Volume 270,
Number 42,
Issue of October 20, 1995 pp. 24753-24760
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification
of a G
Coupling Domain in the Amino Terminus of the Third
Intracellular Loop of the 
-Adrenergic Receptor
EVIDENCE FOR DISTINCT STRUCTURAL DETERMINANTS THAT CONFER GVERSUS G
COUPLING (*)
(Received for publication, March 14, 1995; and in revised form, July 13,
1995)
Margaret G.
Eason
(1),
Stephen B.
Liggett
(1) (2)(§)From the
(1)Departments of Medicine (Pulmonary),
(2)Molecular Genetics, and
(3)Pharmacology, University of Cincinnati College of
Medicine, Cincinnati, Ohio 45267-0564
ABSTRACT
- INTRODUCTION
- EXPERIMENTAL PROCEDURES
- RESULTS
- DISCUSSION
- FOOTNOTES
- ACKNOWLEDGEMENTS
- REFERENCES
ABSTRACT 
-Adrenergic receptors (AR)
functionally couple not only to G but also to
G. We investigated the amino-terminal portion of the third
intracellular loop of the human AR
(C10) for potential G coupling domains
using site-directed mutagenesis and recombinant expression in several
different cell types. A deletion mutant and four chimeric receptors
consisting of the AR with the analogous sequence from
the 5-HT
receptor (a G-coupled receptor) and
the 
AR (a G-coupled receptor) were
expressed in Chinese hamster ovary cells, Chinese hamster fibroblasts,
or COS-7 cells and examined for their ability to mediate stimulation or
inhibition of membrane adenylyl cyclase activity or whole cell cAMP
accumulation.
In stably expressing Chinese hamster ovary cells,
deletion of amino acids 221-231, which are in close proximity to
the fifth transmembrane domain, eliminated
C10-mediated stimulation of adenylyl cyclase activity,
while C10-mediated inhibition was only moderately
affected. This suggested that this region is important for G coupling, prompting construction of the chimeric receptor
mutants. Substitution of amino acids 218-235 with 5-HT receptor sequence entirely ablated agonist-promoted G coupling, as compared with a 338 ± 29% stimulation of
adenylyl cyclase activity observed with the wild-type
C10. In contrast, G coupling for this
mutant remained fully intact (57 ± 2% versus 52
± 1% inhibition for wild-type C10). Similar
substitution with AR sequence had no effect on G coupling but did reduce G coupling. Two additional
mutated C10 containing smaller substitutions of the
amino-terminal region with 5-HT receptor sequence at
residues 218-228 or 229-235 were then studied. While
G coupling remained intact with both mutants, G coupling was ablated in the former but not the latter mutant
receptor. Similar results were obtained using transfected Chinese
hamster fibroblasts (which exclusively display
AR-G coupling) and COS-7 cells (which
exclusively display AR-G coupling). Thus,
a critical determinant for G coupling is contained within
11 amino acids(218-228) of the amino-terminal region of the third
intracellular loop localized directly adjacent to the fifth
transmembrane domain.
Taken together, these studies demonstrate the
presence of a discrete structural determinant for agonist-promoted
AR-G coupling, which is distinct and
separable from the structural requirements for
AR-G coupling.
INTRODUCTION
Activation of cellular signaling pathways by many hormones and
neurotransmitters occurs via interaction with members of a superfamily
of integral cellular membrane receptors that physically bind and
activate heterotrimeric guanine nucleotide binding proteins
(G-proteins). (
)G-protein coupled receptors have an
extracellular amino terminus and intracellular carboxyl terminus and
are thought to span the cellular membrane seven times producing three
extracellular and three intracellular loops. Chimeric
receptor(1, 2, 3, 4, 5, 6) ,
site-directed
mutagenesis(6, 7, 8, 9, 10, 11) ,
and peptide(12, 13, 14, 15, 16, 17) studies
have clearly established that the G-protein coupling domains of these
receptors are located within the intracellular portions, particularly
in the third intracellular loop.
