Coupling Specificity between Somatostatin Receptor sst2A and G Proteins: Isolation of the Receptor-G Protein Complex with a Receptor Antibody
Yi-Zhong Gu and
Agnes Schonbrunn
Department of Integrative Biology, Pharmacology, and Physiology
University of Texas Medical School Houston, Texas 77225
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ABSTRACT
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Somatostatin initiates its actions via a family of
seven-transmembrane domain receptors. Of the five somatostatin receptor
genes cloned, sst2 exists as two splice variants with the sst2A isoform
being predominantly expressed. This receptor is widely distributed in
endocrine, exocrine, and neuronal cells, as well as in hormonally
responsive tumors, and leads to inhibition of secretion, electrical
excitability, and cell proliferation. To investigate the specificity of
signal transduction by the sst2A receptor, we developed antibodies
against two overlapping peptides located within the C terminus of the
receptor protein: peptide 2CSG, containing
amino acids 334348, and peptide 2CER,
containing amino acids 339359. Although antibodies to both peptides
bound the inducing antigen with high affinity, only the antibodies
against peptide 2CER precipitated the receptor.
The best antibody, R288, precipitated about 80% of the sst2A
receptor-ligand complex solubilized from transfected CHO cells and was
specific for the sst2A receptor isotype. Addition of GTP
S (10
µM) to the immunoprecipitated ligand-sst2A
receptor complex markedly accelerated ligand dissociation,
indicating that G proteins remained functionally associated with
the receptor in the immuno-precipitate. Analysis of the G proteins
coprecipitated with the sst2A receptor by immunoblotting with G protein
antibodies showed that both G
and
Gß subunits were bound to the
hormone-receptor complex. Immunoprecipitation of the receptor was not
affected by the presence of bound ligand. However, G protein subunits
were coprecipitated only with the hormone-occupied receptor. Thus, the
unoccupied receptor has low affinity for G proteins, and hormone
binding stabilizes the receptor-G protein complex. Use of
subtype-specific G protein antisera further showed that
G
i1, G
i2, and
G
i3 were complexed with the sst2A receptor
whereas G
o, G
z,
and G
q were not. Together, these studies
demonstrate that the sst2A receptor interacts selectively with
G
i proteins in a hormone-dependent manner.
The finding that this receptor couples to all three
G
i subunits may help explain how
somatostatin can regulate multiple signaling pathways.
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INTRODUCTION
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Somatostatin (SRIF) exists in two biological forms (SRIF-14 and
SRIF-28) and is a widely distributed inhibitor of endocrine, exocrine,
gastrointestinal, and neural functions (1, 2). The actions of the
somatostatin peptides are initiated by a family of seven-transmembrane
domain receptors encoded by five distinct genes (3). The sst2 receptor
subtype mRNA is abundantly expressed in the pituitary, pancreas, brain,
spleen, and gastrointestinal tract as well as in a variety of
hormonally responsive tumors. Its wide distribution is consistent with
important physiological and pathological roles for this receptor in the
control of hormone and exocrine secretion, immune and neural
regulation, and tumor cell growth (3, 4, 5). Previous studies showed that
the primary transcript from the sst2 receptor gene is alternatively
spliced to generate two receptor mRNA isoforms, named sst2A and sst2B,
which diverge in their COOH-terminal sequence (6). Moreover, these
splice variants appear to differ in their coupling to adenylyl cyclase
as well as in their susceptibility to regulation (7, 8) indicating the
importance of the COOH terminus in these receptor functions.
Although the somatostatin receptor family is known to regulate multiple
signal transduction pathways, signaling by endogenously expressed sst2A
receptors have not been clearly identified since this receptor subtype
is seldom expressed in the absence of the other sst receptors, and
specific agonists are not yet available (3, 9). However, when
transfected into receptor-negative host cells, both sst2A and sst2B
receptors inhibit adenylyl cyclase (7, 8, 10). Overexpressed sst2A
receptors have also been shown to inhibit Ca++ channels
(11), stimulate phospholipase C and Ca++ mobilization (12),
and stimulate tyrosine phosphatase activity (13). However, the G
proteins mediating these effects are unknown.
In this study we have used a novel approach to investigate the
specificity of sst2A receptor-G protein coupling. Using a newly
developed sst2A receptor-specific antibody, we immunoprecipitated the
receptor-G protein complex and identified the G protein subunits
associated with the sst2A receptor. This represents the first study to
systematically characterize the spectrum of G proteins coupled to this
sst receptor isotype.
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RESULTS
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Antibody Preparation and Characterization
Two overlapping peptides, derived from a unique sequence in the
carboxy-terminal region of the sst2A receptor, were used as immunogens
(Fig. 1
). The peptide 2CSG corresponds to
amino acids 334348 in the mouse sst2A receptor and differs from both
the rat and human receptors by one residue. The peptide
2CER corresponds to amino acids 339359 in the rat sst2A
receptor and is identical in the human, rat, and mouse sst2A proteins
(14, 15). The two antigen peptides share a nine-amino acid sequence but
contain unique residues at either their amino (2CSG) or
their carboxyl (2CER) termini.

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Figure 1. Alignment of Peptide Antigens with the
Carboxy-Terminal Region of sst Receptors
Amino acids in the carboxy-terminal region of rat sst receptor subtypes
were aligned starting from the seventh putative transmembrane domain.
Single-letter code is used to denote the amino acids.
