(Received for publication, November 18, 1996)
From the Department of Physiology and Medical
Biophysics, Uppsala University, BMC, Box 572, S-75123 Uppsala,
Sweden and the ¶ Department of Pharmacology and Clinical
Pharmacology, University of Turku, FIN-20520 Turku, Finland
The 2-adrenergic receptors
(
2-ARs), which primarily couple to inhibition of cAMP
production, have been reported to have a stimulating effect on adenylyl
cyclase activity in certain cases. When expressed in Spodoptera
frugiperda Sf9 cells the
2A subtype showed only
inhibition of forskolin-stimulated cAMP production when activated by
norepinephrine (NE), whereas the
2B subtype displayed a
biphasic dose-response curve with inhibition at low concentrations of
NE and a potentiation at higher concentrations. To further investigate
the subtype-specific coupling, we expressed a set of chimeric
2A-/
2B-ARs at similar expression levels
in Sf9 cells to determine the structural domain responsible for the difference between the two subtypes. When the third intracellular loops
were interchanged between
2A and
2B
subtypes, the coupling specificity remained unchanged, indicating that
this loop does not confer selectivity toward a stimulating response. A
biphasic dose-response curve, typical for the
2B
subtype, could be seen when the second intracellular loop of the
2B subtype was inserted into the
2A
subtype, suggesting that this loop is important for determining the
subtype-specific coupling of
2-ARs to cAMP production. Site-directed mutagenesis of non-conserved amino acids in the second
intracellular loop of the
2A subtype indicated that
several residues are involved in the coupling specificity.
The 2-adrenergic receptors
(
2-ARs)1 are members of a
large family of heptahelical receptors mediating the extracellular
stimuli to the interior of the cell through G proteins. Three different subtypes of
2-ARs,
2A,
2B,
and
2C, can be distinguished based on the affinity for
selective ligands (1), and this subdivision has been confirmed with the
molecular cloning of three separate genes (2-4). Funtional expression
of cloned cDNAs in different cell types has shown that the
2-ARs can mediate multiple cellular responses including
inhibition or stimulation of adenylyl cyclase activity (5-9),
activation of phospholipase A2 and D (5, 10), stimulation
of phosphatidylinositol turnover (6), and mobilization of intracellular
Ca2+ (11, 12).
In some cells, activation of 2-ARs leads to a biphasic
regulation of adenylyl cyclase activity with an inhibitory phase at low
concentration of agonist and a stimulatory phase at higher concentrations (5, 7, 13, 15), whereas in other cells, the response is
exclusively inhibitory (6, 8) or stimulatory (9, 13-15). The
stimulation of cAMP production with
2-AR agonists has
also been shown in physiological systems with endogenous receptors (16,
17), suggesting that it is not a mere side effect seen in recombinant
systems.
The seemingly controversial effect of 2-AR activation on
stimulation of adenylyl cyclase activity has been attributed to relatively high expression levels of the receptors (7),
cell-type-specific expression of different types of adenylyl cyclases
(14, 15), or overexpression of Gs protein (18). A direct
interaction of
2-ARs with Gs protein has
been shown in Chinese hamster ovary cells (7), while in other cell
types, the mechanism for stimulation seems less clear (9, 15, 19). In
many cases, the stimulatory response has shown subtype selectivity, the
stimulation being more pronounced with the
2B subtype
compared with the
2A and
2C subtypes (9,
13, 15). This suggests that the G protein coupling domains differ
between the subtypes. Different cytoplasmic domains have been
implicated in G protein activation and selection both in adrenergic
receptors (20, 21) and other heptahelical receptors (22, 23). The aim
of the present study was to investigate the apparent subtype-specific
coupling of the mouse
2-AR subtypes,
2A
and
2B, when expressed in Spodoptera
frugiperda (Sf9) cells at comparable receptor levels, and to
delineate the receptor domain leading to potentiation of cAMP
production.
