From the
Among subfamilies of G-protein-coupled receptors, agonists
initiate several cell signaling events depending on the receptor
subtype (R) and the type of G-protein (G) or effector molecule (E)
expressed in a particular cell. Determinants of signaling
specificity/efficiency may operate at the R-G interface, where events
are influenced by cell architecture or accessory proteins found in the
receptor's microenvironment. This issue was addressed by
characterizing signal transfer from R to G following stable expression
of the
Members of the G-protein-coupled receptor superfamily play a
central role in cellular communication mediating the cell response to
numerous hormones and neurotransmitters. Via coupling to heterotrimeric
guanine nucleotide binding proteins (G),
In attempts to further define determinants of
signaling specificity, we focused our efforts on one receptor
subfamily,
Utilizing the
Receptor-mediated
activation of guanine-nucleotide binding proteins was determined by
measuring the increase in [
The apparent differences in signal transfer between
NIH-3T3/DDT
A major determinant of signaling specificity for
G-protein-coupled receptors is the cell-specific expression of the
primary signaling entities, R, G, and E. A secondary line of signaling
specificity lies at the R-G or G-E interface, where these interactions
may be influenced by cell architecture, stoichiometry, and accessory
proteins that regulate signal transfer from R to G or G to E. Through
the use of various experimental systems, we are attempting to define
the relative importance of these factors in determining the efficiency
and selectivity of receptor-effector coupling.
NIH-3T3 and PC-12
cells provide an interesting context in which to address the above
issues. Both cell types express distinct and common signaling entities
and differ in their cell architecture. Both the
Although R, G, and E are the major entities in the signaling
process, other putative regulatory proteins may participate either
indirectly by structural support(33, 34) ,
compartmentalization(35) , and signal cross-talk (36) or
directly by inhibiting or stimulating R-G or G-E coupling
events(37, 38, 39, 40, 41, 42, 43, 44, 45) .
The GTP-bound/GDP-bound state of p21
The augmented
response to agonist in PC-12 versus NIH-3T3 transfectants (in
the absence of PT pretreatment) may be due to expression of different
types/amounts of PT-sensitive G
The signal restoration
system involves 1) inactivation of receptor coupling to endogenous
G-proteins by pertussis toxin pretreatment and 2) restoration of signal
by addition of a purified brain G-protein preparation. The approach is
possible because agonist activation of G-protein in both cell types is
blocked by ADP-ribosylation of G-proteins with pertussis toxin. The
signal restoration system potentially allows the identification of
unknown entities that regulate the transfer of signal from R to G or G
to E. In this system, the transfer of signal from R to G occurs much
more efficiently when the receptor is functioning in the PC-12 versus the NIH-3T3 membrane environment. Furthermore, the
degree of agonist activation of G was ``dissociated'' from
the amount of receptor existing in the R-G-coupled state (high affinity
agonist binding). Thus, Gpp(NH)p-sensitive binding of the selective
A working hypothesis is that signaling
efficiency/specificity is determined in part by proteins found in the
receptor's microenvironment, which together with R, G, and E
contribute to the formation of a signal transduction complex at the
cytoplasmic face of the receptor. The signal transduction network for
this system may parallel that used by receptors with a single membrane
span motif where binding of agonist initiates a series of
protein-protein interactions dependent on protein phosphorylation. This
hypothesis is consistent with data suggesting the existence of
multimeric G-protein subunit complexes and the isolation of receptor or
G-protein subunits together with some
effectors(46, 47, 48, 49, 50) .
Detailed reconstruction of the receptor's microenvironment and
identification of complexed molecules will provide insight as to
mechanisms of signaling specificity and may allow targeting of
therapeutic agents to the R-G or G-E interface as opposed to the
receptor's hormone binding site.
We appreciate the discussion of Dr. Emir Duzic, who
generated the immunoblot shown in Fig. 3A. We thank Bronwyn Tatum
for assistance in the preparation of bovine brain G-protein.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
adrenergic receptor in two different
membrane environments (NIH-3T3 fibroblasts and the pheochromocytoma
cell line, PC-12). Receptor coupling to endogenous G-proteins in both
cell types was eliminated by pertussis toxin pretreatment and R-G
signal transfer restored by reconstitution of cell membranes with
purified brain G-protein. Thus, the receptor has access to the same
population of G-proteins in the two different environments. In this
signal restoration assay, agonist-induced activation of G was
3-9-fold greater in PC-12 as compared with NIH-3T3
-adrenergic receptor transfectants. The cell-specific
differences in signal transfer were observed over a range of receptor
densities or G-protein concentration. The augmented signal transfer in
PC-12 versus NIH-3T3 transfectants occurred despite a
2-3-fold lower level of receptors existing in the R-G-coupled
state (high affinity, guanyl-5`-yl imidodiphosphate-sensitive agonist
binding), suggesting the existence of other membrane factors that
influence the nucleotide binding behavior of G-protein in the two cell
types. Detergent extraction of PC-12 but not NIH-3T3 membranes yielded
a heat-sensitive, macromolecular entity that increased
S-labeled guanosine 5`-O-(thiotriphosphate)
binding to brain G-protein in a concentration-dependent manner. These
data indicate that the transfer of signal from R to G is regulated by a
cell type-specific, membrane-associated protein that enhances the
agonist-induced activation of G.