The adrenergic receptors (AR)
mediate the effects of epinephrine and norepinephrine and are
classified into several types: AR, AR, and
AR. While the AR stimulate
phosphatidylinositol hydrolysis via coupling to a
G
/G
class G-protein, the AR and
AR are predominantly characterized by their abilities
to modulate adenylyl cyclase activity. The AR are coupled to the
stimulatory G-protein G, and thereby evoke stimulation of
adenylyl cyclase activity resulting in the elevation of the
intracellular second messenger cAMP. Conversely, the
AR are primarily coupled to the inhibitory G-protein
G, and in turn, inhibit adenylyl cyclase activity. There
are numerous reports, however, that reveal that under certain
circumstances, the AR elicit stimulation of cAMP
production(18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) ,
and there are several lines of evidence that strongly suggest that
AR are coupled directly to G as well as
G. Cerione et al.(30) demonstrated that
purified human platelet AR (AR)
reconstituted in phospholipid vesicles with purified G stimulates GTPase activity in an agonist-dependent manner. In our
own work (21, 22) and that of others(23) ,
AR-mediated stimulation of adenylyl cyclase activity
has been observed in assays using washed membrane preparations, thereby
eliminating possible stimulatory signals from secondary downstream
intracellular mediators that are independent of G coupling.
In addition, AR-mediated stimulation of adenylyl
cyclase activity in membrane preparations can be blocked both by
cholera toxin (CTX) and by specific antiserum directed against
G
(21) . Finally, we have demonstrated physical
AR-G coupling using immunoprecipitation of
solubilized agonist-AR-G complexes
prepared from transfected Chinese hamster ovary (CHO) cells as
identified by CTX-mediated ADP-ribosylation assays or Western blotting
with G antiserum(21) .
Regardless of
whether AR-G coupling is examined in
intact cells or membranes, the process appears to be less efficient
than G coupling(18, 19, 20, 21, 22, 23, 24) .
Typically, the agonist concentrations necessary to elicit detectable
stimulation of adenylyl cyclase are higher than those for inhibition.
Indeed, the EC
values for agonist stimulation are
10-100-fold higher as compared with inhibition for
AR. In this regard, we have wondered if, in fact,
AR contain distinct and separate structural
determinants for coupling to G or whether a recombinant
system favors promiscuous association and activation of G by predominantly G coupling domains of the receptor.
Delineating a specific region of the AR, which
confers agonist-mediated stimulation of adenylyl cyclase, would also
provide additional confirmation that this signal is in fact the result
of interaction at the level of the receptor itself rather than
secondary effects due to other mechanisms evoked in the whole cell
setting.
Indeed, most mutagenesis studies have been carried out with
receptors that couple to a single G-protein. In some of these studies,
the specificity of G-protein coupling has been attributed to the
amino-terminal portion of the third intracellular loop. Interestingly,
in previous studies, it has been shown that when this region of the
AR is substituted into the analogous position in the
AR that G coupling is not
affected(5, 6) , suggesting that the amino-terminal
portion of the AR can support G coupling
at least within the context of the AR. This is
supported by the studies of Okamoto and Nishimoto(15) , wherein
a synthetic peptide based on this region of the
AR-stimulated GTP
S binding to purified Gin vitro. Accordingly, in the present study, we have
utilized deletion and chimeric mutagenesis of cloned human
AR (C10) to investigate the
amino-terminal region of the third intracellular loop as a potential
specific G coupling domain.
EXPERIMENTAL PROCEDURES
Mutagenesis of
C10
Construction of a deletion mutant
and chimeric AR/C10 or
5-HTR/C10 receptors was carried out using
the human C10 cDNA construct in the mammalian
expression vector pBC12BI(31) . These mutations are illustrated
(see Fig. 1). First, a polymerase chain reaction technique was
used to eliminate the nucleotides encoding amino acids 218-235
and to create a unique BspEI site corresponding to amino acids
217 and 236. Briefly, an oligonucleotide primer corresponding to a
unique BglII restriction site was used in combination with a
mutagenic primer encoding a new BspEI restriction site
(located at amino acids 217-218) to allow for polymerase
extension of a BglII/BspEI fragment. This fragment
was digested with BglII and BspEI and ligated into
digested AR-pBC12BI. Synthetic, complimentary
oligonucleotides with overlapping ends cohesive to BspEI
fragments, which encoded a short linker fragment to produce a mutant
lacking amino acids 221-231 (DEL 221-231) or
AR or 5-HTR sequence analogous to amino
acids 218-235 of C10, were phosphorylated,
annealed, and then ligated into the above mutated
C10-pBC12BI construct digested with BspEI.
These two substitution mutants are denoted as
() and
(5-HT). Two additional mutations (see Fig. 1), which encoded for smaller substitutions of the
5-HTR sequence analogous to amino acids 218-228 and
229-235 (denoted as (5-HT 218-228) and (5-HT 229-235), respectively) were constructed in the same
manner. All mutations were verified by dideoxy sequencing.