Gaps were introduced to maximize alignement. Boxed areas
indicate conserved amino acid residues between the two peptide antigens
and the sst receptor subtypes. Nucleotide sequences for the rat sst
receptors were obtained from Genbank using the following accession
numbers: rat sst1: M97656, rat sst2A: M93273, rat sst3: X63574, rat
sst4: M96544 and rat stt5: L04535 and X74828. sst2B is a splice variant
of sst2A (57)
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Peptides were coupled to keyhole limpet hemocyanin (KLH) through an
amino-terminal cysteine, and each peptide was used to immunize two
rabbits. Immune sera were assayed by enzyme-linked immunosorbent assay,
and the dilution of antiserum giving half-maximal binding to adsorbed
peptide was subsequently used to determine the relative affinity of
each antibody for the inducing antigen. Antisera R287 and R288
bound the immunizing peptide 2CER with ED50
values of 5.1 ± 2.5 nM (not shown) and 4.4 ±
2.9 nM (Fig. 2
), respectively. Antisera
R2204 and R2206 bound the immunizing peptide 2CSG with
ED50 values of 2.7 ± 1.1 nM (not shown)
and 1.5 ± 0.3 nM (Fig. 2
), respectively.
Interestingly, peptide 2CSG did not compete for binding to
antiserum R288 (Fig. 2
), suggesting that this antiserum recognized
the unique C-terminal sequence of the immunizing peptide
2CER rather than the region it shared with the peptide
2CSG.
Specific Immunoprecipitation of sst2A Receptors
CHO-K1 cells stably transfected with the rat sst2A receptor gene
(CHO-R2A) (10) were used to determine which antisera recognized the
receptor protein. After preincubation of CHO-R2A membranes with the
radiolabeled somatostatin analog
[125I-Tyr11]SRIF, the ligand-receptor complex
was solubilized with dodecyl-ß-D-maltoside (DßM) (16).
The soluble [125I-Tyr11]-SRIF-receptor
complex was quantitated by precipitation with polyethylene glycol, and
the amount of the complex bound by each antibody was then determined
(Fig. 3
, upper panel). Previous studies had
shown that the [125I-Tyr11]SRIF-receptor
complex is extremely stable at 4 C (16), and we confirmed that less
than 10% of the bound ligand dissociated from the sst2A receptor
during an overnight incubation with antiserum at 4 C. The results in
Fig. 3
(upper panel) demonstrate that the sst2A receptor was
immunoprecipitated by both antisera against peptide 2CER
(R287 and R288) but was not significantly precipitated by either of
the antisera against peptide 2CSG (R2204 and R2206).
Because antiserum R288 was most effective, it was characterized in
greater detail.

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Figure 3. Immunoprecipitation of the sst2A Receptor
Upper panel, CHO-R2A membranes (37 µg/ml) were
incubated with [125I-Tyr11]SRIF (304, 900
cpm/ml, 0.084 nM) in the absence or presence of 100
nM SRIF for 2 h at 30 C. After binding, membranes were
separated from free ligand by centrifugation and solubilized for 1
h at 4 C. The amount of intact ligand-receptor complex in the soluble
fraction was determined by precipitating an aliquot with 40% PEG as
described in Materials and Methods. Immunoabsorption was
performed by incubating soluble receptor with antisera for 3 h at
4 C followed by precipitation with Protein A-Sepharose. Antiserum
R288 was used at a final dilution of 1:1000 whereas other antisera
were diluted 1:100. The graph shows the amount of the
[125I-Tyr11]SRIF-receptor complex
immunoprecipitated out of 14,400 cpm added to the assay. Lower
panel, CHO-R2A membranes (63 µg/ml) were bound with
[125I-Tyr11]SRIF (792, 800 cpm/ml, 0.22
nM) and then solubilized for 1 h at 4 C. Soluble
receptor was either precipitated with PEG to quantitate the
ligand-receptor complex (Total) or incubated with antiserum R288 at
different dilutions for 3 h at 4 C. After precipitation with
Protein A-Sepharose, specific binding was measured in the
immunoprecipitate. For both panels, the data represent the mean ±
SEM.
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Immunoprecipitation of the sst2A receptor-ligand complex by R288 was
concentration dependent with a maximal 7580% immunoprecipitation
achieved at antiserum dilutions of 1:1000 or less (Fig. 3
, lower
panel). Whereas immune serum precipitated 80% of the soluble
[125I-Tyr11]SRIF-sst2A receptor complex,
preimmune serum precipitated only 2% of the receptor (Fig. 4
, upper panel). Moreover, addition of 10
µM antigen peptide to the incubation with antiserum
completely inhibited receptor immunoprecipitation (Fig. 4
, upper
panel).

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Figure 4. Specificity of the sst2A Receptor Antiserum R288
Upper panel, Membranes from CHO-R2A cells (86 µg/ml)
were incubated with [125I-Tyr11]SRIF (568,
620 cpm/ml, 0.157 nM) in the absence or presence of 100
nM SRIF for 2 h at 30 C. After receptor
solubilization, the amount of ligand-receptor complex in the soluble
fraction (Total) was determined by PEG precipitation. For
immunoabsorption, soluble receptors were incubated with antiserum
R288 (1:100) at 4 C for 3 h. After precipitation of the immune
complex with Protein A-Sepharose, specific binding in the pellets was
measured. Lower panel, Membranes (80130 µg/ml) from
CHO cells expressing sst1, sst2A, sst2B, or sst4 were incubated with
[125I-Tyr11]SRIF for 2 h at 30°C and
then solubilized. The amount of soluble receptor-ligand complex was
determined for each receptor subtype by PEG precipitation (Total) and
ranged from 7,060 to 10,010 cpm. After incubation with R288
(dilution = 1:1001:400) for 3 h at 4 C, specific binding in
the immunoprecipitate was measured and expressed as a percent of the
total soluble receptor-ligand complex added. For both panels the data
represent mean ± SEM for specific binding in
triplicate samples.
