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(Received for publication, May 9, 1995) From the
The possibility that Spodoptera frugiperda (Sf9) cells
can provide an intact cell setting for reconstitution of the human
5-hydroxytryptamine
Receptors for serotonin (5-hydroxytryptamine, or 5-HT) ( The 5-HT Mulheron et al.(19) were the first to express the human 5-HT We verify here a predominantly uncoupled phenotype of the
human 5-HT
Baculoviruses
containing recombinant DNA encoding
For
immunoprecipitation, Sf9 cell membranes were solubilized in 0.5% SDS,
50 mM sodium phosphate (pH 8.0), and 2 mM EDTA at 25
°C for 30 min. Samples were supplemented at 4 °C to achieve 50
mM sodium phosphate (pH 7.2), 0.5% SDS, 1% sodium
deoxycholate, 1% Triton X-100, 150 mM NaCl, 2 mM
EDTA, 1% aprotinin, and 200 mg/ml leupeptin. Selected rabbit antisera
were added (1:10), followed 18 h later by protein
A-Sepharose(37) . Precipitates were collected and washed in
buffered 0.5% Triton X-100.
Expression of the human 5-HT
Figure 1:
Binding of
[
Figure 2:
Effects of GTP on binding of
[
To ascertain the extent to which the 5-HT
Figure 3:
Identification of the 5-HT
Figure 4:
Biosynthetic labeling of the 5-HT
We suspect, based on
previous observations(19, 26) , that a small
proportion of the 5-HT
Figure 5:
Detection of Sf9 cell G protein
Having established expression
of the 5-HT
Figure 6:
Introduction of mammalian G protein
subunits into Sf9 cells. Expression of
Figure 7:
[
In
view of the small effects of To more rigorously define the effects of
To determine the potential of other members
of the
Coupling of G proteins to receptors can also be
measured by GTP-sensitivity of agonist-binding.
[
Figure 8:
Gpp(NH)p sensitivity of
[
Experiments with [
Figure 9:
Binding of
[
Using the human 5-HT The B SDS-PAGE was found to resolve the 5-HT Coupling of 5-HT A coupled phenotype was achieved for the 5-HT Gpp(NH)p sensitivity of agonist-binding is another index
of a coupled phenotype. Partial sensitivity was conferred by Under the conditions of the protocols used for
infection and assay, we found that We
additionally examined the influence of different Previous work with rat hippocampal
membranes showed that [ These data support the conclusion that normal
interactions can be established between a mammalian receptor and a
select array of G proteins when expressed in intact Sf9 cells. Similar
observations can presumably be extended to any seven transmembrane
segment receptor that can be expressed in excess of the small
endogenous pool of G protein to which it might couple. For those
receptors whose functions remain obscure, this type of reconstitution
represents a useful starting point for exploring potential pathways of
transduction. Because Sf9 cells appear to be deficient in seven
transmembrane segment receptors, expression of receptors and
reconstituion with G protein subunits represents an ideal means to
define the specificity of ligands. The reconstitution paradigm is
especially germane to the characterization of agonists, whose
high-affinity interactions with receptors exhibit a strong dependence
on the presence of appropriate G proteins. The overt uniformity in
populations of expressed receptors and G proteins, moreover, lends
itself to the development and optimization of techniques to map
receptor-G protein interactions in mammalian systems.
Volume 270,
Number 31,
Issue of August 04, pp. 18691-18699, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Receptor in Sf9 Cells
RECONSTITUTION OF A COUPLED PHENOTYPE BY CO-EXPRESSION OF
MAMMALIAN G PROTEIN SUBUNITS (*)
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(5-HT
) receptor with
mammalian G protein subunits was explored. The 5-HT
receptor was found to assume an uncoupled phenotype when
expressed alone in Sf9 cells at relatively high levels (5-34 pmol
of receptor/mg of membrane protein), i.e. agonist-binding to
the receptor was characterized by a relatively high K
and an insensitivity to GTP.
Co-expression of the receptor with members of the
``family'' together with various combinations of
and subunits increased the affinity for
agonists to that observed for the coupled form of receptor in mammalian
cells, concomitant with conferrance of guanosine
5`-(
,-imino)triphosphate sensitivity. The agonists employed
were [
H]8-hydroxy-N,
N-dipropyl-2-aminotetralin ([H] 8-OH-DPAT)
and
[I]R(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3`-iodo-2`-propenyl)amino]tetralin
([
I]8-OH-PIPAT). The binding of an antagonist,
[
I]4-(2`-methoxyphenyl)-1-[2`-[N-(2"-pyridinyl)-p-iodobenzamido]ethyl]
piperazine ([
I]p-MPPI), was unaffected
by co-expression of G protein subunits. Both
and
subunits were required for optimal coupling. No differences were
evident among
,
,
,
, and 
when
expressed with 
in this regard, nor
among most permutations of subunits when
expressed with
(
>
). 
and 
expressed with did not
participate in coupling. These data support the conclusion that normal
interactions between a mammalian receptor and a select array of G
proteins can be established in intact Sf9 cells, and extend previous
observations of 5-HT receptor coupling to G
and the pertussis toxin-insensitive G protein G.
)have been classified according to properties of ligand
binding and molecular structure(1) . Of the several subtypes of
5-HT receptors, the 5-HT subtype is among the most
extensively characterized. Assays using the agonist
[
H]8-hydroxy-N,N-dipropyl-2-aminotetralin
([H]8-OH-DPAT) describe the properties of
5-HT receptors in various regions of the rat forebrain,
where the receptor is postsynaptic, and in the dorsal raphe nucleus,
where it is somatodendritic(2) . Activation of postsynaptic
5-HT
receptors leads to inhibition of adenylyl cyclase and
to stimulation of K
channels, while activation of
presynaptic receptors leads only to stimulation of K
channels(3, 4, 5, 6) . Both
actions are inhibited by pertussis toxin (PTX), implying a role for one
or more forms of the G proteins G
and
G(7, 8, 9, 10) . Cloned
DNAs for the 5-HT receptor reveal, as anticipated, a
sequence consistent with a seven transmembrane segment motif common to
G protein-linked receptors(11, 12) . Transfection of
cells normally lacking 5-HT
receptors with the receptor
DNA establishes the expected sensitivity of adenylyl cyclase and
K
channels to serotonin(10, 13) .