Figure 1:
Schematic for
deletion and substitution of amino-terminal residues in the third
intracellular loop of C10. Shown is a schematic
representation of the C10 with the indicated
mutations. Substitution and deletion mutations were undertaken in the
amino-terminal portion of the third intracellular loop, adjacent to
transmembrane domain V (TM V). Amino acid alignment of this
region for both wild-type and mutant C10 appears
below. Underlined residues indicate wild-type
C10 sequence. For the mutant denoted as DEL 221-231,
amino acids 221-231 were deleted. This mutation resulted in the
introduction of an alanine residue that is not present in wild-type
C10. Mutants with substitution of amino acids
218-235 with the analogous sequences from the AR
or 5-HTR are denoted as
() and
(5-HT), respectively. Two additional
mutants were also constructed that contain substitution of amino acids
218-228 and 229-235 with the respective sequence from the
5-HTR and are denoted as (5-HT 218-228) and (5-HT 229-235), respectively.
Cell Culture and Transfection
CHO cells and
Chinese hamster fibroblasts (CHW) were grown in monolayers in
Ham's F-12 media and Dulbecco's modified Eagle's
media, respectively, supplemented with 10% fetal bovine serum, 100
units/ml penicillin, 100 µg/ml streptomycin, and 80 µg/ml G418
(to maintain selection pressure) at 37 °C in a 5% CO atmosphere. CHO and CHW cells were cotransfected with 30-40
µg of wild-type or mutant C10-pBC12BI and 3 µg
of pSVneo using a calcium phosphate precipitation method
described by Cullen(32) . Stably expressing clones were
identified using a [
H]yohimbine binding assay as
described below. COS-7 cells were grown in monolayers in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin at 37 °C in a 5% CO atmosphere. For
transient expression of wild-type and mutant AR, COS-7
cells in monolayers at 
30-50% confluence were transfected
with 10-15 µg of cDNA via a DEAE-dextran method as described
previously(32) . COS-7 cells expressing wild-type and mutant
AR were used for experiments on the second day
following transfection. Mutant receptors DEL 221-231,
(), and
(5-HT) were studied in CHO cells at
expression levels of 10 pmol of receptor/mg of membrane protein
along with wild-type C10 at a matched expression.
Stable transfectants of mutant receptors (5-HT 218-228) and (5-HT 229-235) typically expressed levels of 5 pmol/mg of
receptor in CHO cells and 2 pmol/mg of receptor in CHW cells and
were studied in parallel with wild-type C10 expressing
similar levels of receptor. For experiments, transfected cells in
monolayers at 95% confluence were used, and multiple clones
expressing mutant and wild-type C10 were studied.
Permanent transfection of the cloned human AR in
pBC12BI into CHO cells was performed as above, and stably expressing
clones were identified using a 
I-cyanopindolol binding
assay exactly as described previously(33) . The CHO clonal cell
line expressing the cloned human 5-HTR was obtained from
John R. Raymond (Duke University) and is described
elsewhere(34) .
Cholera and Pertussis Toxin Pretreatment and Membrane
Preparation
For some experiments, AR-G or G coupling in CHO cells was isolated by
pretreatment of cells with either CTX or pertussis toxin (PTX),
respectively. Transfected CHO cells in monolayers were washed twice
with phosphate-buffered saline and incubated in serum-free media
containing either 20 µg/ml CTX or 500 ng/ml PTX for 24 h at 37
°C in a 5% CO atmosphere. We have previously
established that these concentrations of toxin eliminate
agonist-promoted receptor-G or -G coupling,
respectively, without being detrimental to the cells(21) .
After toxin treatment, cells were washed 5 times with
phosphate-buffered saline, and membranes were prepared by scraping with
a rubber policeman in a hypotonic buffer (5 mM Tris-HCl, pH
7.4, 2 mM EDTA) followed by centrifugation at 40,000 
g for 10 min at 4 °C. Crude membrane pellets were then
resuspended in the appropriate buffer for use in the assays below.
