.1 mM PMSF. The receptor then was eluted with 6 ml of the same buffer
prepared with 0.5 M KH
PO
. The
phosphate buffers were prepared from stock solutions of phosphate (1 M KH
PO
, adjusted to pH 7.6 with KOH)
and detergent (4% digitonin (Wako), 0.8% sodium cholate). The first bed
volume of eluate was discarded. The remainder was concentrated and then
processed on an Econo-Pac P6 cartridge as described above in order to
transfer the receptor into buffer C. That fraction of the eluate
containing the purified receptor is designated here as preparation M2.
Both preparations were stored in polypropylene tubes at 2 °C.
The two preparations differed in their complement of G-proteins, which were largely absent from M2 but copurified with the receptor in M2G. All of the procedures described above, from initial homogenization of the tissue to the elution of nonretained material from ABT-Sepharose, were carried out in magnesium-free buffers containing EDTA. Such conditions promote the removal of endogenous guanyl nucleotides (e.g. Chidiac and Wells(1992)), and GDP therefore is expected to be absent from the G-proteins in M2G.
To probe sequentially for different antigens, IgGs were
removed from previously treated nitrocellulose by means of a stripping
buffer (125 mM Tris, 2% SDS, 0.1 M
-mercaptoethanol, adjusted to pH 6.8 with HCl). The buffer was
applied twice for 20 min at 70 °C (Kaufmann et al., 1987),
and the membrane then was rinsed thoroughly at room temperature in PBS
containing 0.2% Tween 20.
All samples containing detergent were
prepared for electrophoresis by extraction with chloroform and
methanol. After separation of the phases by the addition of water, the
insoluble material was sedimented in a microcentrifuge, air-dried, and
sonicated into SDS-polyacrylamide gel electrophoresis loading buffer
containing -mercaptoethanol or dithiothreitol at 0.1 M.
The samples then were boiled for 5 min prior to application on the gel.
In one experiment, the samples were left at room temperature for 20 min
rather than boiled. The extraction procedure served both to concentrate
the material and to remove constituents, such as salt and detergent,
which impaired the performance of the gels. In some cases, the samples
were concentrated at 2 °C on ultrafiltration membranes (Amicon,
Microcon-10) before the extraction.
Cooperativity was modeled according
to Fig. SI, in which a radiolabeled probe (P) and an unlabeled
ligand (A) compete for sites on a tetravalent receptor (R) that
presumably is an oligomer (tetramer). Bivalent (dimer) and trivalent
(trimer) receptors can be described by truncated forms of R. It is
assumed that the oligomeric integrity of R is retained under the
conditions of the binding assays; the model can accommodate processes
in which dissociated monomers regroup without exchanging partners, but
there can be no exchange of individual subunits within the system.
Asymmetry cannot be detected with the present data, and all sites of
the vacant oligomer were assumed to bind P or A with the microscopic
dissociation constant K or K
(e.g.K
=
[P][R]/[PR]). (
)The parameters p
and a
represent the
cooperativity factors for binding of the jth equivalent of P
or A to form RP
or A
R, respectively (j
2) (e.g. [RP
][P]/[RP
]
=
p
K
); the parameters c
, c`
, and c"
represent cooperativity factors in the formation of mixed
complexes, as shown in Fig. SI.
Figure SI:
The model was fitted to the
data by means of , in which B represents total binding of the radioligand; [P]
represents specific binding at a total concentration
[P]
, and NS is the fraction of unbound
radioligand that appears as nonspecific
binding.
The value of [P] was calculated according
to , , or for the bivalent,
trivalent, or tetravalent forms of R, respectively. The concentration
of each complex was obtained as described below. Coefficients greater
than 1 reflect the degree of occupancy by P, since R is defined as
multivalent, and the multiple forms of liganded receptor at each level
of occupancy. Stoichiometrically equivalent species are functionally
indistinguishable with the present data, and the microscopic
dissociation constant therefore was taken as the same for all vacant
sites on partially liganded R (e.g. [P][POOA]/[PPOA] =
[P][POOA]/[POPA], where O represents a
vacant site on tetravalent R). The triliganded species
A
RP
and A
RP
also were
taken as identical (i.e.c
= c`
), as were the tetraliganded species
A
RP
, A
RP
, and
A
RP
(c
= c`
c"
). They are not
necessarily indistinguishable in binding studies, but the additional
parameters were found to be without effect on the sum of
squares.
The values of individual terms in Equations 2a-2c were
calculated from the equilibrium dissociation constants and the free
concentrations of the reactants. The latter were obtained by solving a
set of implicit equations derived from the equations of state for each
ligand and the receptor. Solutions were obtained according to the
Newton-Raphson procedure, but successful convergence often required
initial estimates that were close to the desired roots. Values near or
equal to the answer were computed in an iterative procedure that
involved successive estimates of the free concentration of R on the one
hand and of P and A on the other. The total concentrations of both
ligands were taken as equal to the free concentrations and substituted
in the equation of state for the receptor to obtain the value of
[R] (i.e. [R] = f{[R], [P],
[A], K
, K
, p
, a
, c
}); the latter then was used to obtained
revised estimates of the former (e.g. [P] = f{[P]
, [A],
[R], K
, K
, p
, a
, c
}), and the cycle was repeated until
convergence occurred. Further details regarding the formulation of
cooperative models have been described elsewhere (Wells, 1992).
All parameters were estimated by nonlinear regression, and values at successive iterations of the fitting procedure were adjusted according to the algorithm of Marquardt(1963). The squared residual for each measurement was weighted by the reciprocal of the estimated variance. Most analyses involved multiple sets of data, and the assignment of shared parameters is described where appropriate. Other details related to the statistical procedures have been described previously (Wong et al., 1986; Chidiac and Wells, 1992; Sinkins et al., 1993).