Activation of a phosphoinositide-specific phospholipase C can also be
demonstrated in reconstituted systems, but requires especially high
concentrations of agonist and may not be a physiological
response(10, 14, 15) .
receptor can exhibit different affinities for
[
H]8-OH-DPAT depending on whether the receptor is
coupled to a G protein(s). As assessed with membranes from rat brain or
various transfected cells, the G protein-coupled form of receptor
exhibits a K for
[
H]8-OH-DPAT of about 1 nM, whereas the K of the uncoupled form is about 20
nM(16, 17) . Conversion of the high- to the
low-affinity form can be achieved with GTP or non-hydrolyzable
analogues. GTP promotes dissociation of the G protein into monomeric
and heterodimeric subunits, which in turn destabilizes
the interaction between the G protein and receptor supporting
high-affinity binding of agonists(18) .
receptor in Sf9 cells. Using [
H]8-OH-DPAT,
these investigators documented a B
of 150 fmol
of receptor/mg of Sf9 cell membrane protein and a K of 3 nM. The relatively low value for the K
is consistent with the coupling of the
receptor to endogenous G proteins, an inference confirmed by a
GTP-induced shift in K
to 23 nM and by serotonin-promoted labeling of an
-like
subunit with [P]GTP-azidoanilide.
5-HT
, dopamine D4, and Substance P receptors, when
similarly present at low levels in Sf9 cells, also exhibit
GTP-sensitive binding of
agonists(20, 21, 22) . However, seven
transmembrane segment receptors expressed at higher levels do
not(23, 24, 25) , consistent with the
hypothesis that endogenous G proteins can be limiting. Parker et
al.(26) , expressing the 5-HT
receptor in Sf9
cells at a density of 3 pmol/mg of membrane protein, found that only 7%
of the receptors bound [
H]8-OH-DPAT with high
affinity. receptor when expressed at high levels in Sf9
cells. Using this phenotype as a starting point, we explored the
possibility that Sf9 cells can provide an intact cell setting for
reconstitution of the receptor with mammalian G protein subunits. Sf9
cells carry out a variety of co- and post-translational processing
events which, although not as extensive as those occurring in mammalian
cells, support a viable targeting of receptors and G protein subunits
to the cell membrane. In this respect, Sf9 cells provide a level of
organization beyond that achieved in vitro with proteins whose
purification is difficult or (in the case of bacterially expressed
proteins) that lack relevant covalent modifications. In distinction to
mammalian cell models, Sf9 cells are free of potentially interfering
seven transmembrane segment receptors, and the low level of endogenous
G proteins permits more rigorous testing of mammalian subunits for
their ability to interact with co-expressed receptors. We report here
that human 5-HT
receptors expressed in Sf9 cells can be
induced to assume a coupled phenotype upon co-expression with an
appropriate array of G protein subunits. Both
and
subunits are required for optimal coupling, with the selectivity for
subunits restricted to those of the family, i.e. ,
,
,
, and . An
interaction of the 5-HT receptor with G
and
G, the distributions of which overlap extensively in
mammalian brain, has not been previously reported.
Recombinant Baculoviruses
The XbaI/BamHI fragment of the human 5-HT receptor DNA (the gift of M. Caron and R. Lefkowitz, (27) ) subcloned into the pDP5 vector was provided by Dr. Dolan
Pritchett (University of Pennsylvania). A BglII site was
introduced near the initiating methionine codon, and most of the
5`-untranslated sequence was eliminated, by the polymerase chain
reaction using 5`-GAAGATCTGGCGCGCAGGCATGGAT-3` as a forward primer and
5`-AACCACAACTAGAATGCAGTG-3` (pDP5-specific) as the reverse primer (the BglII site is underlined, and the initiating codon is double
underlined). The polymerase chain reaction product was digested with BglII and EcoRI to yield a 1.3-kilobase fragment with
11 authentic nucleotides 5` to the initiating codon and 34 authentic
(plus 11 pDP5-related) nucleotides 3` to the stop codon. The BglII/EcoRI fragment was subcloned into the transfer
vector pVL1392. Sequencing by the dideoxy chain termination method
verified that no mutations had been introduced by polymerase chain
reaction. Spodoptera frugiperda (Sf9) cells (Invitrogen) were
transfected with a combination of the recombinant transfer vector and a
modified AcNPV DNA (BaculoGold
) according to the protocol
provided by PharMingen (San Diego, CA). Baculovirus containing
recombinant DNA was plaque-purified and amplified.
,
,
, ,
, , ,
, , and 
(28, 29, 30) were kindly provided by
Drs. T. Kozasa and A. Gilman at Southwestern Medical Center, Dallas.