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The receptor specificity of antiserum R288 was characterized using
CHO-K1 cells stably transfected with individual sst receptor subtypes
(Fig. 4
, lower panel). Sst2B receptors lack any sequence
homology with the peptide used for immunization, and, as expected, this
receptor was not recognized by antiserum R288 (Fig. 4
, lower
panel). Moreover, neither sst1 nor sst4 receptors were
immunoprecipitated even though the immunizing peptide 2CER
contained a three- amino acid sequence also present in these receptor
subtypes (Fig. 4
, lower panel). In separate experiments, we
further found that this antiserum did not immunoprecipitate the sst3 or
sst5 receptors (data not shown). Therefore, antiserum R288
specifically recognizes the sst2A receptor and precipitates it with
high efficiency. This antiserum was used in all subsequent
experiments.
Biochemical Characterization of Endogenously Expressed sst2A
Receptors
GH4C1 rat mammosomatotropic pituitary
cells, AtT-20 mouse corticotropic pituitary cells, and AR42J rat
pancreatic acinar cells have been widely used for studies of sst
receptors and SRIF signal transduction (17, 18, 19, 20). However, these cell
lines express mRNA for multiple sst receptor subtypes, and the level of
individual sst receptor proteins is unknown (19, 21, 22). To covalently
radiolabel sst receptors in these cells, membranes from each cell line
were incubated with the photolabile analog
[125I-Tyr11, ANB-Lys4]SRIF
(5'-azido-2'-nitrobenzoyl Lys4, Tyr11) SRIF and
then irradiated with UV light (23). Analysis of the affinity-labeled
membranes by SDS-PAGE and autoradiography showed that
[125I-Tyr11, ANB-Lys4]SRIF
covalently labeled a broad, 60- to 80-kDa band in CHO-R1 cells and a
broad 80- to 100-kDa band in CHO-R2A cells (Fig. 5
, upper panel), confirming previous observations (21).
Addition of 100 nM SRIF inhibited photoaffinity labeling of
these bands as expected for saturable receptors (Fig. 5
, upper
panel). In the three cell lines known to express sst receptors
endogenously, the most heavily labeled proteins also migrated as broad
bands between 80100 kDa, as previously reported (23). Although the
molecular mass predicted for sst receptor proteins from their DNA
sequence is 41 kDa, these receptors contain potential glycosylation
sites. The migration of photoaffinity-labeled sst receptors as broad,
high molecular mass bands is consistent with glycosylation. Two other
bands were weakly labeled in AtT-20 membranes: one at approximately 55
kDa and one at approximately 32 kDa. Photoaffinity labeling of these
bands was also inhibited by excess SRIF consistent with their being
either sst receptors or receptor degradation products.

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Figure 5. Specific Immunoprecipitation of
Photoaffinity-Labeled sst2A Receptors
Top panel, Membranes (2366 µg/ml) from CHO-R1,
CHO-R2, AR42J, AtT-20, or GH4C1 cells were
incubated in the dark for 2 h at 30 C with
[125I-Tyr11, ANB-Lys4]SRIF (253,
410 cpm/ml, 0.07 nM) in the absence or presence of 100
nM SRIF. After centrifugation to remove unbound ligand,
membranes were resuspended in HEPES binding buffer and irradiated with
UV light for 10 min. Membranes were then solubilized in sample buffer
and analyzed by SDS-PAGE and autoradiography. Bottom
panel, Membranes (30120 µg/ml) from each cell line were
incubated with
[125I-Tyr11,ANB-Lys4]SRIF
(326,040 cpm/ml, 0.089 nM) and extracted with DßM/CHS,
and the resulting soluble receptors were then irradiated as described
in Materials and Methods. Photoaffinity-labeled
receptors were subsequently incubated for 3 h at 4°C with
antiserum R288 (1:1000 final dilution) in the presence or absence of
the peptide antigen 2CER (5 µM). After
precipitation with Protein A-Sepharose, the pellets were dissolved in
sample buffer and analyzed by SDS-PAGE and autoradiography.
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We next used the R288 antiserum to isolate the photoaffinity-labeled
sst2A receptor subtype from each cell line. Membranes were incubated
with [125I-Tyr11, ANB-Lys4]SRIF,
solubilized, irradiated with UV light, and incubated with receptor
antiserum in the presence or absence of excess antigen peptide (Fig. 5
, lower panel). No labeled bands were observed in
immunoprecipitates from CHO-R1 cells. However, a broad
85-kDa band
was precipitated from CHO-R2A cells, and immunoprecipitation of this
band was completely inhibited by 5 µM antigen peptide,
confirming that R288 specifically recognized the sst2A receptor.
We next determined whether any of the photoaffinity-labeled receptors
in AR42J, AtT-20, or GH4C1 cells represented
the sst2A receptor. A broadly migrating 85-kDa band was observed in the
immunoprecipitates from all three cell lines (Fig. 5
, lower
panel). Neither of the smaller photoaffinity-labeled membrane
proteins from AtT-20 cells were immunoprecipitated, indicating that
these proteins lacked the sst2A C-terminal epitope. These results
demonstrate that the sst2A receptor is endogenously expressed in
AR42J, AtT-20, and GH4C1 cells and migrates
at 80100 kDa on SDS polyacrylamide gels. The subtle variation in the
apparent molecular mass of this receptor indicates that it may be
glycosylated differently in the three cell types.