Figure 1:
Identification of M
muscarinic receptors and various subunits of G-proteins in purified
fractions from porcine atrial tissue. Freshly prepared samples purified
from solubilized sarcolemmal membranes were concentrated, run on
SDS-polyacrylamide gel electrophoresis, and then blotted onto a
nitrocellulose membrane as described under ``Experimental
Procedures.'' The data are from a single Western blot which was
screened successively with antibodies either individually or in
combination. Specific bands were detected by enhanced chemiluminescence
following incubation with anti-IgG congugated to horseradish
peroxidase, and the signal was recorded onto x-ray film by exposure for
various times. The contents of each lane were as follows (A-D); further details are described under
``Experimental Procedures'': lane 1, first stage of
solubilization; lane 2, second stage of solubilization; lane 3, pass-through from DEAE-Sepharose; lane 4, low
salt wash from DEAE-Sepharose; lane 5, high salt elution from
DEAE-Sepharose after concentration; lane 6, pass-through from
ABT-Sepharose; lane 7, high salt wash from ABT-Sepharose; lane 8, low salt wash from ABT-Sepharose; lane 9,
carbachol-elution from ABT-Sepharose after concentration and filtration
on Econo-Pac P6 (preparation M2G); lane 10, pass-through from
hydroxyapatite; lane 11, low salt wash from hydroxyapatite; lane 12, high salt elution from hydroxyapatite after
concentration and filtration on Econo-Pac P6 (preparation M2). All of
the lanes contained the same amount of protein, which corresponds in lanes 9 and 12 to approximately 50 ng of muscarinic
receptor as measured with 300 nM [
H]NMS. A, M
muscarinic receptor (31-1D1 at 1:1000) and
-G
(QL at 1:3000) were detected by exposure of the
film to ECL for 20 s. The open arrow marks the position of the
receptor, and the black arrow marks the position of
. B,
-G
and common
-subunits (both RM/1 and SW/1 at 1:6000) were detected by exposure
of the film to ECL for 12 min. A lengthy exposure was required to
reveal the small amounts of
in M2G and of
-subunits in M2. The striped arrow marks the position of
, and the gray arrow marks the position of
-subunits. C,
-G
(AS/7 at 1:3000) was
detected by exposure of the film to ECL for 3 min. D,
-G
(GC/2 at 1:10,000) was detected by exposure of the
film to ECL for 40 s. E, relative densities of G-protein
subunits in preparation M2 (solid bars) and preparation M2G (hatched bars). The data were quantified by densitometry of
the bands in lane 9 (M2G) and lane 12 (M2) following
exposure of the blot for 40 s (A, D) or 3 min (C). Each value was divided by that obtained for the M
receptor in the same lane, and the normalized values are shown
relative to the corresponding values in M2G. Densities corresponding to
the
-subunit were measured using a much shorter exposure than that
shown in the figure in order to avoid saturation of the
film.
Fractions obtained at almost every stage of the subsequent
purification yielded a positive signal with each of the
G-protein-specific antibodies used for screening, although the relative
amounts of each subunit or subtype differed from fraction to fraction.
G-protein subunits were retained on the anion-exchange column and were
eluted with the receptor (Fig. 1, lane 5). Most of the
immunoreactivity was removed during subsequent processing on
ABT-Sepharose, when G-protein subunits either were not retained (Fig. 1, lane 6) or tended to come off during the
washing procedure (Fig. 1, lanes 7 and 8).
Elution of the affinity column with the low salt buffer at room
temperature removed a small amount of M receptor, which ran
on the gel only as a large aggregate (Fig. 1A, lane 8).
The receptor was accompanied by
-subunits (Fig. 1B,
lane 8) and by the
-subunit of G
(Fig. 1C, lane 8); other
-subunits were not
detected.
The M muscarinic receptor was recovered from
the affinity column by elution with carbachol (preparation M2G).
Western blots treated with the receptor-specific antibody 31-1D1
revealed a diffuse band of material with the mobility expected of a
globular protein with a molecular mass of 60-75 kDa (Fig. 1A, lane 9); the receptor was accompanied by
-subunits and by the
-subunits of G
,
G
, G
, and G
(Fig. 1, A-D, lane 9). The degree of purification is indicated by
the relative intensities of bands arising from antibodies to the
receptor on the one hand and to the
- and
-subunits on the
other: in contrast to the strong signals obtained with
G-protein-specific probes, the receptor was undetectable or nearly so
in crude solubilized membranes (Fig. 1A, lane 2) and at
each stage of the purification up to and including the high salt wash
from the affinity column (Fig. 1A, lane 7). Bands
identified by different G-protein-specific antibodies differed in their
relative intensity between the eluate from the affinity column (Fig. 1, A-D, lane 9) and that from the
anion-exchange column (Fig. 1, A-D, lane 5). The
different subunits thus appear to differ in their tendency to copurify
with the receptor. Further processing of the affinity-purified receptor
on hydroxyapatite yielded a product in which the relative amount of
each measured G-protein was markedly reduced (preparation M2) (Fig. 1, A-D, lanes 10-12). Smaller,
epitope-containing bands were not detected in M2 or M2G, indicating
that both preparations were free of major proteolytic activity.
The
intensities of the immunoreactive bands identified on Western blots
were quantified by densitometry, and the results are summarized in Fig. 1E. Preparation M2 (Fig. 1, A-D,
lane 12) contained less than 6% of the -subunits and less
than 7% of the
-subunits found in preparation M2G (Fig. 1, A-D, lane 9). The residual
-subunits in M2 were
mostly
(Fig. 1D); there was a lesser
amount of
(Fig. 1C), whereas
(Fig. 1A) and
(Fig. 1B) were undetectable. The amount of
in M2G (Fig. 1B, lane 9) was only 8%
of that found in the fraction eluted from the anion-exchange column (Fig. 1B, lane 5). This value is substantially less
than the mean of 37% estimated for the other
-subunits. G-proteins
identified in M2G presumably copurified as a complex with the receptor,
at least in the case of G
and G
; since the
ratio of total G-protein to receptor was about 20-fold less in M2 than
in M2G, the ratio in M2 is likely to be negligible.