(
)receptor activation regulates numerous intracellular
effector molecules including ion channels, adenylyl cyclases, and
phospholipases. Many of these receptors share common signaling pathways
that are activated with varying degrees of efficiency by a particular
hormone. The specific intracellular signal initiated by agonist and the
strength of signal propagation likely depend on many factors, including
the relative types and/or amounts of the signal-transducing entities
(receptor (R)/G-protein/effector
(E))(1, 2, 3, 4, 5, 6, 7, 8) .
Additional determinants of signaling specificity include cell
architecture and perhaps accessory proteins that regulate events at the
R-G or G-E interface.
-adrenergic receptors
(
-AR)(5, 6, 7) . The
-AR family consists of three genetically defined
receptor subtypes (
,
,
-AR) and shares many properties with other
subfamilies of G-protein-coupled receptors in terms of signaling
flexibility(9) . Based on results in cells expressing the
endogenous receptor gene or in cells stably transfected with receptor
subtypes, members of this receptor subfamily couple to phospholipase
A
(10) , phospholipase C(11) , phospholipase
D(12) , calcium
flux(13, 14, 15, 16) ,
Na
/H
exchangers(17, 18) , p21
(19), or adenylyl
cyclases(6, 20, 21, 22, 23, 24, 25) .
The specific regulation of any of these effector molecules often
depends upon the receptor subtype, receptor density, and the
environment in which the receptor is operating.
-AR subfamily as a representative subgroup of
G-protein-coupled receptors, we have attempted to define how signaling
specificity is engineered in the intact
cell(5, 6, 7) . Critical determinants of
signaling efficiency/specificity may operate at the R-G interface. To
address this possibility, we developed a system to evaluate R-G
coupling in different membrane environments and to identify factors
that may regulate the level of activated G. The present report
indicates that the transfer of signal from R to G is regulated by
cell-specific membrane-associated proteins that alter the nucleotide
binding behavior of G.
Materials
H-Labeled
RX821002 (60 Ci/mmol) was obtained from Amersham Corp.
[
S]GTP
S, G
/G
antisera and
H-labeled UK14304 were purchased from
Dupont NEN. Tissue culture supplies were obtained from JRH Bioscience
(Lenexa, KS). Acrylamide, bisacrylamide, and SDS were purchased from
Bio-Rad. Nitrocellulose and polyvinylidene difluoride (Westran)
membranes were obtained from Schleicher & Schuell.
Propranolol,(-)epinephrine, and pertussis toxin were obtained
from Research Biochemicals Inc. The antisera for
G
/G
were provided by Dr. C. Bianchi
and Dr. C. Homcy (Cardiac Unit, Massachusetts General Hospital).
G
antisera was kindly provided by Dr. Eva Neer
(Harvard Medical School). Ecoscint A was purchased from National
Diagnostics (Manville, NJ). Guanosine diphosphate, thesit
(polyoxyethylene-9-lauryl ether), and guanyl-thiodiphosphate were
obtained from Boehringer Mannheim.
Membrane Preparations, Radioligand Binding Studies,
and Immunoblotting
The rat RG-20 -AR
was stably expressed in NIH-3T3 fibroblasts, PC-12 pheochromocytoma
cells, or DDT
-MF2 smooth muscle cells by cotransfection
with the receptor gene in the expression vector pMSV and pNEO, a
plasmid that confers G418 resistance(7) . None of the cell lines
used for gene transfection expressed the endogenous
-AR gene as determined by radioligand binding
experiments and RNA blot analysis, as previously described(7) .
