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INTRODUCTION |
The two physiologically active somatostatin
(SRIF)1 peptides, SRIF14 and
SRIF28, potently regulate numerous endocrine, exocrine, and neuronal
functions by interacting with a family of six G protein-coupled receptors (sst1, sst2A, sst2B, sst3, sst4, and sst5) (1-4). The cellular changes induced by sst receptors include inhibition of secretion, modulation of neuronal transmission, and smooth muscle contraction, as well as inhibition of proliferation and stimulation of
apoptosis. The sst1 receptor subtype is particularly widely distributed
in the gastrointestinal tract (5, 6) and the brain (7, 8) as well as
being expressed in several other normal tissues (9, 10). Additionally,
sst1 is found in neuroendocrine, prostate, and mammary tumors (11-13).
Thus, this receptor subtype is believed to mediate many of the central
and peripheral actions of SRIF as well as to present a potential
target for cancer therapy and diagnosis (1-4, 14).
The sst1 receptor, like other members of the somatostatin receptor
family, is now known to inhibit adenylyl cyclase by interacting with
pertussis toxin-sensitive Gi/Go proteins
(15-17). Other signaling mechanisms potently regulated by the sst1
receptor in a pertussis toxin-sensitive manner include inhibition of
Ca2+ influx, membrane hyperpolarization, activation of
protein tyrosine phosphatases, and stimulation of the mitogen-activated
protein kinase cascade (18-20). However, the sst1 receptor has also
been shown to act via pertussis toxin-insensitive mechanisms. Both activation of Na+/H+ exchange and potentiation
of
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate current responses to glutamate are unaffected by pertussis toxin pretreatment (21, 22). Thus, sst1 appears capable of
signaling via several classes of G proteins.
Exposure of G protein-coupled receptors to agonist usually leads
desensitization of receptor responsiveness as well as to initiation of
intracellular signaling (23-25). Such receptor desensitization can be
produced by an uncoupling of the receptor from G proteins, by receptor
down-regulation or, most often, by a combination of the two mechanisms.
The molecular events leading to uncoupling are thought to involve
increased phosphorylation of the agonist-activated receptor followed by
the binding of the phosphorylated receptor to arrestins, which then
block further receptor-G protein interactions. Receptor down-regulation
usually results from an increased rate of receptor internalization
following agonist occupancy and this process is also thought to be
triggered by receptor phosphorylation and arrestin binding because of
the observation that arrestin can act as an adaptor to link G
protein-coupled receptors to clathrin-mediated endocytosis (23-25).
Recently, the sst2A and sst3 receptors were shown to be rapidly
phosphorylated following SRIF treatment (26-28). However, little is
known about the molecular mechanisms responsible for the regulation of
the sst1 receptor subtype. Thus, the objective of the present study was
to examine the role of sst1 receptor phosphorylation, uncoupling and
internalization in sst1 receptor desensitization.
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EXPERIMENTAL PROCEDURES |
Hormones and Supplies--
Cell culture medium and G418 were
purchased from Life Technologies, Inc. (Grand Island, NY). The sst1
receptor antiserum (R1-201) has been shown to specifically recognize
only this sst receptor subtype (17). Leupeptin, phenylmethylsulfonyl
fluoride, soybean trypsin inhibitor, bacitracin, Nonidet P-40,
pertussis toxin, bacterial alkaline phosphatase, and Protein A were
obtained from Sigma. N-Dodecyl-
-D-maltoside
was purchased from Calbiochem (La Jolla, CA). CNBr-activated Sepharose
4B was from Amersham Pharmacia Biotech (Uppsala, Sweden). Bradford
reagent and reagents for electrophoresis and Western blotting were
obtained from Bio-Rad. Carrier-free Na125I was from
Amersham Pharmacia Biotech. [
-32P]ATP was obtained
from PerkinElmer Life Sciences. Phosphate-free Dulbecco's modified
Eagle's medium and [32P]orthophosphate were purchased
from ICN Biomedicals (Costa Mesa, CA). All other reagents were of the
best grade available and purchased from common suppliers.
Cell Culture--
The clonal CHO-R1 cell line was generated by
stable transfection of CHO-K1 cells with the rat sst1 receptor followed
by clonal selection (15). The cell line contains 980 ± 90 (n = 4) fmol of receptor/mg of membrane protein with an
affinity (Kd) of 240 ± 100 pM for
[125I-Tyr11]SRIF. Cells were grown in F12
medium containing 10% fetal calf serum and 250 µg/ml G418.
Experimental cultures were plated in medium without G418 and used 3-4
days later with a medium change 18-24 h prior to use. Experiments were
carried out with cells plated in 100-mm dishes except for whole cell
binding experiments, for which 35-mm wells were used.
Membrane Preparation--
Cells were pretreated in a
CO2 incubator at 37 °C with 100 nM SRIF or
carrier in serum-free F12 medium containing 5 mg/ml lactalbumin
hydrolysate and 20 mM HEPES, pH 7.4 (F12LH). The
pretreatment was stopped by washing the cultures with ice-cold HME
buffer (20 mM HEPES, pH 8.0, 2 mM
MgCl2, 1 mM EDTA, 1 mM benzamidine,
10 µg/ml soybean trypsin inhibitor, 0.1 mg/ml bovine serum albumin). Washed cells were scraped into HME plus 10 µg/ml leupeptin, 20 mM tetrasodium pyrophosphate, and 0.1 µM
okadaic acid and homogenized with a Dounce homogenizer. The homogenates
were centrifuged on a step gradient of 23 and 43% sucrose in HE buffer
(20 mM Hepes, pH 8.0, 1 mM EDTA) and the
fraction at the 23:43% interface was collected and stored at
80 °C (29).