The mean
specific binding of [H]NMS in five different
preparations of solubilized atrial membranes was 8 ± 2 pmol/mg
of protein at a radioligand concentration of 300 nM. Receptor
eluted by carbachol from the affinity column and processed further to
obtain the preparations M2G and M2 represented 30-40% of the
sites originally present in the solubilized membranes; accordingly, the
purified material is likely to be representative of the total
population of muscarinic receptors in porcine atria. The apparent
capacity of M2G and M2 for [
H]NMS was 8-12
pmol/ml in a volume of about 2 ml for each preparation. Both M2G and M2
were prepared in parallel from about 200 g of original tissue, and the
combined yield was 3-5 µg of M
muscarinic
receptor. The level of purity was 4-10% based on estimates of
total recovered protein, an assumed stoichiometry of 1 eq of
[
H]NMS/molecule of receptor, and a theoretical
specific activity of 19 nmol/mg for a pure protein with a molecular
mass of 51,670 daltons (Kubo et al., 1986). This is consistent
with the patterns obtained on silver-stained polyacrylamide gels, where
M2G and M2 both yielded a major diffuse band corresponding to
60-75 kDa and several minor bands spaced over the length of the
gel; the latter were more prominent in M2G than in M2. Calculations
based on the apparent capacity for [
H]NMS may
underestimate the actual yield 2-fold or more. As described below,
estimates of maximal specific binding in each preparation differed
among three different radiolabeled antagonists.
The pattern of
immunoreactivity shown by the receptor-specific antibody in Fig. 1A was characteristic of preparations examined
shortly after purification. Additional bands corresponding to discrete
multiples of the monomeric receptor were found in purified material
that had been stored for a period of weeks at 2 °C. The larger
forms exhibited the mobility of trimeric and tetrameric homooligomers (Fig. 2), and they were observed regardless of whether the
sample in the loading buffer was boiled or kept at room temperature for
up to 20 min prior to electrophoresis. Similar results have been
reported previously by Leutje et al.(1987), who also found a
time-dependent appearance of apparent oligomers identified by 31-1D1 on
Western blots of purified porcine M receptor.
Figure 2:
Detection of multimeric forms of M muscarinic receptors purified from porcine atrial tissue. After
storage at 2 °C for 4 weeks, a sample of preparation M2G purified
from solubilized sarcolemmal membranes was concentrated, run on
SDS-polyacrylamide gel electrophoresis, and then blotted onto a
nitrocellulose membrane as described under ``Experimental
Procedures.'' The lane was loaded with approximately 100 ng of
receptor, as estimated by the binding of 300 nM [
H]NMS. The conditions for the probing of
the blot with antibody 31-1D1 were similar to those described in the
legend to Fig. 1; the bands were revealed by exposure of the
film to ECL for 20 s. The arrows mark the position of the
ladder of bands of the M
muscarinic receptor, with the
molecular sizes indicated in kilodaltons.
Cooperativity was assessed in terms of Fig. SIin
order to probe the nature of the effect and the minimum number of
interacting sites. The results of the analyses are illustrated in Fig. 3, and the parametric values are listed in Table 1.
Data acquired with [H]AF-DX 384 can be described
in terms of a dimeric receptor in which the first equivalent of the
radioligand is positively cooperative with respect to the second (i.e. -log p
> 0) (Fig. 3A). Data acquired with
[
H]NMS require a receptor that is at least
trimeric (Fig. 3B), since the sum of squares is 13.5%
lower than that for a dimer. The first equivalent of radioligand is
negatively cooperative with respect to the second (i.e. -log p
< 0), and the second
equivalent is positively cooperative with respect to the third (i.e. -log p
> 0).
Figure 3:
Binding of radiolabeled antagonists to
purified muscarinic receptors in M2. Total binding was measured
following equilibration of the receptor with
[H]AF-DX 384 (A) or
[
H]NMS (B) at the concentrations shown
on the abscissa. The upper and lower sets of data in each
panel represent binding of the radioligand alone and in the presence of
1 mM NMS, respectively. The lines in each panel
represent the best fit of to the pooled data from five
experiments; the value of [P]
was calculated by
assuming a dimeric receptor for [
H]AF-DX 384 (A, ) and a trimeric receptor for
[
H]NMS (B, ). The fitted
estimates of the parameters are listed in Table 1. Values plotted
on the ordinate represent specific binding, calculated as
total binding (B
) less the fitted estimate of
nonspecific binding at the same concentration of unbound radioligand.
Individual estimates of B
were normalized to
the mean values of [R]
and NS according
to the expression B`
= B
[f(x
,
a)/f(x
, a)], as described previously (i.e. Equation 5 in Chidiac and Wells(1992)). Each experiment
included measurements with both radioligands, and the different symbols denote data from different
experiments.
As
illustrated in Fig. 3, the capacities inferred from apparently
saturating concentrations of [H]AF-DX 384 and
[
H]NMS could differ appreciably for the two
radioligands. The disparity varied in magnitude among different
preparations and over time within the same preparation. With freshly
purified receptor, the apparent capacity for
[
H]AF-DX 384 typically was comparable with or
slightly less than that for [
H]NMS; after several
days of storage, however, the capacity for
[
H]AF-DX 384 averaged about one-half of that for
[
H]NMS (Fig. 3, Table 1).