Cells were grown as monolayers on Falcon Primeria dishes at 37 °C
(5% CO
) in Dulbecco's modified Eagle's medium
with high glucose (4.5 g/liter), supplemented with 10% bovine calf
serum (NIH-3T3), 2.5% horse serum, 2.5% bovine calf serum
(DDT
-MF2) or 5% horse serum, 10% fetal bovine serum (PC-12)
plus penicillin (100 units/ml), streptomycin (100 µg/ml), and
fungizone (0.25 µg/ml). In some experiments, cells were pretreated
with 100 ng/ml pertussis toxin for 18 h (37 °C) prior to membrane
preparation. Membranes were prepared and receptor densities and protein
concentration determined as previously described(7) .
Immunoblotting, densitometric determinations, and Gpp(NH)p-sensitive
binding of the selective
-AR agonist
H-labeled UK14304 (1 nM) were performed as
previously described(5, 6) .
Guanine Nucleotide
Binding
[S]GTP
S binding
experiments were performed as described(26) . Briefly, membranes
prepared from transfected cells were resuspended in assay buffer (5
mM MgCl
, 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, 1 µM guanosine
diphosphate, 1 µM propanolol, 50 mM Tris-HCl, pH
7.4), and the reaction initiated by adding membranes (10 µg in 10
µl) to tubes containing 90 µl of assay buffer containing
[
S]GTP
S (0.2 nM, 1000-1500
Ci/mmol) with or without agonist. Incubation was continued at 24 °C
for various times and terminated by rapid filtration through
nitrocellulose filters with 4
4 ml of wash buffer (100 mM NaCl, 50 mM Tris-HCl, 5 mM MgCl
, pH
7.4, 4 °C). Radioactivity bound to the filters was determined by
liquid scintillation counting. Nonspecific binding was defined by 100
µM GTP
S or Gpp(NH)p. Several preliminary experiments
were performed to establish optimal conditions with respect to time,
nucleotide concentration, and buffer (i.e. magnesium and
sodium concentration). Under the incubation conditions used, the
receptor-mediated increases in [
S]GTP
S
binding in
-AR transfectants were PT-sensitive, and
the maximal agonist-induced signal was observed at 30-min incubation
time points. In the intact PC-12 cell, the
-AR couples
to both PT-sensitive and PT-insensitive pathways to regulate cellular
cAMP levels(7) . Detection of PT-insensitive activation of
G-protein in the [
S]GTP
S binding assay may
require different incubation conditions or factors that are lost upon
membrane preparation. Receptor coupling was examined in a number of
clonal transfectants expressing different receptor densities. All
experiments utilized freshly prepared membranes, and receptor density
was determined in each membrane preparation using saturating
concentrations of
H-labeled RX821002, a selective
-AR antagonist.
Membrane/G-protein Reconstitution
To
eliminate receptor coupling to G-proteins endogenous to the cell, we
developed a system in which agonist signal was restored by addition of
bovine brain G-protein membranes prepared from PT-treated cells. Based
upon earlier studies involving signal
reconstitution(27, 28, 29, 30) , several
experimental paradigms were evaluated to achieve optimal conditions for
reconstitution of the agonist-induced signal. Using the agonist-induced
increase in [S]GTP
S binding as a readout,
we evaluated different preincubation times of membranes and G-protein,
different detergent concentrations, and different buffer conditions
(Mg
/GDP). The standard assay system was as follows. A
preincubation mixture was prepared for each assay point. A single assay
point consisted of six test tubes, two for total binding, two for
nonspecific binding, and two for agonist addition. Nonspecific binding
was defined with 100 µM GTP
S or Gpp(NH)p. Basal
specific binding (without agonist) progressively decreased with
increasing concentrations of GDP. Inclusion of GDP (10-100
µM) thus increased the signal to noise ratio and
facilitated detection of agonist activation of G. Unless indicated
otherwise, the final concentration of GDP was 10 µM. The
preincubation mixture consisted of brain G-protein and 60 µg of
membranes in 120 µl of buffer A (50 mM Tris-HCl, pH 7.4, 5
mM MgCl
, 0.6 mM EDTA) containing 0.005%
thesit (polyoxyethylene-9-lauryl ether) and 50 µM GDP. The
concentrations of brain G-protein and GDP in the preincubation mixture
were five times the final concentration desired in the assay tube.
After 1 h of incubation of the preincubation mixture at 4 °C, 20
µl of the mixture was added to the assay tube containing buffer A
plus (final concentrations) 150 mM NaCl, 1 mM
dithiothreitol, 1 µM propanolol,
50,000 cpm (
0.2
nM) [
S]GTP
S, and agonist, vehicle,
or 100 µM GTP
S (total volume = 80 µl).