Effect of Ligand Binding on Receptor Immunoprecipitation
In the previous experiments, receptor immunoprecipitation was
measured by the coprecipitation of either noncovalently or covalently
bound radiolabeled ligand. To determine whether the presence of ligand
affected the ability of the antibody to bind the receptor, we developed
a Western blot procedure for receptor detection (Fig. 6
). Proteins from CHO-R2A membranes were subjected to
SDS-PAGE and immunoblotted with antiserum R288. The antiserum stained
a broad band centered around 85 kDa, and this staining was abolished by
the addition of 1 µM antigen peptide (Fig. 6
, left
panel). The comigration of this immunoreactive band with the
photoaffinity-labeled receptor from CHO-R2A membranes indicates that it
corresponds to the sst2A receptor. Since noncovalently bound ligand is
dissociated from SRIF receptors during SDS-PAGE (23) the R288
antibody can recognize the denatured receptor on immunoblots in the
absence of bound ligand.

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Figure 6. Effect of Ligand Binding on Receptor
Immunoprecipitation
Left panel, CHO-R2A membrane proteins (10 µg/lane)
were separated by electrophoresis in a 10% polyacrylamide SDS gel.
After transfer of proteins to a PVDF membrane, the membrane was
incubated with antiserum R288 (final dilution 1:20,000) in the
absence (-) or presence (+) of 1 µM peptide
2CER. Immunoreactive proteins were visualized as described
in Materials and Methods. Right panel,
CHO-R2A membranes were incubated in the absence (-) or presence (+) of
100 nM SRIF at 30 C for 2 h. After solubilization,
proteins (from 8.5 µg of membranes) were incubated with the
covalently coupled antiserum R288-Protein A-Sepharose complex in the
absence or presence of peptide 2CER (1 µM).
Immunoprecipitated proteins were solubilized in sample buffer,
electrophoresed on a 10% SDS-acrylamide gel, and transfered to a PVDF
membrane. The sst2A receptor was detected by immunoblotting with R288
antiserum (1:10,000 final dilution).
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To determine the effect of hormone binding on receptor
immunoprecipitation, CHO-R2A membranes were incubated in the absence or
presence of 100 nM SRIF, solubilized, and
immunoprecipitated with antibody R288 covalently coupled to Protein
A-Sepharose (Fig. 6
, right panel). Subsequent analysis of
the immunoprecipitated proteins by SDS-PAGE and immunoblotting with
receptor antiserum showed that the 85-kDa receptor protein was present
in the immunoprecipitates from both SRIF-treated and control membranes
(Fig. 6
, right panel). Therefore, antiserum R288
recognized the sst2A receptor either with or without bound ligand.
Coprecipitation of G Proteins with the sst2A Receptor
The sst2A receptor has been shown to couple to inhibition of
adenlyl cyclase via pertussis toxin-sensitive G proteins in the CHO-R2
cell strain used in our studies (10). Consistent with these
observations, we found that pretreating the cells for 16 h with
100 ng/ml pertussis toxin produced an 84% decrease in
[125I-Tyr11]SRIF binding to the sst2A
receptor in membranes (data not shown). To determine whether G proteins
were immunoprecipitated with the sst2A receptor, we took advantage of
the known ability of GTP analogs to stimulate ligand dissociation from
the G protein- coupled form of SRIF receptors (16). After
immunoprecipitation with receptor antibody, the Sepharose-bound
[125I-Tyr11]SRIF-sst2A receptor complex was
resuspended in buffer with or without 10 µM GTP
S and
incubated at 25 C. At various times the radioactive ligand that
remained associated with the antibody-bound receptor was quantitated
after recentrifugation (Fig. 7
). The amount of
receptor-[125I-Tyr11]SRIF complex was
unaltered during a 10-min incubation in the absence of guanine
nucleotide (Fig. 7
). However, GTP
S markedly stimulated ligand
dissociation such that more than 50% of the receptor-bound peptide had
dissociated after 10 min (Fig. 7
). Thus, G proteins are functionally
associated with the receptor-ligand complex in the
immunoprecipitate.

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Figure 7. Effect of GTP S on Ligand Dissociation from
Immunoprecipitated Receptor
CHO-R2A membranes (100 µg/ml) were incubated
[125I-Tyr11]SRIF (308, 950 cpm/ml, 0.085
nM) for 2 h at 30 C. After solubilization, receptors
were incubated with antiserum R288 (1:200 final dilution) and
precipitated with Protein A-Sepharose. The washed immunoprecipitate was
then resuspended and incubated at 25 C in HEPES buffer containing 0.25
mg/ml DßM and 10 nM SRIF, with or without 10
µM GTP S. At the times shown, aliquots were removed
from the reaction and centrifuged at 4 C. The
[125I-Tyr11]SRIF specifically bound in the
immunoprecipitate was measured and expressed as a fraction of the
initial binding (1440 cpm). Each point shows the mean ±
SEM of triplicate samples.
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Specificity and Ligand Dependence of sst2A Receptor-G Protein
Interactions
Before examining the coupling of the sst2A receptor to specific G
proteins, we determined which G protein
-subunits were present in
CHO membranes using immunoblot analysis. Specific immunoreactivity was
detected at 4043 kDa with antibodies to G
i2,
G
i3, and G
q/11 (data not shown). However,
antibodies to G
i1, G
o, and
G
z failed to detect any specific bands in 50 µg of CHO
membrane protein suggesting that, if expressed at all, these G proteins
are present at low levels. These results are consistent with published
observations in CHO cells (10, 24, 25, 26).
To identify the specific G proteins coupled to the sst2A receptor,
membranes from CHO-R2A cells were incubated with or without ligand,
solubilized, and immunoprecipitated with receptor antiserum (Fig. 8
). The G protein subunits copurifying with the receptor
were detected by immunoblotting with antibodies specific for different
- or ß-subunits. A rat brain cholate extract, known to be enriched
in G proteins, was used as a positive control for the blotting
antisera.

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Figure 8. Identification of G Proteins Coprecipitated with
the sst2A Receptor
CHO-R2A membranes were incubated with or without 100 nM
SRIF. After solubilization, receptors were immunoprecipitated in the
absence or presence of 7 µM peptide 2CER with
antiserum R288 covalently coupled to Protein A-Sepharose.