Adenylyl Cyclase Assay
CHO cells stably expressing
mutant and wild-type C10 were untreated or exposed to
CTX or PTX, membranes were prepared as described above, and then
adenylyl cyclase activities were determined using a modification of the
method of Salomon et al.(35) exactly as described
previously(22) . Briefly, membranes (20 µg) were
incubated with 2.7 mM phosphoenolpyruvate, 50 µM GTP, 0.1 mM cAMP, 0.12 mM ATP, 50 µg/ml
myokinase, 0.05 mM ascorbic acid (to prevent oxidation of
epinephrine), and 2.0 µCi of [-
P]ATP in
a buffer containing 40 mM HEPES, pH 7.4, 25 mM NaCl,
1.6 mM MgCl, and 0.8 mM EDTA for 45 min
at 37 °C. Reactions were terminated by the addition of a stop
solution containing excess ATP and cAMP and 100,000 dpm of
[H]cAMP. [P]cAMP and
[H]cAMP were isolated by sequential
chromatography over Dowex and alumina columns, and
[H]cAMP was used to quantitate column recovery.
Activities were measured in the presence of water (basal), 1.0
µM forskolin, and 1.0 µM forskolin with
various concentrations of agonist. To assess the functional responses
of AR or 5-HTR expressed in CHO cells,
transfected cells were incubated with vehicle (untreated) or the
indicated toxin, membranes were prepared, and adenylyl cyclase
activities were determined as described above, with the exception that
forskolin was not included in assays of AR-mediated
stimulation.
Whole-cell cAMP Accumulation Assays
For some
studies examining mutant and wild-type AR-G-protein
coupling, accumulation of cAMP in intact cells was quantitated in
stably expressing CHO and CHW cells or transiently expressing COS-7
cells. Briefly, cells expressing wild-type C10, or the
mutants (5-HT 218-228) and
(5-HT 229-235) growing in
monolayers in multiwell (2 cm/well, 24 wells) plates, were
washed twice with PBS and incubated in serum-free media containing 100
µM 3-isobutyl-1-methyl-xanthine (to inhibit
phosphodiesterase activity) for 30 min at 37 °C in a 5% CO atmosphere. Then, the indicated concentrations of agonist and,
for experiments using CHO or CHW cells, 5 µM forskolin
were added to duplicate wells. Incubations with agonist were carried
out for 45 min (COS-7 cells) or 5 min (CHO and CHW cells), and
reactions were stopped by the addition of 1 N HCl. Total
cellular cAMP was quantitated using a radioimmunoassay as described
previously(33) . Briefly, each sample and a series of cAMP
standards were diluted up to 1.0 ml in 0.1 N HCl, and 40
µl of a 1:2.5 mixture of acetic anhydride/triethylamine was added.
The resultant acetylated products were incubated with specific antisera
for succinyl cAMP and 5 nCi of [I]cAMP tyrosine
methyl ester. Reactions were passed over anion-exchange resin to clear
the mixture of unbound cAMP, and the complexed
[I]cAMP was eluted and counted in a
counter.
Radioligand Binding Assays
Expression levels of
mutant and wild-type C10 were determined using a
[H]yohimbine binding assay as described
previously(22) . Briefly, membranes prepared from cells
expressing mutant or wild-type C10 were incubated in a
Tris buffer (75 mM Tris-HCl, pH 7.4, 12.5 mM MgCl, 2 mM EDTA) with 25 nM
[H]yohimbine in the absence (total binding) and
presence of 10 µM phentolamine (nonspecific binding) for
30 min at 37 °C. Specific binding was defined as the difference
between total and nonspecific binding and was normalized for protein.
For agonist competition studies, membranes were incubated in a buffer
providing 50 mM Tris-HCl, pH 7.4, 10 mM MgSO
, 0.5 mM EDTA with 6 nM
[H]yohimbine, 100 µM GTP, and 12
different concentrations of the agonist epinephrine, ranging from 1
nM to 1 mM for 30 min at room temperature. All
radioligand binding reactions were terminated by dilution with several
volumes of ice-cold 10 mM Tris-HCl, pH 7.4, followed by rapid
vacuum filtration through Whatman GF/C glass fiber filters.
Protein Measurement
Protein concentration was
determined using the bicinchoninic acid method as described by Smith et al.(36) , with bovine serum albumin as standard.
Data Analysis
Adenylyl cyclase and cAMP
accumulation dose-response data and radioligand binding data from
competition experiments were analyzed by iterative least squares
techniques(37) . For adenylyl cyclase and cAMP accumulation
studies, data are reported as mean ± S.E. of the R
(maximal response) and the EC for
epinephrine-mediated stimulation or inhibition from the indicated
number of individual experiments. Comparisons for all experiments were
made by two-tailed t tests, with significance imparted at p < 0.05.