[3H]Adenine (21 Ci/mmol) and [3H]RX821002 (48 Ci/mmol) were from Amersham Corp. (Buckinghamshire, UK). [14C]cAMP (309 mCi/mmol) was from DuPont NEN. Cholera toxin, isobutylmethylxanthine (IBMX), (-)-norepinephrine, pertussis toxin, propranolol, and quinacrine were from Sigma. Phentolamine and UK14,304 were from Research Biochemicals International (Natick, MA).
Cell CultureSf9 cells were maintained as suspension culture at 25-27 °C in TNM-FH medium (pH 6.3) supplemented with 10% fetal calf serum (Life Technologies, Inc., Paisley, UK), 100 units/ml penicillin (Nordvacc Media, Skärholmen, Sweden), and 100 µg/ml streptomycin (Nordvacc Media), and 2.5% amphotericin B (Life Technologies, Inc.). For expression, Sf9 cells were subcultured in monolayer and infected with recombinant baculoviruses at a multiplicity of infection of 2-5 for the indicated times.
Recombinant BaculovirusesThe cDNAs for the mouse
2A- and
2B-ARs and three mouse chimeric
receptors, MCR1, -2, and -3, were a gift from Dr. B. Kobilka (Howard
Hughes Medical Institute, Stanford University). All clones contained a
hemagglutinin tag fused to the amino terminus of the receptor
constructs. For generation of recombinant baculoviruses, the genes were
subcloned into the baculovirus transfer vector plucGRBac1 (9) under the
transcriptional regulation of the polyhedrin gene promoter. The
transfer vectors were then used for cotransfection with wild-type
Autographa Californica nuclear polyhedrosis virus DNA, and
recombinant viruses were purified essentially as described by Jansson
et al. (9).
To generate chimeric receptors MCR4 and MCR5, the cDNA clones
for 2A- and
2B- ARs were transferred into
the SmaI site of pBluescript (Stratagene, La Jolla, CA) in a
KS orientation. A SapI restriction site was introduced into
the
2A sequence at nucleotide position 351 of the coding
sequence (equivalent to the position of a SapI site in the
2B cDNA) using standard PCR techniques (24). The
sequence from the beginning of the
2A gene to the novel
SapI site was then inserted into
pBluescript-
2B cut with the same enzyme to generate
pBluescript-MCR4.
MCR5 was constructed by isolating a DraIII-fragment
(DraIII cuts the cDNAs of 2A and
2B at an equivalent position and the plasmid DNA at one
position) from pBluescript-
2A and ligating it into an
isolated DraIII-fragment of pBluescript-MCR4 to generate pBluescript-MCR5. The sequences encoding MCR4 and MCR5 were cut out
from the pBluescript constructs with EcoRI and
XbaI and ligated into pFastBac1 digested with the same
enzymes. For production of recombinant baculovirus, a BAC-TO-BAC
baculovirus expression system kit (Life Technologies, Inc.) was
used.The correctness of all plasmid constructs were checked by
restriction enzyme mapping and partial dideoxy sequencing (25).
Site-directed mutagenesis of the 2A sequence was
performed using PCR as described (24). Mutated
2A
sequences were subcloned into pBluescript, and the mutations were
verified by sequencing. The mutated sequences were subsequently
transferred to pFastBac1 with EcoRI and NotI.
Recombinant baculoviruses were generated using the BAC-TO-BAC
baculovirus expression system kit.