Incubation was then continued at 24 °C for various times, and the
reaction was terminated by rapid filtration through nitrocellulose
filters (Schleicher & Schuell, BA85) with 4
4-ml washes (50
mM Tris-HCl, 5 mM MgCl
, pH 7.4, 4
°C). Radioactivity bound to the filters was determined by liquid
scintillation counting. High affinity, Gpp(NH)p-sensitive binding of
the
-AR agonist
H-labeled UK14304 in the
signal restoration system was determined in a similar manner using 50
µg of membrane protein and 50-300 nM brain G-protein
with exclusion of sodium and GDP from the preincubation mixture.
Preparation of Membrane Extract
PC-12 or
NIH-3T3 membrane preparations were solubilized with 1% sodium cholate
(detergent:protein ratio of 2:1) in membrane buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl, 0.6 mM EDTA) by incubation at 4 °C for 30 min. The solubilized
material was then centrifuged at 100,000
g for 1 h,
and the supernatant was used as the membrane extract. To determine the
effect of membrane extract on [
S]GTP
S
binding to brain G-protein, membrane extracts (6-12 µg of
protein) were preincubated with brain G-protein, and the reaction was
initiated as described above. In some experiments, the membrane extract
was concentrated by centrifugation using YM10 centricon tubes (AMICON).
Purification of Bovine Brain
G-protein
Bovine brain G-protein heterotrimer was purified
by a modification (28, 31) of the technique of Sternweis
and Robishaw(32) . Based on subsequent fractionation, immunoblot
analysis and protein sequencing, the brain G-protein preparation was
approximately 63% G, 4% G
, 16%
G
, 16% G
, 1% G
. The
heterotrimeric G-protein preparation was isolated in its GDP-liganded
form. Greater than 80% of the heterotrimer was functional based on the
amount of [
S]GTP
S binding expected from
protein determinations.
G-protein Activation by Epinephrine in Receptor
Transfectants
The cell response to -AR
activation in NIH-3T3 versus PC-12 transfectants differs in
events at both the R-G and G-E interface. In NIH-3T3 transfectants,
receptor activation inhibits adenylyl cyclase by coupling to
PT-sensitive heterotrimeric G-proteins. However, in PC-12
transfectants, the receptor couples to PT-sensitive and PT-insensitive
pathways that elicit negative (PT-sensitive) or positive
(PT-insensitive) effects on adenylyl cyclase
activity(5, 7) . The cell type-specific effects of
receptor activation on cAMP in PC-12 versus NIH-3T3 cells are
described elsewhere and reflect, in part, cell-specific expression of
different adenylyl cyclase enzymes (7).
(
)The
present report indicates that cell-specific signaling events also occur
proximal to effector activation as reflected in agonist-induced
activation of G-protein in the two cell types.
S]GTP
S binding
elicited by the
-AR agonist epinephrine in membrane preparations
of the receptor transfectants (Fig. 1). The magnitude of the
increase in [
S]GTP
S binding elicited by
epinephrine was related to receptor density, and the signal was time
dependent with maximal responses observed at 30 min under these
incubation conditions (Fig. 1).
(
)The
agonist-induced signal was determined in both NIH-3T3 and PC-12
-AR transfectants expressing a range of receptor
densities. Epinephrine elicited a greater degree of G-protein
activation in PC-12 as compared with NIH-3T3
-AR
transfectants over the entire range of receptor densities
(800-9000 fmol/mg membrane protein) (Fig. 1B).
Figure 1:
Increase in
[S]GTP
S binding elicited by agonist in
NIH-3T3 and PC-12
-AR transfectants. Clonal
receptor transfectants were generated and receptor densities determined
as described under ``Experimental Procedures.'' In each
experiment, the level of [
S]GTP
S binding
was determined in the presence or absence of epinephrine (10
µM) for various incubation times (30 min in B) at
24 °C. A, receptor density (fmol/mg): NIH-3T3,
1400;
PC-12,
1100. Data presented in A are representative of
three to five separate experiments with different clonal cell lines.
Data in B are presented as the mean ± S.E. of four
experiments with different membrane preparations of the individual
clonal cell lines. A, basal
[
S]GTP
S binding at 2, 5, 10, 30, and 60
min; NIH-3T3, 0.57, 0.88, 1.37, 1.66, and 2.50; PC-12, 0.98, 1.20,
1.91, 3.29, and 4.27.