Those encoding ,
, and
(31) were the gift of Dr. J. Garrison (University of
Virginia). Recombinant baculovirus DNA encoding
was
constructed in our laboratory by subcloning the EcoRI fragment
of the human cDNA (32) into pVL1393 and
transfecting Sf9 cells with a combination of the recombinant transfer
vector and linearized AcMNPV DNA (Invitrogen, San Diego). Baculovirus
containing recombinant DNA was isolated as described above.Cell Culture and Membrane Preparation
Sf9 cells
were maintained in suspension culture in TNM-FH media containing 10%
fetal calf serum, 0.1% pluronic F-68, and 0.01 mg/ml gentamycin at 27
°C. For expression, cells were subcultured in monolayer and grown
to 60% confluency, at which time they were infected with one or
more recombinant viruses at a multiplicity of infection of at least 1
for each virus. Cells were harvested 48 h following infection, washed
twice with 0.9% NaCl, and resuspended in 1 ml of ice-cold 20 mM HEPES (pH 8.0), 2 mM MgCl
, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, and 2 µg/ml aprotinin. After 5 min on ice, cells were
homogenized by repeated passage through a 26-gauge needle. Homogenates
were centrifuged at 100 g for 5 min, and the resultant
supernatants at 16,000
g for 30 min. The final pellets
(membrane) were washed twice with the above buffer and resuspended at
3 mg/ml protein.
[
Binding of [H]8-OH-DPAT Binding
AssayH]8-OH-DPAT was
carried out as described previously(33) . Each tube contained
20 µg of membrane protein in 500 µl of 50 mM Tris (pH
7.7), 0.1% ascorbic acid, 20 µM pargyline, and
0.1-50 nM [H]8-OH-DPAT (220
Ci/mmol, Amersham). Incubations were carried out for 30 min at 25
°C, at which time the contents were filtered under vacuum on
Whatman GF/B filters presoaked in 0.5% polyethylenimine. The filters
were washed 3 times with 5 ml of ice-cold buffer, placed in 5 ml of
Ecolite liquid scintillation mixture, and counted by scintillation
spectrometry. Specific binding was defined as the difference in
radioactivity measured in the presence and absence of 10 µM 5-HT. The percentage of specific binding at the K
was 90%. Maximum binding and K
values were determined by Scatchard (34) transformation of saturation binding data using unweighted
linear regression analysis.
[
Membranes (0.1-1.1 µg of protein per assay
tube) were incubated for 30 min at 37 °C with 9-800
pM
[I]8-OH-PIPAT and
[
I]p-MPPI Binding
Assays
I]R(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3`-iodo-2`-propenyl)amino]tetralin
([
I]8-OH-PIPAT) (2200 Ci/mmol) or 12-1200
pM
[
I]4-(2`-methoxyphenyl)-1-[2`-[N-(2"-pyridinyl)-p-iodobenzamido]ethyl]piperazine
([
I]p-MPPI) (2200 Ci/mmol) in a total
volume of 100 µl. When using [
I]8-OH-PIPAT,
assays were carried out in 50 mM Tris (pH 7.7), 2 mM MgCl
, and 0.1% bovine serum albumin. Assays with
[I]p-MPPI were carried out in 50
mM Tris (pH 7.7), 100 µM Gpp(NH)p, and 0.1%
bovine serum albumin. Assays were terminated by the addition of 5 ml of
ice-cold wash buffer (20 mM Tris, 7.4). Filtration was carried
out using a Brandel cell harvester with glass fiber filters (Schleicher
and Schuell No. 32, previously soaked in 0.3% polyethylenimine)
followed by washing with 15 ml of ice-cold wash buffer. Specific
binding was defined with 10 µM
([
I]8-OH-PIPAT assay) or 100 µM ([
I]p-MPPI assay) 5-HT. The
percentage of specific binding at the K
was 67% for [
I]8-OH-PIPAT and 72%
for [
I]p-MPPI. Maximum binding and K
values were determined by Scatchard
transformation as described above.
Metabolic Labeling
Sf9 cells expressing
5-HT receptors and/or G protein subunits were harvested 44
h following infection and resuspended in methionine-free Graces insect
cell media containing 10% fetal calf serum.
[
S]Methionine was added (50 µCi/ml), and the
cells incubated for an additional 4 h at 27 °C. For studies of
phosphorylation, cells were incubated for 4 h in TNM-FH media
containing 10% fetal bovine serum and
[
P]orthophosphate (50 µCi/ml). For studies
of palmitoylation, the cells were incubated for 4 h in serum-free
TNM-FH media containing [
H]palmitic acid (250
µCi/ml).Immunological Procedures
Antibodies used are
listed in Table 1. Antibodies recognizing G protein and
subunits have been described
previously(35, 36, 37, 38, 39) .
5-HT receptor-directed antibodies were generated using
keyhole limpet hemocyanin-conjugated synthetic peptides as antigens.
Immunotransfer blotting was accomplished by solubilizing membranes
prepared from uninfected or infected Sf9 cells (50 µg of membrane
protein) in Laemmli sample buffer at 37 °C for 30 min. Extracts
were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), proteins transferred to nitrocellulose
membranes, and the membranes incubated sequentially with rabbit
antisera (1:100), biotinylated goat anti-rabbit IgG coupled to
horseradish peroxidase-streptavidin, and H
O plus 4-choro-1-napthol(37) . In some cases, I-protein A was used for detection.
Quantitation of G Protein Subunits
subunits
were quantitated where indicated by Western blots using antiserum 8645.
8645 was generated with a peptide common to ,
,
,
, and
, and also present in and
except for a valine in place of phenylalanine at the
N terminus. G purified from bovine retinal rod outer
segments (40) was typically added to membranes of uninfected
Sf9 cells just prior to SDS-PAGE to provide an external standard.