Precipitated proteins were solubilized in sample buffer and separated
on a 12% acrylamide-SDS gel. After tranfer to PVDF membranes, G
protein subunits were identified by immunoblotting with specific G
protein antibodies (anti-G i1 at 1:1,000; J-883 against
G i2 at 1:1,000; EC against
G i1/ o at 1:2,000; U-46 against
G o at 1:2,000; P-961 against G z at
1:1,000, anti-G q/11 at 1:2, 500, SW1 against
Gß at 1:20,000).
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Figure 8
(top panel) shows that antibody EC, which
recognizes both
i3 and
o (27), detected a
41-kDa band in immunoprecipitates from SRIF preincubated membranes.
This protein was absent when 1 µM antigen peptide was
added during the incubation with antiserum, demonstrating that it was
dependent on receptor immunoprecipitation. Moreover, this protein was
only precipitated when the receptor was preincubated with ligand (Fig. 8
, top panel). Thus, although hormone binding did not affect
receptor immunoprecipitation (Fig. 6
), it did determine whether the
G
subunit was coprecipitated (Fig. 8
). The protein recognized by
antibody EC is G
i3 rather than G
o 1)
because its 41-kDa molecular mass was closer to that of
G
i3 (41 kDa) than that of G
o (39 kDa)
(27), and 2) because it was not recognized by the antibody U-46, which
is specific for G
o (Fig. 8
, bottom panel). As
with the G
subunit, Gß was precipitated with the sst2A receptor in
a ligand- dependent manner (Fig. 8
, top panel). Therefore,
both G protein
- and ß-subunits form a stable complex with the
sst2A receptor only when it is occupied by hormone.
To further characterize the specificity of sst2A receptor-G protein
interactions, we determined whether other G proteins copurified with
the receptor. The results in Fig. 8
(bottom panel) show that
G
i1 and G
i2 also complexed with the
receptor whereas G
q, G
o, and
G
z did not. Hence the sst2A receptor exhibits strong G
protein selectivity.
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DISCUSSION
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The specificity of signal transduction by heptahelical receptors
is determined by the G proteins with which they interact, since it is
the receptor-activated G protein
- and ß
-subunits that regulate
effector enzymes and ion channels (28, 29, 30). SRIF receptors have been
shown to signal via both pertussis toxin-sensitive and -insensitive G
proteins, and sst receptor-G protein coupling specificity has received
considerable attention in the attempt to delineate the molecular
mechanisms involved (4, 31). However, most previous studies were
conducted with tissues or cell lines that are now known to express
multiple sst receptor subtypes (16, 32, 33, 34, 35, 36). Thus, although the
spectrum of G proteins with which the sst receptor family can couple
has been examined, the G protein coupling specificity of individual sst
receptor subtypes remains poorly understood. To investigate sst2A
receptor-G protein coupling, we developed antibodies specific for this
receptor isoform and used these antibodies to isolate and characterize
the sst2A receptor-G protein complex.
Antisera were generated against two overlapping peptide antigens
corresponding to unique sst2A sequences. Although all the antisera
bound the corresponding immunizing peptide with comparable affinities,
the two antisera against peptide 2CER were both able to
immunoprecipitate the receptor, whereas neither of the antisera against
peptide 2CSG could do so. The failure of the
2CSG antisera to recognize the receptor was not due to
interference by receptor-associated G proteins because these antisera
also did not precipitate photoaffinity-labeled receptors preincubated
with GTP
S (data not shown). In the case of the best antiserum,
R288, the epitope region was deduced to be COOH-terminal to amino
acid 348 because the peptide 2CER (amino acids 339359)
bound to the antibody with nanomolar affinity, whereas the overlapping
peptide 2CSG (amino acids 334348) did not bind.
Interestingly, sequence analysis by the method of Hopp and Woods (37)
predicted that the region spanned by amino acids 334348 would be more
hydrophilic/antigenic than that spanned by amino acids 349359. Thus,
although choice of the peptide used for immunization proved critical
for receptor recognition by the antisera, hydrophilicity analysis did
not identify the most useful sequence in this instance.
Several observations proved that antiserum R288 recognized the sst2A
receptor specifically. The antibody immunoprecipitated more than 70%
of the [125I-Tyr11]SRIF-sst2A receptor
complex solubilized from CHO-R2 cells. It also immunoprecipitated the
85-kDa photoaffinity-labeled sst2A receptor and recognized the
denatured receptor on immunoblots. Both the immunoprecipitation and the
immunoblotting of the sst2A receptor were blocked by micromolar
concentrations of antigen peptide, and preimmune serum was inactive.
Finally, the antiserum did not bind any of the other sst receptor
subtypes.
We next identified the endogenous sst2A receptor protein in
differentiated cell lines that had previously been shown to express
sst2 mRNA, along with mRNA for other sst receptors (19, 21, 22).
Photoaffinity-labeled receptors, migrating as broad
85 kDa
bands, were immunoprecipitated from AR42J pancreatic acinar cells,
AtT-20 corticotrophic pituitary cells, and
GH4C1 mammosomatotrophic pituitary cells. Thus
all three of these cell lines express the sst2A receptor protein. The
molecular mass of the sst2 receptor is predicted to be 41 kDa from its
amino acid sequence; however, previous studies with
GH4C1 cells showed that this receptor is
glycosylated (38). Hence, added carbohydrates probably account for the
increased mass of the photoaffinity-labeled sst2A receptor as well as
its broad migration pattern on polyacrylamide gels. The subtle
differences in the apparent size and breadth of the immunoprecipitated
sst2A receptor band in the cell lines that we examined suggest that
receptor glycosylation varies among cell types. More than 70% of the
ligand-receptor complex could be immunoprecipitated from AR42J cells
(data not shown). This is similar to the immunoprecipitation efficiency
observed with receptors from CHO-R2A cells, which express only the
sst2A subtype, and indicates that sst2A is the major sst receptor in
this acinar cell line. Only 3040% of the ligand-receptor complex was
precipitated from AtT-20 and GH4C1 cells (data
not shown), consistent with the known presence of other sst receptors
isotypes in these pituitary cells (19, 21).