Materials
[H]Yohimbine (80
Ci/mmol), [H]cAMP (31 Ci/mmol),
[-P]ATP (30 Ci/mmol), and I-cyanopindolol (2200 Ci/mmol) were from DuPont NEN.
[I]cAMP tyrosine methyl ester (2200 Ci/mmol)
was from Hazleton Washington (Vienna, VA). Forskolin,
(-)-epinephrine, propranolol, and cholera toxin were from Sigma.
Phentolamine was from Research Biochemicals. Pertussis toxin was from
List Biologicals. Synthetic oligonucleotides were purchased from
Oligos, Etc. (Wilsonville, OR). Geneticin (G418) was from Life
Technologies, Inc. All tissue culture reagents were from JRH
Biosciences. Other reagents were obtained from standard commercial
sources.
RESULTS
To examine whether the amino-terminal region of the third
intracellular loop of C10 possesses specific
structural elements required for G coupling, we constructed
mutated C10 cDNAs, recombinantly expressed both mutant
and wild-type genes in CHO cells, and assessed the ability of the
expressed receptors to mediate stimulation and inhibition of adenylyl
cyclase activity. The mutated receptors are illustrated in Fig. 1. The initial approach utilized a deletion mutant that
lacked 10 amino-terminal residues of the third intracellular loop.
Further studies were carried out using substitutions in this region
with the analogous portions of other G-protein-coupled receptors in
order to maintain a greater degree of overall structural integrity of
the loop. Amino-terminal portions of the third intracellular loops of
the G-coupled AR and the
G-coupled 5-HT receptor (5-HTR)
were chosen since these receptors do not undergo dual
G/G coupling. Mutant C10
containing 5-HTR or AR substitutions for
amino acids 218-235 are referred to as
(5-HT) and
(), respectively. Two additional
mutants contained smaller substitutions of amino acids at the most
proximal(218-228) or distal(229-235) portions of this
segment with the analogous sequence from the 5-HTR and are
referred to as (5-HT 218-228) and
(5-HT 229-235), respectively. All
mutant C10 specifically bound the AR
antagonist [H]yohimbine and displayed virtually
identical affinities for the agonist epinephrine as compared with
wild-type C10 (data not shown).
Initially, the
involvement of the amino-terminal region in
C10-G-protein coupling was explored using the mutant
DEL 221-231. As shown in Fig. 2, the most striking result
was found for G coupling. For wild-type
C10, following treatment with PTX,
epinephrine-mediated stimulation of adenylyl cyclase activity was
readily observable with a maximum stimulation of 338 ± 29% over
forskolin-stimulated activity and an EC of 17 ± 1
µM. In contrast, under identical conditions, the mutant
DEL 221-231 failed to stimulate adenylyl cyclase activity (Fig. 2). Following treatment of wild-type
C10-expressing CHO cells with CTX,
epinephrine-mediated inhibition of adenylyl cyclase activity was
observed with a 52 ± 1% decrease from forskolin-stimulated
activity and an EC of 151 ± 23 nM. For the
mutant DEL 221-231, epinephrine-mediated inhibition was retained,
although reduced as compared with wild-type C10, with
a maximum inhibition of 30 ± 2% decrease from
forskolin-stimulated activity, and a significantly greater EC of 19 ± 12 µM (p < 0.02 as
compared with wild-type C10).
Figure 2:
Deletion of residues 221-231 ablates
C10-G coupling. CHO cells stably
expressing wild-type C10 and the mutant DEL
221-231 were pretreated with either CTX or PTX to isolate G coupling or G coupling, respectively, and adenylyl
cyclase activities were measured in washed membranes as described under
``Experimental Procedures.'' Activities were determined in
the presence of 1.0 µM forskolin and various
concentrations of the agonist epinephrine. Results are expressed as the
maximal percent change from forskolin-stimulated activity. Shown are
mean ± S.E. from three experiments. *p < 0.02 as
compared with wild-type
C10.