Colorimetric intensities were linear and bracketed the intensities of
subunits expressed following infection. G contains
as its sole subunit, permitting quantitation of
expression in Sf9 cells with antiserum 8136. Cholate
extraction of membranes was accomplished where indicated by addition of
sodium cholate to resuspended membranes to a concentration of 1%,
followed by incubation for 1 h on ice. The extract was clarified by
centrifugation at 16,000 g for 30 min.
receptor in Sf9
cells was initially evaluated using the agonist
[
H]8-OH-DPAT. Forty-eight hours following
infection of the cells with recombinant baculovirus encoding the
receptor, substantial levels of the receptor were found to exist (Fig. 1). B values ranging from 5 to 22
pmol/mg membrane protein were observed, with K values between 7 and 18 nM. Binding of
[
H]8-OH-DPAT did not occur prior to infection,
and the binding at 48 h was insensitive to GTP (Fig. 2). In
separate experiments with rat hippocampal membranes (B = 230 fmol/mg protein; K of
the high-affinity site = 0.4 nM), GTP caused a 54%
reduction in binding, consistent with published results(41) .
The high K
of the receptor in Sf9 cells
and the GTP insensitivity of agonist binding to this receptor were
consistent with expression of the receptor in a predominantly uncoupled
state.
H]8-OH-DPAT to human 5-HT receptors
in Sf9 cells. Saturation experiments using
[
H]8-OH-DPAT (2.5-50 nM) were
performed on membranes prepared from Sf9 cells 48 h following infection
with recombinant baculovirus encoding the human 5-HT receptor. The figure represents data obtained in one of several
independent experiments, in which the B
was
5-22 pmol/mg membrane protein and the K was 7-18 nM. Each point in the figure
represents the mean of triplicate determinations. Inset, Scatchard transformation of the data. B/F,
bound/free.
H]8-OH-DPAT. Specific binding of
[H]8-OH-DPAT (2.2 nM) was determined in
the presence and absence of 100 µM GTP for membranes
isolated from noninfected Sf9 cells (``-5-HT receptor'') and from Sf9 cells 48 h following infection with
recombinant baculovirus encoding the 5-HT
receptor
(``+5-HT
receptor''). Data represent
the mean ± S.E. of three independent experiments, each performed
in triplicate.
receptor in Sf9 cells was subject to co- or post-translational
forms of processing, expression was analyzed by Western blots and
metabolic labeling. Antibodies recognizing the 5-HT
receptor were generated using sequences present in three
different regions of the receptor, intracellular loop 2 (antiserum
``DP''), outer loop 2 (``RT''), and intracellular
loop 3 (``EV'') (Table 1). Each of the three antibodies
revealed four closely spaced protein bands in Western blots (bands
a-d (39-45 kDa), Fig. 3). None of the four bands was
detected with preimmune sera, nor were any detected in uninfected Sf9
cells, Sf9 cells expressing
-adrenergic or dopamine D3
receptors, or Sf9 cells infected with wild-type virus alone. Thus,
recognition is specific for the 5-HT receptor. The
appearance in Sf9 cells of closely spaced bands near the molecular
weights deduced from cDNAs has been described previously for
-adrenergic (24, 42) and N-formyl
peptide (23) receptors, and is presumed to represent
incomplete, heterogeneous processing of oligosaccharides (23) .
The four bands were also identified by immunoprecipitation of
[S]methionine-labeled protein (Fig. 4).
To determine whether any of the protein species represented by these
bands were substrates for phosphorylation or palmitoylation, cells were
incubated with [
P]orthophosphate or
[
H]palmitate. All four bands were evident as
substrates for phosphorylation. Only two (bands a and b) were substrates for palmitoylation.
receptor by immunotransfer blotting. Membranes isolated from Sf9 cells
expressing the human 5-HT
receptor
(``+5-HT
receptor'') or not
(``-5-HT
receptor'') were subjected to
SDS-PAGE (25 µg of protein/lane) followed by transfer of the
resolved proteins to nitrocellulose for detection using a 1:100
dilution of antiserum DP, RT, or EV, or respective preimmune sera. Four
bands, designated a-d, were observed consistently with DP, RT,
and EV in membranes from cells expressing the 5-HT
receptor.
receptor. Sf9 cells expressing the 5-HT
receptor (44
h post-infection) were incubated for 4 h in media containing
[
S]methionine,
[
P]orthophosphate, or
[
H]palmitate, and immunoprecipitation was
subsequently achieved using solubilized cell membranes and the EV antiserum. Immunoprecipitates were analyzed by fluorography (H; 30-day exposure using ENHANCE) or autoradiography (S and
P; 7- and 10-day exposures,
respectively, using an intensifying screen for the latter). None of the
radiolabeled bands in the 39-45-kDa range were detected when
preimmune serum was used.
receptor expressed in Sf9 cells is
coupled to endogenous G proteins, but that the vast majority is not. In
confirmation of the findings by Mulheron et al.(19) ,
a G protein
subunit that resembles was found in
Sf9 cells. The 40-kDa subunit(s) was detected by Western blotting using
antiserum 1398, which recognizes and
, and 9072, which is specific for (Fig. 5). No protein was detected with 8730, which
recognizes ,
, and, to a lesser
extent,
, thus ruling out appreciable quantities of
these mammalian forms of
in Sf9 cells. An
-like protein, having an apparently larger
molecular size than the
protein, was detected with
0946. Two proteins react with the -directed antibody
1190. No protein was detected with antisera 2919 (),
120 (), and 130 (
) (not shown).
Surprisingly, a 40-kDa protein was recognized by 1521. 1521 is directed
toward
and also recognizes
. Given
the absence of and
as deduced with
8730 and 2919, 1521 must either cross-react with the
-like subunit present in Sf9 cells or recognize
another protein (subunit) altogether.
subunits. Membranes from uninfected Sf9 cells (50 µg of membrane
protein per lane) were analyzed by immunotransfer blotting with the
indicated subunit-directed antisera. A description of the
antisera is provided in Table 1.
receptor in Sf9 cells, and an essentially
uncoupled phenotype, the effects of
(),
,
, and
on the affinity for
[H]8-OH-DPAT were assessed. In these experiments,
a low concentration of agonist was used (0.5 nM) so that an
increase in affinity concomitant with coupling would be detected as an
increase in binding. All four subunits were expressed in Sf9 cells
together with 5-HT receptors as detected with the relevant
antibodies (Fig. 6).
was found to promote a
small increase in the binding of [H]8-OH-DPAT,
while caused a small decrease (Fig. 7).
and were without effect.