Preparation M2G resembled M2 in that the Hill coefficient exceeded 1
for [H]AF-DX 384, but was lower and more variable
for [
H]NMS: the range from six experiments was
0.65-1.05, with a mean of 0.82 ± 0.07. The apparent
capacity for [
H]NMS tended to exceed that for
[
H]AF-DX 384 as the preparation aged. In both
preparations, [
H]QNB revealed Hill coefficients
near 1 and an apparent capacity greater than that associated with
either [
H]NMS or [
H]AF-DX
384. To facilitate a mechanistic description of the system, the binding
of [
H]AF-DX 384, [
H]NMS,
and [
H]QNB was measured concomitantly in fresh
samples of M2 and M2G prepared in parallel from the same batch of
tissue. The apparent capacity for [
H]QNB in M2
was about 1.5-fold that for [
H]AF-DX 384 or
[
H]NMS (Fig. 4A), whereas the
corresponding difference in M2G was about 2-fold (Fig. 4B and Fig. 5A). The same differences were found
repeatedly in experiments with [
H]QNB obtained
from two different manufacturers, with three lots of
[
H]AF-DX 384 and with two lots of
[
H]NMS. Moreover, the data illustrated for M2 and
M2G in Fig. 4were obtained with material from the same lot of
each radioligand. It follows that the differences in maximal observed
binding cannot be attributed to an error in the specific radioactivity
of one or another antagonist.
Figure 4:
Binding of radiolabeled antagonists to
purified muscarinic receptors in M2 and M2G. Total binding was measured
following equilibration of the receptor in preparation M2 (A)
or in preparation M2G (B) with [H]AF-DX
384 (
), [
H]NMS (
), or
[
H]QNB (
) at the concentrations shown on
the abscissa. All of the data were acquired in the same
experiment, which included parallel assays containing 1 mM unlabeled NMS and shown in each panel as the base line. The lines represent the best fit of to all of the
data taken together; the value of [P]
was
calculated by assuming a tetrameric receptor (), and the
fitted estimates of the parameters are listed in Table 2. Values
plotted on the ordinate represent specific binding, calculated
as total binding (B
) less the fitted estimate
of nonspecific binding at the same concentration of unbound
radioligand.
Figure 5:
Noncompetitive inhibition by unlabeled NMS
in M2G. A, total binding of [H]QNB
(
,
) or [
H]NMS (
,
) was
measured following equilibration of the receptor with the radioligand,
either alone (
,
) or together with 1 mM unlabeled
NMS (
,
), at the concentrations shown on the abscissa. The data represent a total of three experiments in
which assays with each radioligand were performed in parallel. B, total binding was measured following equilibration of the
receptor with [
H]QNB (
, 7.8 nM;
, 21 nM) and unlabeled NMS at the concentrations shown
on the abscissa. The solid lines in both panels
represent the best fit of to the pooled data from all five
experiments. The receptor was assumed to be tetrameric, and the value
of [P]
was computed according to .
Single values of K
and a
were common to all of the data acquired at graded
concentrations of unlabeled or labeled NMS, with the assumption that K
= K
and a
= p
for the latter. Single values of K
and p
were common to all of the data acquired
in the presence of [
H]QNB. Single values of c
were common to the two sets of data
acquired at graded concentrations of unlabeled NMS. The fitted
estimates are as follows: for NMS, -log K
= 8.48 ± 0.04, -log a
= -0.66 ± 0.11, -log a
= -2.38 ± 0.46; for QNB, -log K
= 8.47 ± 0.11, -log p
= 0.50 ± 0.29, -log p
= -0.69 ± 0.33, -log p
= -0.02 ± 0.39; for NMS and
QNB, -log c
= 0.25 ± 0.20. The
quantities -log c
, -log a
, and -log c
were
defined only by upper bounds (-log c
<
-2, -log a
< 0, -log c
< 0) and were fixed accordingly to obtain the
fit illustrated in the figure (-log c
= -4, -log a
=
-2, -log c
= -4); they
otherwise were without effect on the sum of squares or on the values of
other parameters. Values of [R]
and NS were common to data from the same experiment. The means for the
data shown in the figure are 134 ± 26 pM for
[R]
and either 0.000049 ± 0.000017
([
H]NMS) or 0.00038 ± 0.00022
([
H]QNB) for NS. Values of B
were adjusted as described in the legend to Fig. 3, and nonspecific binding was subtracted from B`
to obtain the values plotted on the ordinate. Points shown at the lower and upper ends of the abscissa in B represent binding in the absence of
unlabeled ligand and in the presence of 1 mM NMS; the former
were included in the analysis at log [A] = -15.
The dashed line in A represents the inferred binding
of NMS when the data acquired at graded concentrations of
[
H]QNB (A) and NMS (B) were
analyzed in terms of distinct and mutually independent sites (i.e. Equations 76 and 77 in Wells(1992)). Single values of K
([
H]QNB), K
(NMS), and F
(j = 1, . . . n) were common to all of the data, and
values of [R]
and NS were common to data
from the same experiment. The fit was marginally better with two
classes of sites rather than one (p = 0.067), and the
parametric estimates are as follows: -log K
= 8.84 ± 0.07, -log K
= 7.57 ± 0.56, -log K
= 8.29 ± 0.11, -log K
= 6.84 ± 0.80, F
= 0.20
± 0.09. The line was calculated from the values of K
and F
.