Two other groups have described the biochemical properties of sst2
receptors by immunoblotting with antipeptide antibodies. Patel et
al. (19) generated an antiserum (EE704) to an extracellular
amino-terminal segment common to the sst2A/B receptors and detected a
broad, 72-kDa protein in membranes from sst2-transfected CHO and AtT-20
cells (19). This size is close to that of the photoaffinity-labeled
receptor immunoprecipitated from CHO-R2 and AtT20 cells by our
sst2A-specific antibody. In contrast, Theveniau et al. (39)
stained distinctly different molecular mass proteins with an antibody
(2e3) generated to amino acids in the third extracellular domain common
to the sst2A/B receptors. The 2e3 antibody detected a sharp 93-kDa band
in sst2-expressing CHO membranes and a sharp band of 148 kDa in AR42J
cell membranes. These bands differ from the broad,
photoaffinity-labeled 85 kDa band that we identified as the sst2A
receptor in CHO and AR42J cells by immunoprecipitation. Since the 93-
and 148-kDa proteins were not shown to bind SRIF, their identity as sst
receptors, rather than related proteins, remains to be established. The
antibody described here is the first to specifically recognize the
sst2A receptor subtype in immunoprecipitation and immunoblotting, as
well as immunohistochemical, assays (40) and provides a valuable new
reagent for studies of the sst2A receptor protein.
To determine whether immunoprecipitation could be used to elucidate the
G protein-coupling specificity of the sst2A receptor, we first
determined the effect of ligand and G protein association on antibody
binding to the receptor. Our results showed that the antibody
efficiently immunoprecipitated both the hormone-receptor complex and
the unoccupied receptor. To determine whether G proteins were
physically associated with the receptor in the immune complex, we
examined the effect of GTP
S on the rate of ligand dissociation from
the immunoprecipitated receptor. To favor the formation of the
agonist-receptor-G protein ternary complex in the native membrane
environment, membrane receptors were first incubated with radiolabeled
hormone in the absence of guanine nucleotides (28) and then solubilized
under conditions that maintain a functional interaction between
receptor and G proteins (16). Since GTP
S markedly stimulated hormone
dissociation from the immunoprecipitated receptor, G proteins must
remain coupled, and the carboxy-terminal receptor epitope recognized by
the antibody cannot be essential for functional interaction between the
sst2A receptor and G proteins. We previously found that this was also
true for the sst1 receptor subtype (21). Further analysis of the
immunoprecipitated complex showed that both
- and ß- G protein
subunits were coprecipitated with the sst2A receptor. Since the
ß-subunit is usually found tightly complexed with the
-subunit, it
is likely that the latter is also present. The physical association
between these G protein subunits and the receptor was absolutely
dependent on ligand occupancy, showing that hormone binding stabilizes
the receptor-G protein complex and that little of the receptor is
precoupled to G proteins in the absence of agonist activation.
The G protein
-subunits physically associated with the sst2A
receptor were subsequently identified by immunoblotting of the
immunoprecipitated complex. Immunoblotting of CHO membranes indicated
that G
i2 and G
q/11 were abundantly
expressed whereas G
i3, although readily detectable, was
less intensely stained (data not shown). G
i1,
G
o, and G
z were not detected. Our results
are consistent with the quantification of G
i subunits
reported by Gettys et al. (26) who showed that
G
i1 was not detectable (<0.1 pmol/mg) in CHO-K1
membranes and that G
i2 (5 pmol/mg) was present at much
higher levels than G
i3 (0.6 pmol/mg). Other groups have
also reported that G
o and G
z are not
detectable in CHO-K1 cells (25).
Whereas the three G
i subunits copurified with the sst2A
receptor, G
q/11, G
o, and
G
z were not found in the receptor complex. The
observation that G
o and G
z were not
associated with the receptor probably results from the absence of these
G proteins from CHO cells, although we cannot rule out the possibility
that they are expressed at levels below the limits of immunodetection.
However, because G
q/11 is abundant, its absence from the
receptor complex demonstrates that the sst2A receptor exhibits strong G
protein specificity. Although the receptor was physically associated
with all three G
i subunits, it had the highest affinity
for G
i1, since this is the least abundant
G
i in CHO-K1 membranes and showed the greatest
enrichment in the receptor complex.
In contrast to our observations, Law et al. (41) concluded
that the sst2A receptor couples to G
i3 but not to
G
i1 or G
i2 when expressed in CHO-DG44
cells, a cell line that contains all three G
i subunits.
This difference may reflect functional differences in coupling in the
two cell lines. In the same transfected CHO-K1 cell line used in our
study, the sst2A receptor was shown to inhibit adenylyl cyclase via
pertussis toxin-sensitive G proteins (10). In contrast, the transfected
sst2A receptor did not inhibit adenylyl cyclase in CHO-DG44 cells (42).
However, critical differences between the protocols used in the two
studies could also explain the disparate results obtained. In our
experiments, hormone binding was carried out in membranes to stabilize
specific receptor-G protein complexes within the normal lipid
environment of the receptor. The ternary complex was then solubilized
under conditions in which it is known to remain stable (16). By
comparison, Law et al. (41) solubilized the unoccupied
receptor and then examined the ability of G protein antibodies to
inhibit hormone binding in the detergent extract. The presence of
detergent in the binding reaction may well alter the specificity of
receptor-G protein coupling from that which occurs in the plasma
membrane.