,
,
, , and
were analyzed 48 h following coinfection with
recombinant baculoviruses encoding the subunits and the 5-HT receptor. Shown are immunoblots using membranes (25 µg of
protein/lane) prepared prior to and following infection (left and right lanes of each panel), using antisera 1190,
8730, 2921, 0946, and 5357.
H]8-OH-DPAT binding
to the 5-HT receptor co-expressed with G protein subunits in Sf9 cells.
Specific binding of [H]8-OH-DPAT at 0.5 nM was determined for membranes prepared from Sf9 cells expressing
the 5-HT receptor,
subunits, and
as indicated. Data represent the
mean ± S.E. of 5-24 individual experiments assayed in
triplicate. As a point of reference, the dashed line indicates
[H]8-OH-DPAT binding where the 5-HT receptor is expressed alone. Statistical significance (*, p < 0.01) was determined using Student's t test.
subunits on binding of
[H]8-OH-DPAT, we explored the possibility that
the inclusion of and subunits would promote more
substantive coupling.
can stabilize
subunit
conformation(29) , help to target subunits to
membranes(29, 43, 44) , and otherwise
facilitate receptor- subunit
interactions(45, 46, 47, 48) . In
preliminary experiments, we had not detected subunits in Sf9
cells with antibodies generated against bovine (5357; Fig. 6) or a peptide sequence present in
and
(and, with a one-residue difference, in and 
) (8136; not shown). We therefore introduced
and subunits, a combination of
subunits expressed in mammalian brain. and
co-expressed with the 5-HT receptor
resulted in a small increase in the binding of
[
H]8-OH-DPAT (Fig. 7). When or were expressed in addition to
, binding of
[H]8-OH-DPAT was increased 3-4-fold above
control and well above that observed with or subunits
alone. The potentiating effect of
was not observed for or . As a
point of reference, in these experiments was
expressed at about 30 pmol of subunit/mg of Sf9 cell membrane as
assessed by 8645 reactivity. Greater than 90% of the subunit was
cholate-extractable. Levels of
and were the same or severalfold higher. Levels of were not calculated, since this subunit is not equivalently
recognized by 8645. is expressed at much higher
levels, about 320 pmol of subunit/mg of Sf9 cell membrane as assessed
by 8136 reactivity, but only about 10% of the subunit was
cholate-extractable, indicating that the remainder was denatured and/or
nonspecifically aggregated(30) . expression
was not quantitated.
and subunits on agonist binding to the 5-HT
receptor, Scatchard analysis was carried out using
expressed with or without
.
Expression of alone caused a moderate 3-fold
increase in the affinity of the receptor for
[
H]8-OH-DPAT (Table 2). Co-expression of
with
resulted in
a 7-fold increase in the affinity of the receptor for
[H]8-OH-DPAT. Changes in the B were not observed. In separate experiments, we found to have the opposite effect on affinity. Consistent with the
results shown in Fig. 7, alone caused a 35%
increase in the K of the receptor for
[
H]8-OH-DPAT with no change in B (data not shown).
family to couple with the 5-HT receptor, and to assess the effects of different
combinations, we tested the ability of selected combinations of
subunits to increase binding of [
H]8-OH-DPAT (0.5
nM, Table 3). By Western blots with 8645, membrane
levels of the different subunits were within severalfold of each
other, generally in the rank order of
. All
the
subunits tested (but for , used as a
negative control) increased binding of
[H]8-OH-DPAT to a comparable extent. All
combinations of tested with
supported increases in binding of
[
H]8-OH-DPAT. The effect of
combinations was
; ,
, > . Functional comparisons
of and were not pursued since
was expressed considerably less well than in our hands.
H]8-OH-DPAT binding observed with the
5-HT receptor alone was unaffected by Gpp(NH)p (Fig. 8). Co-expression of
with the receptor
had no effect on this response. Also consistent with previous data is
the reversal by Gpp(NH)p of the slight decrease in binding induced by
(Fig. 7), evident here as a Gpp(NH)p-induced
increase relative to the control of alone. Similarly,
the increases in binding promoted by and
were reversed by Gpp(NH)p. The small increase in binding
supported by was reversed by
Gpp(NH)p. When and
were expressed
together with , 60-70% of
[H]8-OH-DPAT binding became sensitive to
Gpp(NH)p.
H]8-OH-DPAT binding to the 5-HT
receptor co-expressed with G protein subunits. Specific binding of
[
H]8-OH-DPAT at 0.5 nM was determined
for membranes from Sf9 cells expressing the 5-HT receptor,
subunits, and as indicated.
Data represent the mean ± S.E. for binding obtained in the
presence of 100 µM Gpp(NH)p expressed as percent of
binding obtained in the absence of the nucleotide for each permutation
of receptor and subunit(s). The dashed line represents 100% (i.e. no change in binding with Gpp(NH)p). The data were
obtained from three to ten individual experiments performed in
triplicate. Comparisons were made using the two tailed Student's t test. * (p < 0.05) and** (p < 0.01)
refer to differences in binding in the absence and presence of
Gpp(NH)p; (p < 0.05) and (p < 0.01) refer to
differences in binding between the receptor plus subunit alone
and the receptor plus the combination of subunit and
, both in the presence of
Gpp(NH)p.
H]8-OH-DPAT
suggest that the increase in binding as a result of co-expression of G
protein subunits was due to an increase in the affinity of 5-HT receptors for the agonist. To corroborate this interpretation,
experiments were carried out using two newly developed radioligands,
[
I]8-OH-PIPAT and
[
I]p-MPPI.