As introduced
earlier, subsequent studies were carried out utilizing
5-HTR/C10 and
AR/C10 chimeras, since we were
concerned about potential nonspecific consequences of deletion
mutations. To be certain that AR and
5-HTR sequences were appropriate for such studies (i.e. that these receptors do not display dual
G/G coupling), adenylyl cyclase studies were
carried out using the AR and 5-HTR
permanently expressed in CHO cells under the same conditions as those
used for the C10 mutants. These results are shown in Fig. 3. In membranes prepared from CHO cells stably expressing
the AR, the agonist isoproterenol elicited stimulation
of adenylyl cyclase activity that was essentially eliminated (95%
loss) following treatment with CTX. Note that following CTX,
isoproterenol did not elicit AR-mediated inhibition of
adenylyl cyclase activity. Similarly, in membranes prepared from CHO
cells expressing the 5-HTR, serotonin promoted inhibition
of adenylyl cyclase activity that was entirely ablated following
pretreatment of the cells with PTX. After PTX, agonist-promoted
stimulation of adenylyl cyclase activity was not detected with the
5-HTR (Fig. 3).
Figure 3:
AR-G coupling
and 5-HTR-G coupling. CHO cells expressing
AR or 5-HTR were incubated in the absence (UNTREATED) or presence of either CTX or PTX to ablate
AR-G coupling or
5-HTR-G coupling, respectively. Membranes were
prepared, and adenylyl cyclase activities were determined in the
presence of the indicated concentrations of the AR
agonist isoproterenol or the 5-HTR agonist serotonin.
Shown are results from a single experiment representative of four
performed. The functional responses of the AR and
5-HTR were entirely eliminated by pretreatment with CTX
and PTX, respectively, which demonstrates that these two receptors are
not dually coupled to G and
G.
CHO cells permanently expressing
(),
(5-HT), and wild-type
C10 were exposed to CTX and PTX to dissect the
G- and G-coupling pathways, respectively. Then,
washed membranes were prepared, and adenylyl cyclase activities were
determined in the presence of the agonist epinephrine as before.
Substitution of the amino-terminal portion of the third intracellular
loop with 5-HTR sequence resulted in a complete loss of
C10-mediated stimulation of adenylyl cyclase activity (Fig. 4). In contrast, G coupling remained intact
and displayed the wild-type phenotype with a 57 ± 2% decrease in
adenylyl cyclase activity and an EC for
epinephrine-mediated inhibition of 220 ± 3 nM (p is not significant as compared with wild-type
C10, Fig. 4). The results with the mutant
consisting of substituted AR sequence supported the
above concept that the amino terminus of the third intracellular loop
of C10 is critical for G coupling. The
() mutant did display G coupling, although the maximal response was diminished by
75% as compared with wild-type C10. Similar to
what was found above, AR substitution did not reduce
G coupling; in fact, the maximum inhibition was slightly
greater than that of wild-type C10 (67 ± 2 versus 52 ± 1% decrease in adenylyl cyclase activity,
respectively, p < 0.05, Fig. 4). Thus, the
preservation of G coupling with both mutations suggests
that the loss of G coupling observed with substitution of
this region is due to loss of a specific G coupling domain.
Figure 4:
Effects of substitution of amino acids
218-235 on C10-G and -G coupling. CHO cells expressing wild-type C10 and
the mutants () and
(5-HT) were incubated with either CTX (closed symbols) or PTX (open symbols) to isolate
G or G coupling, respectively. Squares, wild-type C10; diamonds,
(); circles,
(5-HT). Adenylyl cyclase activities were
determined in washed membranes in the presence of 1.0 µM forskolin and the indicated concentrations of the agonist
epinephrine. Results are expressed as the percent of
forskolin-stimulated activity. Shown are the mean ± S.E. from
three to five experiments.
Inasmuch as G coupling was completely removed by
substitution of amino acids 218-235 with 5-HTR
sequence, we concluded that the key residues within this region that
are critical for C10-G coupling are
contained within these 18 amino acids. Interestingly, the first 11
residues(218-228) of this 18-amino acid sequence are relatively
conserved (>80%) among the three human AR subtypes,
while there are virtually no identities among the next 7 amino
acids(229-235). Hence, we considered whether the requirements for
C10-G coupling are contained within these
11 amino acids and represent a G coupling domain that is
conserved among all AR. In this regard, we constructed
two additional mutants containing smaller substitutions with
5-HTR sequence within this 18 amino acid region (Fig. 1). One mutant, termed (5-HT 218-228), contained substitution of the first 11 amino
acids with the analogous sequence from the 5-HTR. The
other mutant, termed (5-HT
229-235), contained substitution of only the last seven amino
acids within this region with the analogous 5-HTR
sequence.
CHO cells expressing matched expression levels of the
mutants (5-HT 218-228) and
(5-HT 229-235) and wild-type
C10 were studied under the same conditions as before.