Transfected sst2 receptors stimulate tyrosine phosphatases and
phospholipase C as well as inhibit calcium channels and adenylyl
cyclase (11, 12, 13). Whereas the inhibition of cAMP accumulation is
completely reversed by pertussis toxin treatment, SRIF-induced
activation of tyrosine phosphatase and phospholipase C are either
insensitive or only partially inhibited by the toxin (12, 13). Hence,
the sst2 receptor has been proposed to act via pertussis
toxin-insensitive as well as -sensitive G proteins. We did not detect
any interaction between the sst2A receptor and the pertussis toxin-
insensitive G proteins G
q/11 and G
z, even
though the former is abundant in the CHO-K1 cells. Thus, the pertussis
toxin-insensitive pathways activated by the sst2A receptor remain to be
identified.
It is now widely recognized that seven-transmembrane domain receptors
are capable of coupling with multiple but selected G proteins either of
the same or different subgroups, thus permitting activation of several
distinct signal transduction pathways. This coupling is believed to
play an important role in determining cell-specific receptor functions.
Our studies demonstrate that in a normal membrane environment, hormone
binding causes the sst2A receptor to couple specifically to the three
Gi proteins, with highest affinity for G
i1.
The availability of an antibody that selectively immunoprecipitates
this receptor subtype now makes it possible to explore the coupling of
endogenously expressed sst2A receptors irrespective of the presence of
other sst isoforms and allow identification of its signal transduction
pathways in different cellular environments.
 |
MATERIALS AND METHODS
|
---|
Antisera Preparation and Assay
Antigen peptides were synthesized with an N-terminal cysteine
and conjugated to KLH through the cysteine sulfhydryl with
m-maleimidobenzoyl-N-hydroxysuccinimide (43). New Zealand
white male rabbits (Ray Nichols, Lumberton, TX) were immunized with
each KLH-coupled peptide antigen as previously described (21). The
titers and binding affinities of each antiserum for the corresponding
peptide antigen were measured by ELISA (21).
Covalently coupled Protein A-Sepharose was prepared by incubating
Protein A (20 mg) with CNBr-activated Sepharose 4B (1.75 g) according
to manufacturers instructions (Pharmacia-LKB Biotechnology,
Rockville, MD). IgG and Protein A-Sepharose 4B were coupled with
dimethyl pimelimidate as described by Harlow and Lane (44). Any
noncovalently bound IgG was eluted from the Protein A-Sepharose by
incubating the beads in 10 volumes of 0.1 M glycine, pH
2.8, at room temperature for 5 min. After centrifugation, the pellet
was extensively washed with PBS, pH 7.5, and then stored at 4 C in PBS
with 0.05% NaN3.
Cell Culture and Membrane Preparation
CHO-K1 cell lines transfected to stably express individual sst
receptor subtypes were obtained from the following sources: rat sst1
(CHO-R1) and rat sst2A (CHO-R2A) receptor-expressing cells were
provided by Dr. Philip Stork (10), mouse sst2B receptor-expressing
cells (CHO-R2B) were provided by Dr. Voker Hollt (6), human sst3
receptor-expressing cells were provided by Dr. Yogesh Patel (45), and
rat sst4- expressing cells were provided by Drs. Jack Bruno and Michael
Berelowitz (46). CHO-K1 cells stably expressing the human sst5 receptor
were generated from a plasmid provided by Dr. Susumu Seino (47). A
1.7-kb DNA fragment containing the entire human sst5 coding sequence
was excised from the pCMV6c plasmid by digestion with
EcoRI/SalI and ligated into the
EcoRI/XhoI sites of plasmid pcDNA3. Cells were
selected in 250 µg/ml G418 and cloned by serial dilution.
GH4C1, AR42J, AtT-20, RINm5F, and CHO cell
lines were grown and subcultured as previously described (10, 23, 48).
Pertussis toxin treatment was performed by incubating cells with fresh
medium containing 100 ng/ml pertussis toxin for 1624 h before
membrane preparation.
Cell membranes were prepared and stored as previously published (23).
Briefly, cells from either monolayer or suspension cultures were
collected, washed with PBS (10 mM
Na2HPO4, 150 mM NaCl, pH 7.4), and
homogenized at 4 C in Tris buffer (10 mM Tris-Cl, 2
mM MgCl2, 2 mM EDTA, 0.5
mM phenylmethylsulfonyl fluoride, pH 7.6). After
centrifugation at 500 x g for 10 min, the supernatant was
collected and centrifuged again at 10,000 x g for 30
min. The membrane pellet was stored at -70 C.
Ligand Binding and Receptor Photoaffinity Labeling
[Tyr11]SRIF and the photoactive SRIF analog
[Tyr11, ANB-Lys4]SRIF were iodinated with
Chloramine-T, and the radiolabeled peptides were purified by
reverse-phase HPLC to a specific activity of 2,200 Ci/mmole (23).
Binding reactions were performed as previously described (23). Briefly,
membranes were incubated at 30 C for 2 h in HEPES binding buffer
(50 mM HEPES, 7 mM MgCl2, 2
mM EDTA, and 2 U/ml of bacitracin) containing radiolabeled
peptide (0.050.15 nM) with or without 100 nM
unlabeled SRIF. After dilution with cold binding buffer, samples were
centrifuged at 30,000 x g for 15 min, and the
radioactivity associated with the pellets was measured. Specific
binding was calculated as the difference between the amount of
radioactivity bound in the absence and in the presence of 100
nM unlabeled SRIF. Curve-fitting and data analysis was
carried out as described previously (23).