[
I]8-OH-PIPAT was developed as a 5-HT
receptor agonist, and [
I]p-MPPI
as a receptor antagonist (49, 50, 51) .
Specific binding of [
I]8-OH-PIPAT was not
detected on membranes obtained from Sf9 cells expressing the
5-HT
receptor alone (Fig. 9). When the 5-HT
receptor was co-expressed with
,
, and subunits, however, binding of
the agonist was clearly evident. The B was
14-34 pmol/mg of Sf9 cell membrane protein, and the K was 0.35 ± 0.06 nM (n = 4). In contrast, binding of the antagonist
[
I]p-MPPI was observed regardless of G
protein subunit expression. The density and affinity of binding sites
for [
I]p-MPPI were the same in the
presence or absence of
,
, and
subunits (Fig. 9, inset). These data
confirm those obtained with [H]8-OH-DPAT that
introduction of G protein subunits does not alter the level of
expression of 5-HT receptors, but that the increase in
agonist binding is attributable to a shift in affinity.
I]8-OH-PIPAT and
[
I]p-MPPI to 5-HT
receptors in Sf9 cells. Increasing concentrations of
[
I]8-OH-PIPAT were incubated with membranes
prepared from Sf9 cells expressing 5-HT
receptors alone or
together with
,
, and subunits as indicated. Shown is a representative Scatchard plot.
Each data point represents the mean of three determinations.
Nonspecific binding was determined using 10 µM 5-HT. The
experiment was repeated four times with similar results. The inset shows the effect of co-expressing ,
, and subunits with the 5-HT receptor on the density of
[
I]p-MPPI binding sites. The data is
expressed as a percentage of the density determined in membranes
prepared from Sf9 cells expressing only 5-HT
receptors.
Shown is the mean ± S.E. of four independent experiments
performed with triplicate determinations. By Scatchard analysis the K
values for binding of
[
I]p-MPPI to 5-HT
receptors in the absence and presence of co-expressed G protein
subunits were 0.63 ± 0.12 and 0.57 ± 0.14 (n = 4), respectively. Nonspecific binding was determined
using 100 µM 5-HT.
receptor and co-expressed G
protein subunits, we have tested the hypothesis that Sf9 cells can
serve as an intact cell reconstitution system and have determined that
the 5-HT
receptor can interact not only with subtypes of
G
, but with G and G. Sf9 cells,
unlike mammalian cells or tissues, have so far proven to be free of
endogenous seven transmembrane segment receptors and to contain only
small amounts of G proteins in relation to the amount of receptor or G
protein subunits that can be introduced. Sf9 cells therefore constitute
a relatively well defined setting upon which the expression of
mammalian receptors and G protein subunits can be superimposed. The
absence of potentially interfering receptors becomes particularly
important in the classification of receptor ligands. The specificity of
a ligand for a particular receptor can be determined unambiguously, and
its properties as an agonist or antagonist can be inferred without
knowledge of the effectors that are regulated. Although not explored
here in detail, reconstitution in Sf9 cells can also be used to
determine if the properties of a receptor vary according to the G
protein present. Reconstitution of this nature may also be used to
validate emerging techniques of mapping receptor-G protein pathways in
mammalian cells, e.g. co-immunoprecipitation, agonist-promoted
binding of [S]GTP
S, or photoaffinity
labeling.
for the 5-HT receptor expressed here was 5-34 pmol of receptor/mg of
membrane protein. This value was higher than those reported elsewhere, i.e. 0.15 and 3 pmol/mg(19, 26) . The reason
for the differences in expression among the different laboratories is
unclear, but may be related to the nature of the DNA constructs
employed. For example, small changes in nucleotide sequences,
particularly near the initiation codon, can have a marked effect on
protein expression(52) . In the present study, the
5`-untranslated sequence was shortened to 11 authentic bases to which 5
bases required for introduction of a BglII site were added. In
the work by Mulheron et al.(19) , nucleotides encoding
a FLAG epitope together with an NheI site were introduced
adjacent to the normal initiation codon. The amount of 5`-untranslated
sequence in the construct employed by Parker et al. (26) was not described. Regardless of the reason for the
differences in expression, sufficiently high levels of receptor were
achieved in our studies to permit reconstitution with co-expressed
mammalian G proteins.
receptor into several closely migrating bands. The heterogeneity
in migration probably represents differences in the extent of
glycosylation(23) . Whether the different receptor species
differ in functionality was not ascertained. Several closely spaced
bands were observed for the turkey
-adrenergic receptor, for
example, but only one could be labeled with the photoaffinity analogue
iodocyanopindolol-diazirine(24) . Similar results were obtained
in studies of human -adrenergic receptors expressed in
Sf9 cells(42) . Only substrates for photoaffinity labeling in
the latter studies were subject to palmitoylation. In the present
study, two of the four resolved species of 5-HT receptor
were palmitoylated. The palmitoylation perhaps reflects transport of an
appropriately folded receptor to a membrane compartment. The two other
forms may represent biosynthetic precursors or improperly
folded/processed receptors.