As shown in Fig. 5, in membranes expressing wild-type
C10, epinephrine elicited stimulation of adenylyl
cyclase activity with a maximum stimulation of 192 ± 18% of
forskolin-stimulated activity and an EC of 16 ± 0.4
µM. Similarly, for the mutant
(5-HT 229-235),
epinephrine-mediated stimulation occurred with a maximum stimulation of
185 ± 7% of forskolin-stimulated activity and an EC of 24 ± 3 µM. In contrast, the mutant
(5-HT 218-228) appeared not to
couple to G in that no epinephrine-mediated stimulation of
adenylyl cyclase activity was detected (Fig. 5). As with
previous mutations, both substitutions had no effect on
C10-G coupling. Wild type
C10 displayed epinephrine-mediated inhibition with a
52 ± 3% decrease in adenylyl cyclase activity and an EC of 182 ± 26 nM. For (5-HT 218-228) and (5-HT 229-235), epinephrine-mediated inhibition of adenylyl
cyclase was virtually identical to wild-type C10 with
a maximum inhibition of 49 ± 3% and 56 ± 5% decrease from
forskolin-stimulated activity, respectively, and ECs of
151 ± 10 nM and 83 ± 12 nM,
respectively. These data, with the maximal G or G responses normalized to wild-type C10 for all
the mutations, are summarized in Table 1.
Figure 5:
(5-HT
218-228), (5-HT 229-235), and
wild-type C10-G coupling. Adenylyl cyclase
activities were determined in membranes prepared from PTX-treated CHO
cells expressing wild-type C10 and the mutants
(5-HT 218-228) and
(5-HT 229-235). Activities were
measured in the presence of 1.0 µM forskolin and the
indicated concentrations of epinephrine. Shown are the mean ±
S.E. from three to four experiments.
In additional
experiments, we also assessed both membrane adenylyl cyclase assays and
cAMP accumulation studies in intact CHO cells expressing wild-type
C10 and the mutant (5-HT 218-228), which had not been pretreated with either CTX or
PTX. Shown in Fig. 6A are the results of membrane adenylyl
cyclase assays in the presence of the agonist epinephrine. As we have
previously reported(21) , without pretreatment with either
toxin, C10-mediated modulation of adenylyl
cyclase activity in CHO cells was complex and biphasic, consisting of
both an inhibitory (G coupling) and stimulatory (G coupling) component (Fig. 6A). In contrast, the
mutant (5-HT 218-228) displayed
only monophasic inhibition, revealing a loss of G coupling
but not G coupling. In complimentary studies assessing
whole-cell cAMP accumulation, similar results were obtained. Wild-type
C10 mediated a biphasic cAMP accumulation response,
while a predominantly inhibitory response was found with the mutant
(5-HT 218-228) (Fig. 6B).
Figure 6:
Membrane adenylyl cyclase and whole-cell
cAMP accumulation with wild-type and mutant C10 in CHO
cells without pretreatment with toxin. A, adenylyl cyclase
activities were determined in membranes prepared from CHO cells
expressing wild-type C10 and the mutant
(5-HT 218-228) in the presence of
1.0 µM forskolin and the indicated concentrations of
epinephrine. B, cAMP accumulation in intact CHO cells
expressing wild-type C10 and the mutant
(5-HT 218-228) was determined as
described under ``Experimental Procedures.'' Shown are the
mean ± S.E. from four to five experiments. Absent error bars
denote standard errors that were obscured by the size of the symbol and
were < 5%.
Finally, we examined the ability of
wild-type C10 and the mutant
(5-HT 218-228) to couple to both
G and G using two transfected cell lines in
which C10-modulation of adenylyl cyclase activity has
been observed to be either exclusively inhibitory (CHW cells) or
stimulatory (COS-7 cells). For these studies, intact cAMP accumulation
studies were performed in the presence of the specific
AR-agonist UK-14304. In COS-7 cells transiently
expressing wild-type C10, UK-14304 elicited a
10-fold stimulation of cAMP accumulation with an EC of 319 ± 74 nM (Fig. 7). Conversely, the
mutant (5-HT 218-228) in COS-7
cells displayed no detectable stimulation of cAMP (Fig. 7). As
was found above, substitution of amino acids 218-228 did not
reduce AR-G coupling. In CHW cells, both
wild-type C10 and (5-HT 218-228) inhibited adenylyl cyclase activity similarly with
EC values for agonist-mediated inhibition of 5.6 ±
1.7 versus 3.6 ± 0.9 nM, respectively, and R values of 69.2 ± 1.9 versus 85.5 ± 1.7% decrease in forskolin-stimulated activity,
respectively (Fig. 7).