Receptor photoaffinity labeling was performed with either membranes or
solubilized receptors (21, 23). Membranes were incubated in the dark
with [125I-Tyr11, ANB-Lys4]SRIF,
centrifuged, and then resuspended in cold HEPES binding buffer to a
final concentration of 0.1 mg/ml. Irradiation was at 254 nm on ice for
10 min (Mineralight model R-52, Ultraviolet Products Inc., San Gabriel,
CA). After the addition of 1 M Tris·Cl, pH 7.6, membranes
were pelleted, solubilized in sample buffer (62.5 mM
Tris·Cl, pH 6.8, 2% SDS, 20% glycerol, and 50 mM
dithiothreitol), and subjected to SDS-PAGE according to the method of
Laemmli (49). The dried gels were exposed to Amersham Hyperfilm at -70
C. For immunoprecipitation experiments,
[125I-Tyr11, ANB-Lys4]SRIF
binding was carried out in the dark as described above, but irradiation
was performed after solubilization of the ligand-receptor complex with
DßM/cholesterol hemisuccinate (CHS). The photoaffinity-labeled
receptor was then subjected to immunoprecipitation and analysis by
SDS-PAGE and autoradiography.
Immunoprecipitation
Unless stated otherwise, cell membranes, prebound with a
radiolabeled SRIF analog, were solubilized for 1 h at 4 C in HEPES
binding buffer containing 1 mg/ml DßM, 0.2 mg/ml CHS, 10 µg/ml
soybean trypsin inhibitor, 50 µg/ml bacitracin, and 10 µg/ml
leupeptin as described previously (16). After centrifugation at
100,000 x g, the solubilized ligand-receptor complex
was either quantitated by polyethylene glycol 8000 (PEG) precipitation
(16) or subjected to immunoprecipitation.
For immunoprecipitation, antiserum was added to the solubilized
receptor to the final concentration indicated and incubated at 4 C for
320 h. Protein A-Sepharose-4B was then added, and the incubation was
continued for another hour at 4 C. After centrifugation at 10,000
x g for 2 min, the pellet was washed with cold HEPES
binding buffer containing 0.25 mg/ml DßM, and the precipitated
radioactivity was measured.
To determine the rate of ligand dissociation from the
immunoprecipitated receptor, cell membranes were incubated with
[125I-Tyr11]SRIF in the absence and presence
of 100 nM SRIF for 2 h at 30 C. After solubilization
and immunoprecipitation, the precipitate was washed, resuspended in
HEPES binding buffer containing 10 nM SRIF, and 0.25 mg/ml
DßM with or without 10 µM GTP
S, and then incubated
at 25 C. At indicated times, duplicate aliquots of the suspension were
removed and centrifuged, and the specifically bound ligand remaining
associated with the immunoprecipitated receptor was quantitated.
Immunoblotting
After receptor immunoprecipitation, the pellet was resuspended
in sample buffer and subjected to SDS-PAGE. Rat brain cholate extract
(50 µg) was used as a positive control for all G protein blotting
experiments. The resolved proteins were transferred to polyvinylidene
difluoride (PVDF) membrane at 100 mA for 30 min and 500 mA for 1.5
h. The membrane was then blocked with 5% nonfat dry milk at room
temperature for 2 h and subsequently incubated at 4 C overnight
either with receptor antiserum or with G protein subtype-specific
antisera. PVDF membranes were washed twice with PBS containing 0.05%
Tween 20, incubated with goat anti-rabbit antibody conjugated with
horse radish peroxidase for 1 h, and washed again. Immunoreactive
proteins were detected by chemiluminescence using enhanced
chemiluminescence (ECL) substrate according to manufacturers
instructions (Amersham, Arlington Heights, IL).
Antisera against different G protein subunits were obtained from the
following sources: G
i1 (internal 159168) (51) and
G
i3 (C-terminal 345354) (52) purchased from Calbiochem
(San Diego, CA); antiserum J-883 against G
i2 and
antiserum U-46 against G
o from Dr. Suzanne Mumby (53);
the G
z antiserum P-961 from Dr. Patrick Casey (54);
antiserum EC against G
i3/G
o and antiserum
SW1 against Gß from Dr. Allen Spiegel (27, 55); antiserum
against G
q/11 from Dr. Thomas F. J. Martin (56).
 |
ACKNOWLEDGMENTS
|
---|
We thank Ms. Fengyu Jiang for excellent technical assistance,
Ms. Yining Wang for generating the CHO-R5 cells, Dr. Philip Stork for
providing CHO-R1and CHO-R2A cells, Drs. Jack Bruno and Michael
Berelowitz for providing CHO-R4 cells, Dr. Volker Hollt for providing
CHO-R2B cells, Dr. Yogesh Patel for providing CHO-R3 cells, and Dr.
Susumu Seino for providing human sst5 receptor plasmid. We also thank
Drs. Suzanne Mumby, Patrick Casey, Paul Sternweis, Allen Spiegel, and
Thomas F. J. Martin for providing G protein subtype-specific antibodies
and Dr. William T. Moore of the Analytical Chemistry Center at the
University of Texas Medical School and Drs. Kaijun Ren and Timothy P.
Kogan of Texas Biotechnology Corporation for peptide synthesis.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Agnes Schonbrunn, Department of Pharmacology, University of Texas Health Sciences Center, Houston, P.O. Box 20708, Houston, Texas 77225.
This investigation was supported by Research Grant DK-32234 from the
National Institute of Arthritis, Diabetes, Digestive, and Kidney
Diseases.
Received for publication January 16, 1997.
Revision received February 7, 1997.
Accepted for publication February 10, 1997.
 |
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