Neither a dimeric nor a trimeric
receptor is sufficient for Fig. SIto describe the behavior of
all three ligands in both preparations, but excellent agreement was
obtained with a tetramer. The fitted curves are shown in Fig. 4,
and the parametric values are listed in Table 2. The need for a
tetravalent receptor derives in part from the multiple states of
affinity apparent in the binding of [H]AF-DX 384
and [
H]NMS (e.g.n
1), which imply at least two interacting sites in terms of
cooperativity, and in part from the relative apparent capacities for
[
H]QNB and either [
H]AF-DX
384 or [
H]NMS in preparations M2 (i.e. 1.5:1) and M2G (i.e. 2:1). Since the oligomeric
composition of the receptor is assumed to be the same throughout, the
pooled data cannot be described with fewer than four interacting sites.
In the context of Fig. SI, the differences between M2 and M2G
can be attributed entirely to differences in the degree of
cooperativity between successive equivalents of the radioligand.
Estimates of K were similar or the same in both
preparations, and the weighted sum of squares was not increased
significantly with a single value for each antagonist (Table 2).
The comparatively higher binding of [
H]QNB in M2G
emerges as a manifestation of reduced negative cooperativity between
the last two equivalents of the radioligand. The macroscopic
dissociation constants for the second and third equivalents of
[
H]QNB differ by 3-fold or less between the two
preparations; in contrast, the corresponding value for the fourth
equivalent is 27-fold higher in M2 than in M2G owing to a 35-fold
difference in the value of p
. (
)The
receptor in M2G therefore is saturated at concentrations of
[
H]QNB that occupy only about three-quarters of
the sites in M2. Similarly, a high degree of negative cooperativity can
account for the comparatively low level of maximal binding observed
with [
H]NMS and [
H]AF-DX
384 in both preparations: that is, the values of p
and p
are sufficiently large to exclude the
third and fourth equivalents of either radioligand at the
concentrations used.
The notion that apparently
unreactive sites reflect cooperativity is consistent with the
inhibitory effect of unlabeled NMS on the binding of
[H]QNB. The data illustrated in Fig. 5B were obtained in M2G at two concentrations of
the radioligand, 7.8 and 21 nM. The corresponding levels of
occupancy in the absence of unlabeled NMS were 66 and 81%,
respectively; [
H]QNB therefore occupied
26-55% more sites than were labeled by tritiated NMS at 0.56
µM, the highest concentration used (Fig. 5A). Unlabeled NMS nevertheless achieved 95%
inhibition at concentrations of only 0.48 and 1.4 µM,
respectively (Fig. 5B). Comparatively low
concentrations of unlabeled NMS therefore inhibited the binding of
[
H]QNB to more sites than were occupied by
labeled NMS at similar or higher concentrations.
A good description
of the data can be obtained in terms of Fig. SI, as illustrated
by the solid lines shown in Fig. 5. In assays with
unlabeled NMS and [H]QNB, the noncompetitive
effect of NMS reflects the formation of a biliganded species (i.e. NMS
R
[
H]QNB) with low affinity
for additional equivalents of either antagonist (-log c
< -2, -log c
< 0). To confirm the noncompetitive nature of the effect shown
in Fig. 5B, the data acquired at graded concentrations
of [
H]QNB and unlabeled NMS also were analyzed by
assuming that both ligands compete for a heterogeneous population of
mutually independent sites. The binding profile inferred for NMS alone
diverges from that measured independently with radiolabeled NMS, as
shown by the dashed line in Fig. 5A; moreover,
the inferred concentration for half-maximal binding exceeds the
measured concentration by 2-3-fold. Noncompetitive effects
therefore account at least in part for the inhibitory effect of NMS,
and a similar pattern has been observed in the binding of AF-DX 384.
Figure 6:
Inhibition of
[H]AF-DX 384 by oxotremorine-M in M2. Total
binding was measured following equilibration of the receptor with
[
H]AF-DX 384 (20-33 nM) and
oxotremorine-M at the concentrations shown on the abscissa.
Each point represents the mean (±S.E.) from four
experiments. The line represents the best fit of to the pooled data from nine experiments: the four
represented in the figure and five experiments in which binding was
measured at graded concentrations of [
H]AF-DX
384. The data from the latter are illustrated in Fig. 3A. The receptor was assumed to be tetrameric, and
the value of [P]
in was computed
according to . Single values of K
and p
were common to all of the data, and
single values of K
, a
, and c
were common to data acquired at graded concentrations of the
agonist. The fitted parametric values are as follows: -log K
= 6.24 ± 0.22, -log K
= 7.13 ± 0.03, -log a
= -2.72 ± 0.24, -log a
= 0.32 ± 0.30, -log p
= 0.29 ± 0.10, and -log c
= -2.17 ± 0.06. The values
of -log p
and -log p
were fixed at -4 and 0, respectively, in accord with the
failure of [
H]AF-DX 384 to label more than about
67% of the sites labeled by [
H]QNB or more than
50% of the presumed total capacity (e.g.Fig. 4). The
quantities -log a
and -log c
were defined only by upper bounds, and
-log c
was defined only by a lower bound.
They otherwise were without effect on the sum of squares or the values
of other parameters, except for a negative correlation between c
and c
. The values therefore
were fixed appropriately to obtain the fit illustrated in the figure (i.e. -log a
= -4,
-log c
= 2, -log c
= -4). Values of
[R]
and NS were common to data acquired
within the same experiment; the means for the data shown in the figure
are 128 ± 35 pM and 0.00014 ± 0.00006,
respectively. The mean concentration of [
H]AF-DX
384 was 28 nM. Values of B
were
adjusted as described in the legend to Fig. 3, and the resulting
values of B`
at the same concentration of
agonist were averaged to obtain the mean (±S.E.) plotted on the ordinate. Points shown at the lower and upper ends of the abscissa represent binding in the absence of agonist and in
the presence of 1 mM unlabeled NMS, respectively; the data
were included in the analysis, with log [A] taken as
-15 and 0.