Figure 7:
Whole-cell cAMP accumulation in COS-7 and
CHW cells expressing wild-type C10 and the mutant
(5-HT 218-228). Whole-cell cAMP
accumulation studies were performed in the presence of the indicated
concentrations of the AR agonist UK-1304 using COS-7
or CHW cells expressing wild-type C10 and the mutant
(5-HT 218-228) as described under
``Experimental Procedures.'' For observation of
AR-mediated inhibition in CHW cells, 1.0 µM forskolin was included in the assay. Shown are the mean ±
S.E. from four experiments. Absent error bars denote standard errors
that were obscured by the size of the symbol and were <
5%.
DISCUSSION
The seemingly paradoxical ability of AR to
mediate stimulation of cAMP production has been reported in pancreatic
islet cells(28) , cerebral cortical brain slices(29) ,
and a number of recombinantly expressing clonal cell lines including
CHO
cells(18, 19, 21, 22, 23) ,
COS-7 cells(20) , HEK-293 cells(20, 24) ,
PC-12 cells(25) , JEG-3 cells(26) , and the S115 mouse
mammary tumor cell line(27) . Although the underlying mechanism
for these AR-mediated increases in cAMP has been a
matter of some debate, recent studies, as outlined earlier (see the
Introduction), have established compelling evidence that
AR directly couple to G and thereby elicit
stimulation of adenylyl cyclase activity. While coupling to multiple
signaling transduction pathways is not uncommon among G-protein-coupled
receptors, the ability of AR to couple to
G as well as to G is particularly intriguing in
that, as such, the AR are capable of simultaneously
evoking both stimulatory and inhibitory regulation of the activity of a
single effector. The primary aim of the current work was to explore the
nature of AR-G coupling to determine if,
indeed, AR contain specific structural elements for
Gversus G coupling. Based on studies
of other G-protein coupled
receptors(3, 4, 5, 6, 7, 8, 9, 10, 12) , in vitro peptide studies (15) , and the results from
our initial deletion mutation (Fig. 2), we focused on the
amino-terminal region of the third intracellular loop of
C10 as a potentially selective G coupling
domain.
In early studies, we found that deletion of amino acids
221-231 entirely ablated C10-mediated
stimulation of adenylyl cyclase activity, yet
C10-mediated inhibition was still present, albeit
somewhat diminished and less efficient as compared with wild-type
C10 (Fig. 2). In order to further determine the
specificity of these effects on functional
C10-G-protein coupling with less drastic mutations
than such a deletion, we constructed and assessed the coupling
characteristics of a series of chimeric
5-HTR/C10 and
AR/C10 receptors (Fig. 1).
With this approach, the amino-terminal region was replaced with
sequence from the AR, which only couples to
G, and sequence from the 5-HTR, which only
couples to G (Fig. 3). Thus, with the
AR substitution, we could discern losses in
C10-G coupling, and conversely, with
5-HTR substitution we could ascertain losses in
C10-G coupling. We found that substitution
of amino acids 218-235 with the analogous 5-HTR
sequence entirely removed the ability to elicit epinephrine-mediated
stimulation of adenylyl cyclase activity (Fig. 4). Smaller
substitutions with the analogous 5-HTR sequence further
revealed that the necessary requirements for
C10-G coupling in this region are confined
to a small stretch of 11 amino acids (RIYQIAKRRTR) directly adjacent to
the fifth transmembrane domain (Fig. 5Fig. 6Fig. 7). This loss of functional
C10-G coupling appears to be due to
removal of specific G coupling domains, in that the
G-coupled pathway remained fully intact with both
5-HTR and AR substitutions (Table 1, Fig. 4, Fig. 6, and Fig. 7).
Although no studies have explored G coupling domains of
AR, there are several reports that support our
findings. In intact receptor studies, O'Dowd et al.(6) and Liggett et al.(5) have reported
that substitution of the amino-terminal domain of the third
intracellular loop of the AR with the analogous
sequence from the C10 has very little, if any, effect
on AR-mediated stimulation of adenylyl cyclase
activity. Moreover, consistent with our current results that this
region is not critical for C10-G coupling,
C10 substitution in this region of the
AR does not confer G coupling to the
AR(5) . Interestingly, unlike what is found
with AR substitution of this region in the
AR, we found that substitution of this region in
C10 with AR sequence reduces