Receptor-mediated activation of G-protein was also evaluated in
another cell type stably expressing the receptor protein. In
DDT-MF2
-AR transfectants, the
epinephrine-induced increase in [
S]GTP
S
binding was similar to that observed in NIH-3T3 transfectants (Fig. 2). In each cell type, the agonist-mediated signal required
receptor expression and was blocked by pretreatment of cells with PT,
implicating a subgroup of G-proteins involved in signal transfer (Fig. 2). Although the agonist-induced activation of G was
greater in PC-12 versus NIH-3T3 and DDT
-MF2
-AR transfectants, the population of receptors
exhibiting high affinity for agonist (the R-G-coupled state) was
2-4-fold higher in the latter two cell types compared with PC-12
cells (Fig. 2), indicating that two separate events are required
for productive R-G coupling: R-G interaction and G-activation (subunit
dissociation and guanine nucleotide exchange). Experiments were then
designed to investigate the mechanism of the cell-specific differences
in G activation by agonist.
Figure 2:
Comparison of agonist-induced activation
of G and Gpp(NH)p-sensitive binding of the selective
-AR agonist
H-labeled UK14304 in NIH-3T3,
DDT
-MF2, and PC-12
-AR transfectants.
Membranes were prepared from control or PT-treated cells (PT
) as described under ``Experimental
Procedures.'' Aliquots of the membrane preparations were then
evaluated for high affinity agonist binding and agonist-mediated
effects on [
S]GTP
S binding. Results are
presented as the mean ± S.E. of four experiments. Receptor
density (fmol/mg): NIH-3T3,
5200; PC-12,
4550;
DDT
-MF2,
4000. Gpp(NH)p = 100 µM;
H-labeled UK14304 = 1 nM; epinephrine
= 10 µM. A, basal
[
S]GTP
S binding (fmol): NIH-3T3, 1.61
± 0.105; PT+, 0.27 ± 0.045; PC-12, 2.12 ±
0.14; PT+, 0.47 ± 0.08; DDT
-MF2, 1.56 ±
0.1; PT+, 0.19 ± 0.04. B, specific binding of
H-labeled UK14304 (1 nM) in control cells (without
pertussis toxin pretreatment) in the absence of Gpp(NH)p: 859 ±
183 cpm, NIH-3T3; 289 ± 32 cpm, PC-12; 489 ± 80 cpm,
DDT
-MF2. Data are presented as the total amount of specific
ligand binding that is inhibited by Gpp(NH)p. Counting efficiency
=
50%. In A and B, DDT refers to
DDT
-MF2 cells.
Cell Type-specific Expression of
G
Within the family of
PT-sensitive G-proteins, NIH-3T3 fibroblasts and PC-12 cells express
G, G
and G
, G
,
G
, G
, respectively.
(
)Differences in the type or amount of G-protein
expressed in the two cell types may contribute to augmented signal
transfer in PC-12 transfectants either by enhanced R-G coupling
efficiency or the GTP binding properties of the G
subunit itself.
-MF2 cells and PC-12 cells were not due to
greater levels of G
and G
per µg of
membrane protein in PC-12 cell membranes (Fig. 3A). The
levels of G
per µg of membrane protein were actually
lower in PC-12 cell membranes compared with the other two cell types.
To determine if the expression of G
in PC-12 but not
NIH-3T3 transfectants accounted for the cell type difference in signal
transfer, we re-analyzed R-G coupling after stable expression of
G
in NIH-3T3 fibroblasts. In NIH-3T3 fibroblasts
stably cotransfected with the receptor and
G
(6) , the agonist-induced signal was
augmented relative to cells transfected with
pMSV.
-AR alone (Fig. 3B). However,
despite the expression of G
in NIH-3T3 transfectants
at levels 7-fold higher than the amount of immunoreactive G
found in PC-12 cells (Fig. 3B, inset),
the effect of epinephrine on [
S]GTP
S
binding in NIH-3T3 receptor/G
cotransfectants was
still less than that observed in PC-12 transfectants. These data
indicate a more productive coupling of R and G in PC-12 cells.
Figure 3:
G-protein expression in NIH-3T3,
DDT-MF2, and PC-12 cell membranes and agonist activation of
G-proteins in NIH-3T3 fibroblasts cotransfected with the
-AR and G
. A,
identification of G
/G
or
G
/G
in NIH-3T3, DDT
-MF2, and PC-12
membranes. B, agonist-induced activation of G-proteins in
NIH-3T3 and PC-12
-AR transfectants and in NIH-3T3
-AR/G
cotransfectants.
Epinephrine = 10 µM. Immunoblot-membrane protein
(50 µg for each cell type, 20 µg of brain) from different cell
types were electrophoresed and transferred to Westran membranes; the
blots were incubated with antisera selective for
G
/G
or G
/G
in A or G
in B (inset) (7). The
doublet in the G
/G
immunoblot of PC-12
membranes in A is due to antibody cross-reactivity with
G
. Receptor density (fmol/mg): NIH-3T3,
1100;
NIH-3T3/G
,
1200; PC-12,
1200.