The binding of
oxotremorine-M in M2G is illustrated in Fig. 7, where the data
were obtained at concentrations of [H]AF-DX 384
in excess of its EC
. In the absence of guanyl nucleotide,
the agonist described a strictly inhibitory pattern similar to that
illustrated for preparation M2 in Fig. 6. In contrast, the
pattern obtained at a saturating concentration of GMP-PNP was
bell-shaped at intermediate concentrations of oxotremorine-M. An
agonist-dependent increase in the specific binding of
[
H]AF-DX 384 is inconsistent with any scheme
based on mutually independent sites in a system at equilibrium; rather,
the data point to cooperative effects between multiple equivalents of
agonist and radioligand bound concurrently at interacting sites. The
effect of GMP-PNP suggests that G-proteins in M2G interacted with the
receptor and retained the functionality that is characteristic of
native membranes and reconstituted preparations.
Figure 7:
Effect of GMP-PNP and oxotremorine-M on
the binding of [H]AF-DX 384 in M2G. Total binding
was measured following equilibration of the receptor with
[
H]AF-DX 384 (36-100 nM) and
oxotremorine-M at the concentrations shown on the abscissa;
each experiment included parallel assays carried out in the absence of
guanyl nucleotide (
) and in the presence of 0.1 mM GMP-PNP (
). Each point represents the mean
(±S.E.) from five experiments performed with receptor from three
batches of M2G. The line represents the best fit of to the pooled data from eight experiments: the five
represented in the figure and three experiments in which binding was
measured at graded concentrations of [
H]AF-DX 384
(not shown). The latter did not include GMP-PNP, which was found in
separate experiments to be without discernible effect on the binding of
the radioligand alone. The receptor was assumed to be tetrameric, and
the value of [P]
in was computed
according to . Single values of K
and p
were common to all of the data. A
single value of K
was common to all data acquired
at graded concentrations of the agonist; values of a
and c
were common to those
data acquired either with or without GMP-PNP. The fitted parametric
values are as follows (±GMP-PNP): -log K
= 6.46 ± 0.62 (±), -log K
= 7.15 ± 0.04 (±),
-log a
= -0.86 ±
0.97(-) and -1.39 ± 0.71 (+), -log a
= -0.26 ± 0.56(-) and
0.29 ± 0.53 (+), -log p
=
-0.09 ± 0.12 (±), -log c
= 1.45 ± 0.83(-) and 0.22 ± 0.94
(+), -log c
= -0.45
± 0.09(-), and 0.98 ± 0.45 (+). The parameters a
, p
, and c
tend to be correlated, and most are not
well defined by the present data. There was a significant increase in
the sum of squares if GMP-PNP was assumed to be without effect on the
value of c
(p = 0.00089), and a
smaller increase occurred in the case of c
(p = 0.025); there was no appreciable change in case of a
or a
(p >
0.07). The values of -log p
and -log p
were fixed at -4 and 0, respectively. The
values of -log a
and -log c
were fixed at -4, which is below the value
at which either parameter affects the sum of squares or the values of
other parameters. Values of [R]
and NS were common to data acquired within the same experiment; the means
for the data shown in the figure are 128 ± 41 pM and
0.00030 ± 0.00010, respectively. The mean concentration of
[
H]AF-DX 384 was 58.7 nM. Further
details are described in the legend to Fig. 6.
In terms of Fig. SI, an agonist-dependent increase in binding can occur only
at concentrations of the radioligand that are half-saturating or less
with respect to all sites. Since the concentration of
[H]AF-DX 384 was 36-100 nM in the
experiments represented in Fig. 7, approximately 53-77% of
the observable sites were labeled in the absence of agonist. It follows
that an equal or greater number of coupled sites must remain unlabeled
by the radioligand at the concentrations used, in accord with the
suggestion that negative cooperativity accounts for the 2-fold
difference in maximal observable binding between
[
H]QNB and [
H]AF-DX 384. A
tetrameric receptor yields good agreement between Fig. SIand
the data, as illustrated by the fitted curves in Fig. 7. There
was no appreciable effect of GMP-PNP on the affinity of the agonist for
the vacant receptor, and all data therefore were assigned a single
value of K
. As indicated by the fitted parametric
values derived from the analysis (Fig. 7, legend), the
nucleotide acts primarily by affecting the degree of cooperativity
between the agonist and the radioligand (i.e.c
).
All of the data can be accounted
for by cooperative effects within a receptor that is at least
tetravalent (i.e.Fig. SI). A 4-fold stoichiometry or
multiple thereof is consistent with the ratios of about 1.5:1 and 2:1
obtained in M2 and M2G, respectively, for the maximal observed binding
of [H]QNB and [
H]AF-DX 384.
Heterogeneity implied by Hill coefficients greater than 1 is
interpreted as positive cooperativity between two or more equivalents
of the same ligand. Sites labeled by [
H]QNB but
not by [
H]AF-DX 384 or
[
H]NMS emerge as a consequence of pronounced
negative cooperativity in the binding of the latter; similarly,
differences in negative cooperativity can account for relative
differences in the apparent capacities of M2 and M2G for different
probes. Multiphasic effects of oxotremorine-M on the binding of
[
H]AF-DX 384 are the net result of cooperativity
between the two ligands and between successive equivalents of the
agonist alone; bell-shaped effects and strictly inhibitory behavior
differ only in the degree of cooperativity between agonist and
radioligand. The complete inhibition of [
H]QNB by
unlabeled NMS and the bell-shaped effect of oxotremorine-M on
[
H]AF-DX 384 both were observed with the
radioligand at high levels of occupancy relative to apparent capacity,
thereby confirming the existence of functionally linked sites not
labeled at practicable concentrations of [
H]NMS
or [
H]AF-DX 384.