receptors
to G proteins endogenous to Sf9 cells was not evident with the assays
used in the present study, i.e. the receptors exhibited a K
for [
H]8-OH-DPAT
similar to that of the low affinity form of brain 5-HT receptors(16) , and the binding of the agonist was not
sensitive to GTP. These observations are consistent with data obtained
for
-adrenergic, m1- and m2-muscarinic cholinergic(24) ,
dopamine D3(25) , and N-formyl peptide
receptors(23) . Given the results of Mulheron et al.(19) and Parker et al.(26) , we
nevertheless suspect that a small fraction of the 5-HT receptors are coupled to one or more G proteins but that the
coupling is obscured by the large number of uncoupled receptors. The
work by Parker et al.(26) , in particular, would place
an upper limit of about 200 fmol/mg of membrane protein for coupled
receptor. Mulheron et al.(19) identified an
agonist-activated Sf9 G
-like protein through
[P]GTP-azidoanilide labeling. Our results with G
protein-directed antibodies confirm the existence of an
-like subunit and the probable absence of at least
three species of in Sf9 cells. The array of
endogenous subunits probably also includes homologs of and . G would account for
isoproterenol-stimulated adenylyl cyclase activity in Sf9 cells
expressing -adrenergic receptors(24) , and G may underlie the reported Gpp(NH)p sensitivity of agonist binding
to expressed substance P receptors(22) . Although Sf9 cells
contain as demonstrated following purification of the
subunit on
-agarose(30) , the inability to
detect the subunit by Western blotting with antibodies such as
5357 and 8136 (the latter antiserum is comparable to
K-521(30) ) suggests that the subunit is not or , and probably not or
. Alternatively, a homolog may exist in quite small
amounts. receptor by co-expression of the receptor with mammalian G
protein subunits, i.e. a substantial increase in affinity of
the receptor for agonist (together with Gpp(NH)p sensitivity in
binding, see below) was supported by co-expression of appropriate
combinations of
, , and subunits. Thus, the desired
functional reconstitution of receptor and G protein was realized. These
results are consistent with the large body of data, obtained with
native and reconstituted mammalian membranes, and with purified
receptors and G proteins reconstituted into liposomes, that supports a
ternary complex model of interaction (18, 53, 54) . Interestingly, when an
subunit was expressed alone with the 5-HT receptor, the
receptor assumed an affinity for [
H]8-OH-DPAT
intermediate between that of its high- and low-affinity forms. That
subunits alone can interact to some extent with receptors has
been demonstrated previously for and rhodopsin in
retinal rod outer segment disc membranes(45, 55) .
Alternatively, the subunit may utilize endogenous ,
although with a lower affinity or effectiveness. In the former
instance, consistent with our data, the interaction is stabilized by
introduction of
(55) . In terms of mechanism, the
and subunits may form a heterotrimeric complex or may
act separately to enhance interaction with the receptor. It is also
possible that
prevents denaturation of
subunits, or
promotes a preferred placement of the subunits with respect to the
receptors. or
subunits alone. Maximal sensitivity to the nucleotide (60%)
was achieved with the coordinate expression of both
and
. These data are consistent with the measured increases in
affinity for agonist described above, and with the Gpp(NH)p sensitivity
observed by us and others for 5-HT
receptors in membranes
from rat brain.
,
,
,
, and
, when expressed together with
, all have the capacity to interact
with the 5-HT receptor. The data with respect to subtypes
of
are in general agreement with those of Raymond et al. (56) and Bertin et al.(57) ,
in which coupling to all three subtypes could be demonstrated for the
receptor expressed in HeLa cells and Escherichia coli,
respectively. A selectivity for coupling to
G(56) , however, was not evident. In contrast to
Bertin et al.(57) , moreover, we find that G
has the capacity to couple with the co-expressed receptor. This
result is important in that it provides direct evidence for an
interaction that has, at best, only been intimated for the mammalian
protein. The reason for the difference between our observation and that
achieved with bacterially expressed receptor/G protein subunits is not
clear, but may be related to covalent modification. Our results are
consistent with the fact that 5-HT receptors can activate
an
-like subunit endogenous to Sf9 cells(19) ,
and the recent reports that G underlies several
serotonin-controlled behaviors exhibited by Caenorhabditis
elegans(58, 59) . Transduction pathways employed
by the 5-HT receptor in mammalian cells have so far proven
to be sensitive to PTX, consistent with the utilization of G
or
G(8, 9, 10, 14, 15) .
Interactions of the receptor with , which is
PTX-insensitive, however, were not unanticipated. Wong et
al.(60) , by co-transfecting 293 cells with DNA encoding
stimulatory and inhibitory receptors and ,
demonstrated an interaction between G and receptors that
normally communicate with G when the latter was inactivated
with PTX. Parker et al.(24) have similarly
demonstrated an interaction between the m2-muscarinic cholinergic
receptor and G when both are reconstituted into liposomes.
It is interesting to note that the amount of G is
relatively high in the hippocampus, where a high density of 5-HT receptors is also found(61) . The behavior of
, which tends to suppress high-affinity agonist
binding, is unusual. may interact directly with the
receptor to lower affinity for agonist in a fashion reversed by
Gpp(NH)p and . It is also possible that
sequesters from an endogenous
subunit that
interacts (albeit poorly) with the 5-HT receptor.
subunits on the
coupling of 5-HT
receptors to G proteins. Kisselev and
Gautam (62) had demonstrated that
, ,
and could interact equally well with
, but only supported interaction of with rhodopsin.
Iiguez-Lluhi et al.(30) and
Ueda et al.(63) had demonstrated, at the level of
effectors, that is less good than
, ,
, and (when examined)
at inhibiting calmodulin-stimulated
type-I adenylyl cyclase, potentiating -stimulated
type-II adenylyl cyclase, and stimulating phospholipase
C. Four of the subtypes of subunit tested here
(in combination with
and
),
, , , and
, proved to be equivalent to each other in their
support of coupling to the 5-HT receptor.