Signal Reconstitution with Purified Brain G-protein
Heterotrimer
The results of the preceding experiments did
not entirely eliminate a role for cell-specific expression of
G types in the differences in R-G coupling in
the two cell types. Therefore, the transfer of signal from R to G was
further characterized by evaluating the ability of each cell membrane
preparation to support signal restoration by exogenous heterotrimeric
G-proteins. In this series of experiments, receptor coupling to
endogenous G-proteins was eliminated by pertussis toxin treatment of
the cells prior to membrane preparation. Pertussis toxin-treated
membranes were reconstituted with a purified preparation of brain
heterotrimeric G-protein, and thus the receptor accesses the same
population of G-proteins. As shown in Fig. 4, the agonist-induced
response (eliminated by PT) was restored by preincubation of membranes
and brain G-protein. The agonist-induced increase in
[
S]GTP
S binding was dose dependent,
inhibited by the
-AR antagonist rauwolscine, and
required receptor expression as it was not observed in control
pMSV.neo-transfected cells (Fig. 4).
Figure 4:
Agonist-induced activation of G-protein in
the membrane/brain G-protein reconstitution system using PC-12
-AR transfectants. Membranes were prepared from
cells pretreated with pertussis toxin and reconstituted with 25 nM bovine brain G-protein as described under ``Experimental
Procedures.'' A, agonist-induced activation of G-protein
in membrane preparations from cells transfected with
pMSV.
-AR or resistance plasmid alone in the
presence and absence of added G-protein. Data are presented as the mean
± S.E. of four experiments using different membrane
preparations. B, effect of increasing epinephrine
concentrations on [
S]GTP
S binding in the
presence and absence of the
-AR antagonist
rauwolscine. Receptor density =
5400 fmol/mg. The results
are representative of three experiments using different clonal cell
lines and are expressed as the percent of the epinephrine-induced
increase in [
S]GTP
S binding at 100
µM agonist ([
S]GTP
S binding:
basal = 0.81 fmol, epinephrine (100 µM) =
3.45 fmol).
The signal restoration
system was then used to compare NIH-3T3 and PC-12 transfectants in
terms of their ability to support the agonist signal. As is the case in
non-PT-treated membranes, the receptor-mediated activation of G-protein
in the signal restoration system was up to 8-fold greater in PC-12 versus NIH-3T3 transfectants ( Fig. 5and Fig. 6),
and the augmented signal transfer in PC-12 transfectants actually
occurred with a 2-4-fold lower amount of receptors (relative to
NIH-3T3 fibroblasts) existing in the R-G-coupled state (Fig. 5).
Reconstitution of high affinity Gpp(NH)p-sensitive binding in NIH-3T3
transfectants indicates that exogenous G-protein has access to the
receptor in both cell types and that the transfer of signal from R to G
is more efficient in PC-12 transfectants. Further analysis of signal
transfer in the two cell types indicated that the cell-specific
differences were observed over a range of heterotrimer or magnesium
concentrations (Fig. 6) as well as in membrane preparations that
were washed with 1 M KCl to remove proteins loosely associated
with the cell membranes.(
)
Figure 5:
Comparison of agonist-induced activation
of G and Gpp(NH)p-sensitive binding of the selective
-AR agonist
H-labeled UK14304 in NIH-3T3
and PC-12
-AR transfectants in the
membrane/G-protein reconstitution assay. Membranes were prepared from
pertussis toxin-treated cells and reconstituted with 25 (A) or
50 and 300 (B) nM brain G-protein as described under
``Experimental Procedures.'' A, basal
[
S]GTP
S binding (fmol) ranged from 0.75 to
1.24 in NIH-3T3 transfectants and from 1.19 to 2.14 in PC-12
transfectants. Data are presented as the mean of duplicate
determinations using different clonal cell lines. Experiments were
repeated twice in each transfectant with similar results. Epinephrine
= 10 µM. See legend to Fig. 2 for additional
details.