Ligand-dependent differences
in binding capacity have been reported previously for G-protein-linked
receptors in native membranes. The dopamine antagonist
[H]nemonapride has been found to label
1.5-2-fold more D
receptors than were labeled by
[
H]spiperone in membranes from Sf9 cells,
mammalian cell lines, and mammalian brain (Seeman et al.,
1992; Ng et al., 1994b and references cited therein).
Similarly, more muscarinic receptors were labeled by
[
H]QNB than by [
H]NMS or
[
H]AF-DX 384 in homogenates of various tissues
(Lee and El-Fakahany, 1985; Castoldi et al., 1991; Entzeroth
and Mayer, 1991). The differences were attributed to the comparatively
high lipophilicity of QNB or to a heterogeneous population of receptors
differentiated only by [
H]AF-DX 384. Since the
affinity of [
H]AF-DX 384 differs by less than
10-fold among four of the five subtypes of muscarinic receptor (e.g. Dorje et al.(1991)), it seems unlikely that a
subpopulation of one subtype or another could be overlooked.
Differences related to the lipophilic nature of QNB imply a
subpopulation of receptors that are less accessible to the more
hydrophilic ligand. Unless access is denied absolutely, however, all
ligands are expected to yield the same capacity in a system at
equilibrium.
The present results recall earlier suggestions of
cooperativity in the binding of ligands to -adrenergic and
muscarinic receptors in native membranes (Limbird et al.,
1975; Mattera et al., 1985; Boyer et al., 1986).
Since there appears to be one binding site per polypeptide chain (e.g. Dixon et al.(1988)), cooperativity is
indicative of an oligomeric array comprising multiple equivalents of
the M
protein. Oligomers of muscarinic receptors also have
been inferred from binding studies on native and affinity-alkylated
membranes from rat brainstem, where M
receptors have been
suggested to occur in functionally asymmetric pairs (Potter et
al., 1991). Furthermore, complementary chimeras of
-adrenergic and M
muscarinic receptors (e.g.
/m3 and m3/
) in
membranes from COS-7 cells were found to bind adrenergic or muscarinic
antagonists only when the two recombinant proteins were coexpressed
(Maggio et al., 1993). Kinetic studies on the binding of
[
H]oxotremorine-M to recombinant m2 receptors
expressed in CHO cells have been interpreted in terms of an
agonist-regulated interconversion between monomers and asymmetric
dimers (Hirschberg and Schimerlik, 1994).
It is implicit in analyses
with Fig. SIthat the receptors were exclusively tetrameric.
This arrangement may be unjustified, at least in principle, for
material that is potentially heterogeneous despite a comparatively high
degree of purity. Denaturation or differences in post-translational
processing may affect function while having little or no effect on
electrophoretic mobility. Also, differences in the apparent capacity
for [H]NMS and [
H]AF-DX 384
and in the occurrence of oligomers on Western blots tended to emerge
over time. It follows that the purified material used in this study may
contain oligomers of different size, and the prevalence of one or
another form may depend on the age of the preparation. Cooperativity
nevertheless is required by the data, and a tetrameric receptor emerges
as the simplest acceptable arrangement. Since the model accounts for
all of the results, any other structural forms apparently can be
accommodated within that framework; tetramers may have predominated, or
a mixture of forms may have been indistinguishable from tetramers
alone. Alternatively, time-dependent changes in relative apparent
capacity may have arisen from changes in the cooperative properties of
an otherwise stable tetramer.
In contrast to the oligomeric form
predicted by cooperative effects, G-protein-linked receptors typically
appear as monomers upon examination of their hydrodynamic properties.
In some studies, however, the monomeric form has been accompanied by
larger species that suggest the existence of oligomers. Molecular
weights inferred from radiation inactivation point to dimers of
- and
-adrenergic receptors (Venter
and Fraser, 1983). Also, glucagon receptors cross-linked with
radiolabeled agonist appeared to migrate as dimers during
ultracentrifugation on sucrose density gradients (Herberg et
al., 1984). What appear to be dimers of metabotropic glutamate
receptors (Pickering et al., 1993), c-myc-5-HT
receptors, c-myc-D
dopamine receptors, and
D
dopamine receptors (Ng et al., 1994a, 1994b)
have been identified on Western blots following electrophoresis of
solubilized Sf9 membranes on SDS-polyacrylamide gels. Muscarinic
receptors tagged with a labeled antagonist have exhibited the
electrophoretic mobility expected of dimers (Avissar et al.,
1983) or of dimers and larger aggregates (Dadi and Morris, 1984).
Similarly, immunoreactive bands corresponding to dimers and larger
oligomers have been found with M
muscarinic receptors
purified from Sf9 membranes (Parker et al., 1991) and, in the
present study, from porcine atria (Fig. 2).
Multimeric forms identified by their molecular size may be artifacts unrelated to those inferred from the binding studies. Oligomers found after electrophoresis in discontinuous buffers probably are stabilized by intermolecular disulfide bonds; while the samples typically are pretreated with a reducing agent, the receptor and the reagent can separate during the initial moments of migration (Dadi and Morris, 1984). Such cross-links may be of no biological relevance, but the nature of the products suggests a process that is specific nonetheless. Disulfide bonds arising from random interactions among receptors and other proteins might be expected to yield large aggregates of indeterminate molecular size, particularly with the mixtures of components obtained from native membranes. In contrast, the observed oligomers are typically of defined size and appear to comprise multiples of a single gene product. This pattern suggests that intermolecular cross-linking occurs subsequent to the formation of specific complexes in the highly concentrated environment of the stacking gel. If the process is specific, oligomers that survive or reassemble during electrophoresis may have some interactions in common with those that mediate the cooperative effects. It seems unlikely, however, that the patterns observed on polyacrylamide gels approximate the state of the system in the binding assays.