was less effective. In this regard, the interaction between
and may not be
particularly strong, as suggested in experiments testing the ability of
combinations to support PTX-catalyzed
ADP-ribosylation(30, 63) . This may be attributable to
the fact that
, unlike (and
presumably the other subunits(64, 65) ), is in
part farnesylated instead of uniformly geranylgeranylated in Sf9
cells(66) , (
)or to differences in structure. The
pattern established by combinations to support
coupling of the 5-HT
receptor to
is the
converse of that reported for the coupling of rhodopsin to
(62) but is analogous to that observed for effector
regulation(30, 63) . In our studies, we did not test
permutations involving since expression of this
subunit was far less robust than that of . Differences
in expression between and have been
reported previously(63) .I]8-OH-PIPAT, but not
[
I]p-MPPI, displays guanine
nucleotide-sensitive binding to 5-HT
receptors, consistent
with identification of [
I]8-OH-PIPAT as an
agonist and [
I]p-MPPI as an
antagonist(49, 50) . p-MPPI completely
antagonizes the inhibition of forskolin-stimulated adenylyl cyclase
activity caused by 8-OH-DPAT and 8-OH-PIPAT in hippocampal
membranes(51) . Detectable binding of
[
I]8-OH-PIPAT in Sf9 cells was observed only
when 5-HT
receptors were co-expressed with G protein
subunits. In contrast, binding of
[
I]p-MPPI occurred regardless of G
protein subunit expression, as would be anticipated for an antagonist.
The results with [
I]p-MPPI corroborate
that co-expression of infected Sf9 cells with additional recombinant
virus did not alter the level of expression of the 5-HT
receptor.
,-imino)triphosphate; p-MPPI,
4-(2`-methoxyphenyl)-1-[2`-[N-(2"-pyridinyl)-p-iodobenzamido]ethyl]piperazine;
PTX, pertussis toxin; PAGE, polyacrylamide gel electrophoresis.
We acknowledge Drs. T. Kozasa and A. G. Gilman
(Southwestern Medical Center, Dallas), and Dr. J. C. Garrison
(University of Virginia), for the kind gifts of G protein recombinant
baculoviruses, Drs. M. Caron and R. Lefkowitz (Duke University) for the
human 5-HT receptor DNA, and Drs. H. F. Kung, M.-P. Kung,
and Mu Mu (University of Pennsylvania) for
[
I]8-OH-PIPAT and [
I] p-MPPI.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 R. Grosse, T. Schöneberg, G. Schultz, and T. Gudermann Inhibition of Gonadotropin-Releasing Hormone Receptor Signaling by Expression of a Splice Variant of the Human Receptor Mol. Endocrinol., August 1, 1997; 11(9): 1305 - 1318. [Abstract] [Full Text]
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D. S. Cowen, P. B. Molinoff, and D. R. Manning 5-Hydroxytryptamine1A Receptor-Mediated Increases in Receptor Expression and Activation of Nuclear Factor-kappa B in Transfected Chinese Hamster Ovary Cells Mol. Pharmacol., August 1, 1997; 52(2): 221 - 226. [Abstract] [Full Text]
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 U. Martin, D. Bock, L. Arseniev, M. A. Tornetta, R. S. Ames, W. Bautsch, J. Kohl, A. Ganser, and A. Klos The Human C3a Receptor Is Expressed on Neutrophils and Monocytes, but Not on B or T Lymphocytes J. Exp. Med., July 21, 1997; 186(2): 199 - 207. [Abstract] [Full Text] [PDF]
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M. N. Garnovskaya, T. W. Gettys, T. van Biesen, V. Prpic, J. K. Chuprun, and J. R. Raymond 5-HT1A Receptor Activates Na+/H+ Exchange in CHO-K1 Cells through Gialpha 2 and Gialpha 3 J. Biol. Chem., March 21, 1997; 272(12): 7770 - 7776. [Abstract] [Full Text] [PDF]
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A. J. Barr, L. F. Brass, and D. R. Manning Reconstitution of Receptors and GTP-binding Regulatory Proteins (G Proteins) in Sf9 Cells. A DIRECT EVALUATION OF SELECTIVITY IN RECEPTOR·G PROTEIN COUPLING J. Biol. Chem., January 24, 1997; 272(4): 2223 - 2229. [Abstract] [Full Text] [PDF]
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D. S. Cowen, R. S. Sowers, and D. R. Manning Activation of a Mitogen-activated Protein Kinase (ERK2) by the 5-Hydroxytryptamine1A Receptor Is Sensitive Not Only to Inhibitors of Phosphatidylinositol 3-Kinase, but to an Inhibitor of Phosphatidylcholine Hydrolysis J. Biol. Chem., September 13, 1996; 271(37): 22297 - 22300. [Abstract] [Full Text] [PDF]
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S. Moffett, L. Adam, H. Bonin, T. P. Loisel, M. Bouvier, and B. Mouillac Palmitoylated Cysteine 341Modulates Phosphorylation of the beta 2-Adrenergic Receptor by the cAMP-dependent Protein Kinase J. Biol. Chem., August 30, 1996; 271(35): 21490 - 21497. [Abstract] [Full Text] [PDF]
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H. Yasuda, MargaretA. Lindorfer, KarenA. Woodfork, JuliaE. Fletcher, and JamesC. Garrison Role of the Prenyl Group on the G Protein gamma Subunit in Coupling Trimeric G Proteins to A1 Adenosine Receptors J. Biol. Chem., August 2, 1996; 271(31): 18588 - 18595. [Abstract] [Full Text] [PDF]
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C. A. Chen and D. R. Manning Regulation of Galpha i Palmitoylation by Activation of the 5-Hydroxytryptamine-1A Receptor J. Biol. Chem., July 28, 2000; 275(31): 23516 - 23522. [Abstract] [Full Text] [PDF]
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X. Fan, L. F. Brass, M. Poncz, F. Spitz, P. Maire, and D. R. Manning The alpha Subunits of Gz and Gi Interact with the eyes absent Transcription Cofactor Eya2, Preventing Its Interaction with the Six Class of Homeodomain-containing Proteins J. Biol. Chem., October 6, 2000; 275(41): 32129 - 32134. [Abstract] [Full Text] [PDF]
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