Figure 6:
Agonist-induced activation of G-protein in
the membrane/brain G-protein reconstitution system using PC-12 and
NIH-3T3 -AR transfectants and the effect of
magnesium and G-protein concentration. Membranes were prepared from
cells pretreated with pertussis toxin to eliminate receptor coupling to
endogenous G and then reconstituted with heterotrimeric G-protein (25
nM in A) purified from bovine brain. Epinephrine
= 10 µM. Receptor density (fmol/mg): NIH-3T3,
5600 (A) and
1100 (B); PC-12,
5100 (A) and
950 (B). Data are presented as the mean
of duplicate determinations and are representative of two (A)
or five (B) separate experiments using different membrane or
G-protein preparations. GDP concentration in A = 30
µM and in B = 100 µM. A, basal [
S]GTP
S binding (fmol) at
0.3, 1, 3, and 10 mM MgCl
: NIH-3T3, 0.08, 0.2,
0.24, 0.28; PC-12, 0.09, 0.31, 0.67, 0.81.
Effect of Cell Membranes and Membrane
Extracts on [
The results of the
signal reconstitution experiments suggest the existence of a membrane
factor in PC-12 or NIH-3T3 cells that alters guanine nucleotide binding
to heterotrimeric G-protein. This issue was addressed by analysis of
the guanine nucleotide binding behavior of purified brain G-protein
alone or following preincubation of brain G-protein with membrane
preparations or membrane extracts from nontransfected PC-12 or NIH-3T3
cells. In each experiment, [S]GTP
S Binding to
Purified Brain Heterotrimeric G-protein
S]GTP
S binding
was measured in the brain G-protein preparation and the membrane
preparation/extract alone and then compared with the amount of
nucleotide bound when the two preparations were co-incubated. In the
absence of any ``regulatory factors,'' the
[
S]GTP
S binding in the co-incubations would
be additive and represent the sum of nucleotide binding in either
preparation alone. However, in the presence of PC-12 membrane
preparations, [
S]GTP
S binding to purified
brain G-protein was increased by 50-120% above the value expected
from summation of nucleotide binding in the two preparations alone (Fig. 7). The greater than additive effect was augmented at lower
GDP concentrations and was not observed when purified brain G-protein
was incubated with PC-12 cytosol.
(
)In contrast,
incubation of purified brain G-protein with NIH-3T3 membrane
preparations resulted in a level of nucleotide binding that was close
to additive, relative to results obtained with brain G-protein or
NIH-3T3 membranes alone.
Figure 7:
[S]GTP
S
binding to brain G-protein in the presence and absence of cell membrane
preparations. Membranes were prepared from nontransfected PC-12 or
NIH-3T3 cells as described under ``Experimental Procedures.''
[
S]GTP
S binding (2 nM) to brain
G-protein (12.5 nM) was determined in the absence and presence
of 10 µg of cell membrane protein. Brain G-protein and membrane
were preincubated for 1 h at 4 °C, and the binding reaction was
initiated as described under ``Experimental Procedures.''
Data are presented as the mean of duplicate determinations and are
representative of three experiments with different membrane
preparations.
As indicated above, the cell type-specific
differences in signal transfer were maintained following
``stripping'' of peripheral membrane proteins (1 M KCl wash), suggesting that the regulatory activity is tightly
associated with the membrane. Subsequent experiments indicated that the
regulatory activity was extracted from the membrane by detergent
solubilization with sodium cholate. PC-12 membrane extract increased
the binding of [S]GTP
S binding to brain
G-protein by 65-75%, whereas NIH-3T3 membrane extract was without
effect (Fig. 8A). The PC-12 membrane extract also
augmented basal and receptor-mediated activation of G-protein in
NIH-3T3
-AR transfectants using the signal
reconstitution assay.
Membrane extracts prepared from
NIH-3T3 fibroblasts were without effect in this system. The effect of
PC-12 membrane extract (
15% increase) on receptor-mediated
activation of G in NIH-3T3
-AR transfectants was less
than the effect of the extract on [
S]GTP
S
binding to brain G-protein alone in the solution-phase assay, likely
reflecting the experimental conditions of the two different assay
systems.
Figure 8:
Effect of detergent-solubilized membrane
extracts on [S]GTP
S binding to brain
G-protein. [
S]GTP
S binding (2 nM)
to brain G-protein (12.5 nM) was determined in the absence and
presence of membrane extract (A, 5.5 µg of protein).
Membrane extracts were prepared from nontransfected PC-12 or NIH-3T3 by
membrane solubilization with sodium cholate as described under
``Experimental Procedures.'' Brain G-protein and membrane
extract or vehicle were preincubated for 1 h at 4 °C, and the
binding reaction was initiated as described under ``Experimental
Procedures.'' A, concentrate: the membrane
extract was concentrated 5-fold by centrifugation in a centricon
concentrater with a 10,000 molecular size exclusion membrane and then
diluted to the original volume with solubilization buffer prior to
assay. 95 °C: an aliquot of the retentate was placed in a
boiling water bath for 5 min and cooled to 4 °C before
preincubation with brain G-protein. filtrate: flow-thru
fraction obtained by centrifugation of the membrane extract in a
centricon concentrater with a 10,000 molecular size exclusion membrane.