Sf9 cells were plated on tissue culture dishes and allowed to attach for 1 h before infection with respective recombinant baculovirus for the indicated times. The cells were incubated with 5 µCi/ml [3H]adenine for 2-3 h and thereafter were scraped off, pelleted, and washed in MES-buffered medium (130 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl2, 4.2 mM NaHCO3, 7.3 mM NaH2PO4, 20 mM MES, 63 mM sucrose, 10 mM glucose, and 1 mM CaCl2, pH 6.3). The pellet was resuspended in the same buffer and devided into aliquots of about 106 cells/0.8 ml. The cells were preincubated with 0.5 mM IBMX, 100 µM propranolol, and 150 µM quinacrine for 10 min at 26 °C, and then forskolin (30 µM) and agonist were added. After 10 min of incubation, the cells were spun down, the supernatant was removed, and the pellet was resuspended in 1 ml of 0.33 M perchloric acid containing about 2000 cpm [14C]cAMP. Cyclic AMP was isolated by sequential Dowex/alumina ion exchange chromatography (26), and radioactivity was determined in a liquid scintillation counter (Wallac, Turku, Finland). The conversion of [3H]ATP to [3H]cAMP was calculated as a percentage of total cellular [3H]ATP and normalized to the recovery of [14C]cAMP.
Receptor Binding AssayInfected cells from monolayer cultures were harvested in phosphate-buffered saline solution and centrifuged 1500 × g for 5 min. The cell pellet was resuspended in cold potassium phosphate buffer (40 mM K2HPO4, 10 mM KH2PO4, pH7.4) and homogenized with an Ultra-Turrax homogenizer (Janke and Kunkel, Germany). 100-200 µg of protein of the homogenate was incubated with 10 nM [3H]RX821002 in a volume of 0.3 ml potassium phosphate buffer at 25 °C for 30 min. 10 µM phentolamine was used to determine nonspecific binding. The reactions were terminated by filtration through printed filtermat B filters (Wallac) using a Harvester 96 (Tomtec Inc., Orange, CO). After the filters had dried, a MeltiLex B/HS scintillator sheet was melted on them, and radioactivity was determined in a Microbeta scintillation counter (Wallac).
Infection of
Sf9 cells with baculovirus harboring the genes for the mouse
2A- or
2B-AR resulted in a
time-dependent increase in receptor density as determined
by specific binding of [3H]RX821002 (data not shown).
Cells infected with wild-type virus did not show specific binding of
[3H]RX821002. When assayed for functional coupling to
regulation of cAMP production, the
2A subtype mainly
inhibited the forskolin stimulation (Fig.
1A), whereas the
2B subtype
showed a biphasic response with inhibition at low concentration (1 µM) of norepinephrine (NE) and a potentiating effect at
higher concentrations (100 µM) (Fig. 1B). The
potentiation appeared to reach a maximum around 38 h postinfection
(p.i.) and then to decline with longer infection times. For further
characterization, 48 h p.i. was chosen because the magnitudes of
inhibition versus potentiation of the forskolin response
between the two subtypes were similar at this time point.
In Fig. 2, the dose-response curves for the two receptor
subtypes with two different agonists, NE and UK14,304, are shown. UK14,304 was used as a control agonist for inhibition since this compound has been shown to be very weak in potentiating forskolin stimulation with the 2B subtype while being full agonist
for the inhibition (27). Both NE and UK14,304 inhibited cAMP production in
2A-expressing cells to the same extent and with
similar potencies. In cells expressing the
2B subtype,
NE displayed a biphasic response with an inhibition at low
concentrations, that reached a maximum at 1 µM, and that
potentiated the forskolin stimulation at higher concentrations.
UK14,304 elicited mainly inhibition with the
2B subtype,
confirming that UK14,304 is much less effective in eliciting a
stimulation of cAMP production compared with the endogenous agonist
NE.
Pertussis toxin (PTX) has been used in Chinese hamster ovary cells to
abolish the inhibition of cAMP production through Gi proteins and thus reveal a less efficient coupling of
2-ARs to a stimulatory component, presumably
Gs (7, 19, 27). When Sf9 cells were treated with PTX for
48 h, the inhibition was abolished in both
2A- and
2B-expressing cells, and the stimulatory response with
the
2B subtype was enhanced over 2-fold (Fig.