The total amount of G, G
, and
G
in M2 was only about 6% of that in M2G, yet both
preparations displayed multiple affinities for antagonists and for the
agonist oxotremorine-M. Similar results have been described previously,
in that purified muscarinic receptors apparently devoid of G protein
have revealed multiphasic binding of agonists in preparations from
brain (Haga et al., 1986) and heart (Peterson et al.,
1984; Ikegaya et al., 1990). Multiple classes of sites
therefore appear to exist independently of G-proteins, particularly in
preparation M2 but also in M2G. It follows that G-proteins in the
latter preparation act by modulating properties that are intrinsic to
the receptor. Fig. SIcan account in full for the properties of
M2G, including the bell-shaped effect of oxotremorine-M in the presence
of GMP-PNP; the differences between M2 and M2G emerge as nucleotide- or
G-protein-related changes in the degree of cooperativity between
successive equivalents of ligand. In contrast, the properties of M2G
are difficult to rationalize in terms of ligand-regulated equilibria
involving uncoupled and G-coupled receptors. The assays were performed
in solution at subnanomolar concentrations of the labeled sites. As
described above, it is expected that such a system would be shifted
predominantly to the uncoupled state under those conditions. Also, the
strictly inhibitory effect of oxotremorine-M in M2 is strikingly
different from the bell-shaped profile induced by GMP-PNP in M2G. If
coupling were precluded by the nucleotide, receptors devoid of
G-protein ought to be indistinguishable from receptors plus G-protein
at saturating concentrations of the nucleotide. The observed difference
suggests that, in preparation M2G, receptors and G-proteins remained
coupled in the presence of GMP-PNP.
G-proteins that copurify with
M receptors, and the nature of their participation in
allosteric effects, suggest that the RG complex is more stable than
implied by exchange-based models for binding and transduction. That
stability differs from the general observation, first demonstrated for
the
-adrenergic receptor (Limbird et al., 1980), that the
RG complex survives solubilization only if the membranes are
preincubated with agonist. A ligand-stabilized complex also is
suggested by the observation that muscarinic agonists increase the
amount of G
and G
immunoprecipitated
by antibodies specific for the M
muscarinic receptor
(Matesic et al., 1989; Matesic and Luthin, 1991; Matesic et al., 1991; Offermanns et al., 1994). The
requirement for an agonist is not universal, however, as illustrated by
the binding properties of nucleotide-sensitive receptors labeled by
[
H]histamine in solubilized preparations from rat
cortex (Wells and Cybulsky, 1990). Also, it has been shown previously
that guanyl nucleotides do not necessarily promote the dissociation of
G-proteins from M
muscarinic receptors (Matesic et
al., 1989; Poyner et al., 1989).
It remains unclear
which of the G-proteins in preparation M2G accounts for the sensitivity
to GMP-PNP or for the differences between M2G and M2. Both G and G
have been shown to interact with purified
M
muscarinic receptors in an agonist- and
nucleotide-sensitive manner (Ikegaya et al., 1990; Parker et al., 1991). G
is believed not to associate with
M
receptors (Hulme et al., 1990), but the amount
is comparatively small and may represent coelution of a nonspecific
nature. The amount of copurifying G
is comparatively
larger, but its occurrence also may be an artifact. G
is
believed to mediate the regulation of phospholipase C (Blank et
al., 1991), and M
receptors generally have been found
not to affect the hydrolysis of phosphoinositides unless expressed at
high density (Hulme et al., 1990); moreover, M
receptors have been shown to activate G
but not
G
in recombinant systems (Offermanns et al.,
1994). The multiple subtypes of G-protein found in preparation M2G
recall the observation that cardiac muscarinic receptors can be
immunoprecipitated with roughly equal amounts of G
and
G
(Matesic et al., 1991). The stoichiometry of the
components in M2G remains unresolved, but at least two explanations for
the observed heterogeneity come to mind: a single equivalent of one or
the other G-protein may associate with each equivalent of receptor, or
the G-proteins themselves may form a heterooligomeric array. The latter
possibility could account for the presence of G
, which
may couple with the receptor via G
or G
, and it
suggests a vehicle for the participation of G
in
M
-initiated signaling.
The suggestion of an oligomeric
array is consistent with hydrodynamic evidence that G-proteins occur as
multimeric complexes comparable in size with cross-linked tubulin
(Coulter and Rodbell, 1992; Jahangeer and Rodbell, 1993). A functional
role for such an arrangement is suggested by evidence that
cooperativity accounts for the agonist-sensitive, multiple states of
affinity recognized by guanyl nucleotides at G-proteins linked to
cardiac muscarinic receptors (Chidiac and Wells, 1992). Furthermore,
there is a striking similarity between the effect of GMP-PNP on the
binding of oxotremorine-M to M receptors (Fig. 7)
and the effect of carbachol on the binding of GDP to receptor-linked
G-proteins ( Fig. 4in Chidiac and Wells(1992)). The reciprocal
nature of these effects suggests a mechanism in which the functional
unit of transduction is a heterooligomer comprising multiple
equivalents of receptor and G-protein. In such a scheme, the allosteric
interactions between agonists and guanyl nucleotides derive from the
effects of one ligand on the degree of cooperativity in binding of the
other.
Earlier objections to the notion of a transient RG complex arose in large part from the failure of such exchange-based schemes to describe the binding properties of G-protein-linked receptors (Lee et al., 1986; Chidiac and Wells, 1992; Graeser and Neubig, 1993). If binding is governed by cooperativity, however, the notion of exchange re-emerges in a different light: the RG heterooligomer may coexist in exchange with oligomers of receptors on the one hand and of G-proteins on the other, or it may retain its oligomeric integrity throughout the signaling process (Chidiac and Wells, 1992). The latter possibility is favored by the stability, at least in some circumstances, of the RG complex upon solubilization and during purification.