The protein concentration in the filtrate was below detectable levels,
and thus the amount of filtrate used was equivilant to the volume of
unconcentrated membrane extract added to the assay. In A, data
are presented as the mean ± S.E. of three experiments and
represent the increase above the summed value of nucleotide binding in
extract and G-protein preparation alone. The effect of NIH-3T3 membrane
extract was not statistically different from control. In B, a
constant detergent concentration was maintained for each assay point.
Data are presented as the mean of duplicate determinations from two
separate experiments, and the values in the open and closedsquares correspond to increases above the
summed value of nucleotide binding in extract and G-protein preparation
alone. The GDP concentration was 1 µM in both A and B. Under the experimental conditions used, the
amounts of [
S]GTP
S bound in PC-12 or
NIH-3T3 membrane extracts alone were 3-4 and 1-2 fmol,
respectively. In such assay conditions,
25 fmol of
[
S]GTP
S was bound to purified brain
G-protein alone.
The stimulatory action of the detergent-solubilized PC-12
membrane preparation was related to the protein concentration of the
extract (Fig. 8B). The regulatory activity was found in
the retentate following centrifugation of the PC-12 membrane extract in
a YM10 membrane (10,000 molecular size exclusion) centricon, and the
stimulatory effect of PC-12 membrane extract was eliminated by heating
of the extract at 95 °C for 5 min (Fig. 8A). Both
observations suggest that the ``regulatory factor'' is a
protein and not a lipid/nucleotide derivative of low molecular size (Fig. 8A).
- and
-AR subtypes differ in their coupling to adenylyl
cyclase when expressed in the two cell types(6) . The present
report indicates that the PC-12 and NIH-3T3
-AR
transfectants also differ in the efficiency/magnitude with which the
agonist-occupied receptor activates both endogenous and exogenous
heterotrimeric G-proteins. In PC-12 transfectants, a neuron-like cell
line, the receptor appears to transfer the agonist-occupation event to
G-protein activation with greater efficiency than it does when
expressed in NIH-3T3 fibroblasts or DDT
-MF2 smooth muscle
cells.
-related
monomeric G-proteins is regulated by specific proteins that stimulate
dissociation of bound GDP or GTPase activity. Analogous functions for
heterotrimeric G-proteins may be subserved by receptor,
G
, or effector as indicated by experiments in
which purified R, G, or E is reconstituted in phospholipid vesicles.
However, a role for additional proteins (i.e. arrestin,
phosducin, neuromodulin, kinases) in regulating cell signaling by
G-protein-coupled receptors may be lost in reconstitution experiments
using purified proteins and could only be observed in the natural cell
environment as described in the present report.
or
G
.
Thus, the more efficient response
in PC-12 membranes may reflect either a preferred and more effective
coupling of the receptor to a heterotrimer (i.e. G
) not found in NIH-3T3 fibroblasts or the GTP/GDP
binding properties of the G
activated by the receptor
in the two cell types. However, despite 7-fold greater expression of
G
in NIH-3T3 cells relative to the levels of
immunoreactive G
expressed in PC-12 cells, the
epinephrine-induced signal in NIH-3T3 receptor/G
cotransfectants did not reach that elicited by the agonist in
PC-12 transfectants. These data and the lower amount of receptors
actually coupled to G (high affinity agonist binding) in PC-12 versus NIH-3T3 or DDT
-MF2 membranes suggest that
the transfer of signal from R to G is regulated by other entities in
the receptor's microenvironment. Such a possibility is supported
by the results of the signal reconstitution system in which the
receptor is coupling to the same population of G
within the two membrane environments.
-AR agonist
H-labeled UK14304 in NIH-3T3
transfectants is 2-4-fold higher than that observed in PC-12
transfectants in both PT-untreated membranes and the membrane/brain
G-protein reconstitution system. These data indicate that factors other
than the presence or absence of a particular G
heterotrimer contribute to the difference in signal transfer in
the two cell types and that the transfer of signal from R to G is
regulatable. Indeed, the results of subsequent experiments indicated
the existence of a cell type-specific membrane-associated protein that
directly activates G and influences the propagation of agonist-induced
signals.
-AR,
adrenergic receptor; PT, pertussis toxin; GTP
S, guanosine
5`-3-O-(thio)triphosphate; Gpp(NH)p, guanosine
5`-(
,
-imido)triphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.