3). Treatment of Sf9 cells with cholera toxin (CTX),
which is known to activate Gs proteins persistently,
drastically reduced the inhibitory response with both subtypes (Fig.
3).
The influence of Ca2+ on the cAMP production was tested by
chelating extracellular Ca2+ with EGTA. The chelation of
extracellular Ca2+ did not effect the responses to any
larger extent in cells expressing either the 2A subtype
or the
2B subtype (Fig. 3).
To try to delineate the domain in the
2B subtype responsible for a potentiation of cAMP
production, we expressed a set of chimeric
2A-/
2B-receptors (Fig.
4).
MCR1 and MCR2, 2A and
2B with interchange
of the third intracellular loop (i3-loop), respectively, show similar
dose-response curves with NE as the parent receptor (Fig.
5). MCR1 showed a reduced maximal inhibition compared
with the
2A subtype, but no biphasic response mode could
be seen. MCR2 displayed a typical biphasic response but with a reduced
potentiating effect. Employing UK14,304 as the agonist increased the
maximal inhibition with MCR2 compared with the parent
2B
subtype, whereas the response with MCR1 did not deviate from the parent
2A subtype (data not shown).
MCR3, in which the carboxyl-terminal part from the end of the i3-loop
of the 2A subtype was exchanged with
2B
sequence, responded in the same way as the native
2A
subtype (Fig. 6). MCR4, with
2B-sequence
from the beginning of the second intracellular loop (i2-loop) to the C
terminus, behaved as the intact
2B subtype in displaying
a biphasic dose-response curve with NE (Fig. 6), indicating that the
second intracellular loop might be involved in the potentiation seen
with the
2B subtype.
Effect of Second Intracellular Loop on Coupling Specificity
To test the involvement of the i2-loop in the
different coupling modes, we expressed a chimeric 2A
receptor in which the i2-loop had been exchanged to
2B
receptor sequence, MCR5. This construct exhibited a similar biphasic
dose-response curve with NE as the
2B subtype 48 h
p.i. (Fig. 7). Since this viral construct expressed
receptors at somewhat higher density compared with the other
constructs, we measured the dose-response relationship at 38 h
p.i. when the receptor level was comparable to the parent
2A subtype (see Fig. 4.). At 38 h p.i., this
construct stimulated cAMP production very potently, showing almost no
inhibition with NE (Fig. 7), which indicated that the change in
coupling specificity could not be attributed to differences in the
expression levels of the chimeric receptor.
Site-directed Mutagenesis of the Second Intracellular Loop
There are six amino acid residues that differ between the
2A and
2B subtypes in the second
intracellular loop (Fig. 4.). Three of these residues are essentially
similar (Ile-135 in
2A versus Val at the
corresponding position in
2B, Thr-136 versus
Ser, and Ile-139 versus Leu). The three other non-conserved
residues differ in terms of polarity (Ser-134 in
2A
versus Ala at the corresponding position in
2B, Gln-137 versus Arg, and Leu-143 versus Ser). Site-directed mutagenesis of each of these
three amino acids of the
2A subtype to corresponding
residues of the
2B subtype did not give clear
indications which residues might be responsible for the coupling
specificity (Fig. 8.). These mutants all showed a lower
degree of inhibition with NE compared with the
2A
subtype, and no marked biphasic response could be seen. On the
contrary, a double mutant, S137A, L143S, exhibited a biphasic response
with NE similar to the
2B subtype although with a
smaller magnitude of stimulation. All of the mutated constructs were
expressed at receptor levels comparable with the
2A
subtype, and the inhibition with UK14,304 was 40-50% of forskolin
stimulation with all four constructs (not shown).
