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Department of Integrative Biology, Pharmacology, and Physiology University of Texas Medical School Houston, Texas 77225
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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
Gi1, Gi2, and
Gi3 were complexed with the sst2A receptor
whereas Go, Gz,
and Gq were not. Together, these studies
demonstrate that the sst2A receptor interacts selectively with
Gi proteins in a hormone-dependent manner.
The finding that this receptor couples to all three
Gi subunits may help explain how
somatostatin can regulate multiple signaling pathways. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The peptide 2CSG corresponds to
amino acids 334–348 in the mouse sst2A receptor and differs from both
the rat and human receptors by one residue. The peptide
2CER corresponds to amino acids 339–359 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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), respectively. Antisera
R2–204 and R2–206 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 R2–88 (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.
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, 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
(R2–87 and R2–88) but was not significantly precipitated by either of
the antisera against peptide 2CSG (R2–204 and R2–206).
Because antiserum R2–88 was most effective, it was characterized in
greater detail.
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, 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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, lower panel). Sst2B receptors lack any sequence
homology with the peptide used for immunization, and, as expected, this
receptor was not recognized by antiserum R2–88 (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 R2–88
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 AR4–2J 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 80–100 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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, 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 R2–88 specifically recognized the sst2A receptor.
We next determined whether any of the photoaffinity-labeled receptors
in AR4–2J, 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
AR4–2J, AtT-20, and GH4C1 cells and migrates
at 80–100 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 R2–88. 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 R2–88
antibody can recognize the denatured receptor on immunoblots in the
absence of bound ligand.
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, 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 R2–88
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 GTPS 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, GTPS 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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-subunits were present in
CHO membranes using immunoblot analysis. Specific immunoreactivity was
detected at 40–43 kDa with antibodies to Gi2,
Gi3, and Gq/11 (data not shown). However,
antibodies to Gi1, Go, and
Gz 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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(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 Gi3 rather than Go 1)
because its 41-kDa molecular mass was closer to that of
Gi3 (41 kDa) than that of Go (39 kDa)
(27), and 2) because it was not recognized by the antibody U-46, which
is specific for Go (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
Gi1 and Gi2 also complexed with the
receptor whereas Gq, Go, and
Gz did not. Hence the sst2A receptor exhibits strong G
protein selectivity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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- 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 GTPS (data not shown). In the case of the best antiserum,
R2–88, the epitope region was deduced to be COOH-terminal to amino
acid 348 because the peptide 2CER (amino acids 339–359)
bound to the antibody with nanomolar affinity, whereas the overlapping
peptide 2CSG (amino acids 334–348) did not bind.
Interestingly, sequence analysis by the method of Hopp and Woods (37)
predicted that the region spanned by amino acids 334–348 would be more
hydrophilic/antigenic than that spanned by amino acids 349–359. 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 R2–88 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 AR4–2J 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 AR4–2J 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 30–40% 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 AR4–2J cell membranes. These bands differ from the broad, photoaffinity-labeled 85 kDa band that we identified as the sst2A receptor in CHO and AR4–2J 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 GTPS 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 GTPS 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 Gi2 and Gq/11 were abundantly
expressed whereas Gi3, although readily detectable, was
less intensely stained (data not shown). Gi1,
Go, and Gz were not detected. Our results
are consistent with the quantification of Gi subunits
reported by Gettys et al. (26) who showed that
Gi1 was not detectable (<0.1 pmol/mg) in CHO-K1
membranes and that Gi2 (5 pmol/mg) was present at much
higher levels than Gi3 (0.6 pmol/mg). Other groups have
also reported that Go and Gz are not
detectable in CHO-K1 cells (25).
Whereas the three Gi subunits copurified with the sst2A
receptor, Gq/11, Go, and
Gz were not found in the receptor complex. The
observation that Go and Gz 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 Gq/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 Gi subunits, it had the highest affinity
for Gi1, since this is the least abundant
Gi 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 Gi3 but not to
Gi1 or Gi2 when expressed in CHO-DG44
cells, a cell line that contains all three Gi 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 Gq/11 and Gz, 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 Gi1.
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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Covalently coupled Protein A-Sepharose was prepared by incubating Protein A (20 mg) with CNBr-activated Sepharose 4B (1.75 g) according to manufacturer’s 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, AR4–2J, 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 16–24 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 (0.05–0.15 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 3–20 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 GTPS, 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 manufacturer’s
instructions (Amersham, Arlington Heights, IL).
Antisera against different G protein subunits were obtained from the
following sources: Gi1 (internal 159–168) (51) and
Gi3 (C-terminal 345–354) (52) purchased from Calbiochem
(San Diego, CA); antiserum J-883 against Gi2 and
antiserum U-46 against Go from Dr. Suzanne Mumby (53);
the Gz antiserum P-961 from Dr. Patrick Casey (54);
antiserum EC against Gi3/Go and antiserum
SW1 against Gß from Dr. Allen Spiegel (27, 55); antiserum
against Gq/11 from Dr. Thomas F. J. Martin (56).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) or peptide
2CSG (
) was adsorbed onto each well of a 96-well plate
and incubated with antiserum and the concentrations of competing
peptide shown on the abscissa. ELISA assays were performed as described
in Materials and Methods. Antiserum R2–88 (• and 