During the last few years, it has become evident that G
protein-coupled receptors can couple to multiple G proteins to elicit different cellular responses (7, 28-30). The 2-ARs have
been shown to couple to both negative and positive regulation of
adenylyl cyclase activity (5, 7, 9, 13-15). If coupling to both pathways occurs simultaneously but with different potencies, one could
expect to obtain a biphasic dose-response curve. With the
2-ARs, this is often the case; an inhibition of
forskolin-stimulated cAMP production is seen with low concentrations of
agonist and a stimulation or potentiation of cAMP production with
higher concentrations (5, 7, 13, 15). Earlier studies have indicated
that the stimulatory effect of
2-ARs is
cell-type-specific (15) and/or dependent on the expression level of the
receptors (7). This response also seems to be subtype-specific, the
2B subtype having a more pronounced stimulatory effect
than the
2A and
2C subtypes when
expressed in the same cells (8, 9, 13, 15, 31). In the present study,
we expressed the mouse
2A- and
2B-AR subtypes in Sf9 cells at comparable expression levels and obtained a
marked difference in the coupling specificity between the subtypes. The
2B subtype showed a biphasic dose-response curve with a
potentiation of the forskolin stimulation at high concentration of NE,
whereas the
2A subtype displayed a monophasic inhibitory
dose-response curve. With prolonged infection times, which parallel an
increase in receptor density, the maximal inhibition with
2A was increased. This is in contrast to the finding by
Eason et al. (7) that an increase in receptor density
promotes the stimulatory pathway with the human
2A
subtype (
2-C10). The reason for this discrepancy is
unclear but might involve interspecies variation of the receptor subtypes or differences in the types of G proteins expressed by the two
different cell lines used. A reduction in the maximal stimulation with
longer infection times for the
2B subtype was also seen,
which indicates a more efficient coupling to the inhibitory component
at higher expression levels. The imidazoline-like agonist UK14,304 was
very weak in eliciting a biphasic response with the
2B
subtype. The ability of UK14,304 to promote coupling to a stimulatory
pathway with the
2B subtype has been shown to be very
weak in Chinese hamster ovary cells (27).
PTX treatment, which is known to reveal a stimulatory pathway to cAMP
production with the 2-ARs (7, 19, 27), increased the
stimulation over 2-fold with the
2B subtype. This
supports the hypothesis that coupling to both pathways occur
simultaneously, and thereby, an elimination of either pathway would
result in an enhancement of the other. PTX treatment of cells
expressing the
2A subtype did not reveal any significant
coupling to a stimulatory pathway although the inhibition was
abolished. This confirms that the ability of the receptors to
potentiate cAMP production is, apart from being related to expression
levels, also subtype-specific.
Treatment of the cells with CTX, which would abolish the stimulation if
it was Gs-mediated, drastically reduced the inhibition with
both subtypes but did not seem to affect the stimulatory component with
the 2B subtype. These data are difficult to interpret, however, since CTX stimulated the adenylyl cyclase activity
several-fold over the forskolin-stimulated activity, and this activity
may be difficult to inhibit (9, 32).
2-AR-mediated stimulation of adenylyl cyclase activity
has been suggested to occur through a rise in intracellular calcium concentration in PC12 cells (15). Since we observed a small but
significant elevation of intracellular Ca2+ in Sf9 cells
expressing the
2B subtype when assayed with fura-2 fluorescence (data not shown), we measured the change in cAMP production in the prescence of EGTA. The potentiating response did not
differ significantly from the control experiment while EGTA largely
prevented the Ca2+ elevation in the fura-2 assay. This
suggests that Ca2+ elevation is not the stimulating factor
in the
2B-mediated potentiation of cAMP production in
these cells although Ca2+ may enhance the stimulatory
response.
The third intracellular loop of G protein-coupled receptors has been
implicated in G protein selectivity and activation (for review, see
Ref. 33). When we expressed chimeric
2A-/
2B-ARs with interchange of the
i3-loops, the responses with NE were very similar to the parent
receptor subtypes. A reduction of the potentiating response of
2B was seen with MCR2, and a reduction of the inhibition of
2A could be seen with MCR1. This may be related to a
more efficient coupling to Gi proteins through the i3-loops
of the receptors. Another possibility is that the change of the i3-loop might alter the general conformation of the coupling device leading to
slightly altered responses.
In an earlier study, where part of the carboxyl-terminal tail of the
2A subtype was introduced into the
2-AR,
a small reduction in isoproterenol-stimulated adenylyl cyclase activity
was seen, suggesting an involvement of the carboxyl-terminal tail in G
protein coupling (21). In this study, the exchange of the
carboxyl-terminal tail in
2A subtype for
2B sequence (MCR3) did not alter the coupling mode for
the
2A subtype (Fig. 6), indicating that this domain, if
involved in coupling to G proteins in the
2-ARs,
probably interacts with a Gi protein. The chimeric receptor
MCR4 displayed a typical biphasic dose-response curve. This was also
expected since this chimera contains
2B sequences in all
the proposed G protein-coupling domains. We also constructed a chimeric
2A receptor with
2B sequence from the
amino terminus to the distal end of the i2-loop, but this construct was
not expressed at such a density that a functional characterization
could be accomplished.
When the i2-loop from 2B was introduced into the
2A subtype, the chimeric receptor displayed a biphasic
dose-response curve with NE similar to the
2B subtype
when assayed 48 h p.i. At an earlier time point (38 h p.i.), the
chimeric receptor stimulated cAMP production even more potently than
did the native
2B subtype at the same time
postinfection. There are two possible explanations for this. First, the
chimeric construct was expressed at higher receptor levels than the
2B construct, about 3.8 pmol/mg of protein for MCR5
compared with about 1.5 pmol/mg of protein for the
2B subtype. A higher expression could increase coupling to the stimulatory component and thereby mask a coupling to the inhibitory component. Second, a Gs-coupling domain in the third intracellular
loop of the human
2A subtype has been identified based
on studies with chimeric
2/5-HT1A receptors
(34). Exchange of the i2-loop from the
2B subtype might
result in a receptor that couples more tightly to a stimulating G
protein.
The i2-loop has been implemented in G protein selectivity in a study
using muscarinic m1:-adrenergic receptor chimeras (22). The role of
the i2-loop in G protein activation has also been studied using
synthetic peptides derived from
2-ARs and muscarinic receptors (35, 36). In the study by Okamoto and Nishimoto (35), the
peptide derived from the i2-loop of the human
2A subtype
(
2-C10) potently stimulated GTP
S binding to
Gs proteins. Although this finding is in contrast to our
results that the
2A receptor is strictly inhibiting,
other regions of the receptor are probably also involved in governing
the selectivity.
The i2-loop sequences of the 2A and
2B-ARs differ at six positions when predicted from the
cDNA sequences. There are three non-conserved amino acid
substitutions between the two subtypes that will change the polarity of
the i2-loop, Ser-134, Gln-137, and Leu-143, in the
2A
subtype. Single point-mutated receptors with each of these
non-conserved residues substituted with corresponding
2B
residues showed no clear biphasic responses with NE. In contrast, a
double mutant with S134A and L143S responded in a similar way to the
2B subtype. The interpretation of this is that at least these two residues are needed to maintain the integrity of the coupling
domain. Interestingly, in the
2-adrenergic receptor, the
equivalent residues (Ser-134 and Leu-143) are the same as in the
2B subtype.
In conclusion, we have presented evidence that the coupling of
2-ARs to cAMP production is subtype-specific and that
the second intracellular loop of the
2B subtype
determines the different coupling specificity leading to stimulation of
cAMP production. Site-directed mutagenesis of non-conserved amino acid
residues indicates that the whole structure of the second intracellular loop is important for efficient coupling.
We thank Dr. Brian Kobilka for providing the cDNA clones, Katariina Pohjanoksa for preparing the cDNA inserts, Kent Rönnholm for technical assistance, and Jyrki Kukkonen for constructive criticism.