Adenylyl Cyclase Assay--
Membranes (5-10 µg of
protein/tube) were assayed in triplicate for adenylyl cyclase activity
at 30 °C for 10 min in the presence of 4 mM added
MgCl2 (29).
Radioligand Binding and
Internalization--
[Tyr11]SRIF was radioiodinated
using chloramine T and subsequently purified by reverse-phase high
performance liquid chromatography as described previously (17). CHO-R1
cells were incubated either at 37 or 4 °C in F12LH containing
~100,000 cpm of [125I-Tyr11]SRIF in the
absence or presence of 100 nM unlabeled SRIF (30). Following 4 °C binding incubations cells were rinsed free of unbound trace and subsequently incubated in fresh 37 °C buffer to allow internalization of the receptor-bound ligand. To determine the distribution of bound radioligand, cells were incubated on ice for 5 min in acetic acid-buffered saline (200 mM acetic acid, 500 mM NaCl, pH 2.5). After collection of the acidic buffer the cells were dissolved in 0.1 N NaOH. The radioactivity in
both the acid wash, representing surface-bound ligand, and in cell lysates, representing internalization ligand, was measured (30). Specific binding was calculated as the difference between the amount of
radioligand bound in each fraction in the absence (total binding) and presence of 100 nM SRIF
(nonspecific binding).
Purification and Deglycosylation of
32PO4-Labeled Receptor--
Metabolic labeling
of cells with 32PO4 and
subsequent immunoprecipitation of the sst1 receptor was carried out as
described previously for the sst2A receptor (26). Briefly, cells were incubated for 3 h in phosphate-free Dulbecco's modified Eagle's medium containing 1 mCi of [32P]orthophosphate. Hormones
and pharmacological agents were then added directly to the labeling
medium, and the cells were further incubated at 37 °C under 5%
CO2 for the indicated times. The cells were then scraped
into cold Hepes-buffered saline with protease and phosphatase
inhibitors (HBS: 150 mM NaCl, 20 mM Hepes, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml soybean
trypsin inhibitor, 10 µg/ml leupeptin, 50 µg/ml bacitracin, 5 mM EDTA, 3 mM EGTA, 10 mM sodium
pyrophosphate, 10 mM sodium fluoride, 0.1 mM
orthovanadate, 100 nM okadaic acid). Following
centrifugation, the cell pellet was solubilized in lysis buffer (HBS
containing 4 mg/ml dodecyl-
-D-maltoside) at 4 °C. The
detergent lysates were centrifuged at 100,000 × g for
30 min, and the protein content of the supernatant was assessed by the
method of Bradford (31).
Sst1 receptors were usually subjected to a three-step purification
consisting of: 1) adsorption to wheat germ agglutinin-agarose (26); 2)
enzymatic deglycosylation (32); and 3) immunoprecipitation with
receptor antibody (26). Briefly, equal amounts of lysate proteins were
incubated at 4 °C for 90 min to 16 h with washed wheat germ
agglutinin-agarose (WGA-agarose, Vector Laboratories, Inc., Burlingame,
CA). Following centrifugation, the WGA-agarose was washed vigorously
with lysis buffer. In some experiments adsorbed glycoproteins were then
eluted at 37 °C for 30 min with lysis buffer containing 3 mM
N,N",N'''-triacetylchitotriose (TACT)
(Sigma) and 0.5% SDS (v/v). In most experiments, however, adsorbed
glycoproteins were deglycosylated by incubating the washed WGA-agarose
at 37 °C overnight with lysis buffer (pH 7.4) containing 0.1% SDS
(v/v) and 10 units/ml of peptide-N-glycosidase F (PNGase F,
Roche Molecular Biochemicals, Indianapolis, IN) (32). This enzyme,
which catalyzes the cleavage of N-glycosidically linked
carbohydrate chains between N-acetylglucosamine and
asparagine, caused the release of most of the receptor from the
WGA-agarose. After either the TACT or glycosidase treatment, the
supernatant containing the dissociated receptor was incubated with a
1:200 dilution of the anti-sst1 receptor antibody R1-201 (17) at
4 °C for
90 min followed by incubation at 4 °C for 60 min with
25 µl (packed volume) of protein A-Sepharose 4B. Following
centrifugation, the Sepharose beads were washed as described previously
(26) and the immunoprecipitated proteins were solubilized in sample
buffer (62.5 mM Tris-HCl, 2% SDS, 10% 2-mercaptoethanol
(v/v), 6 M urea, 20% glycerol, pH 6.8) at 37 °C for 60 min, and resolved on 12% SDS-polyacrylamide gels.
Purification and Deglycosylation of Unlabeled Receptor and
Detection by Immunoblotting--
CHO-R1 cells were incubated in growth
medium in the presence or absence of SRIF, PMA, or forskolin at
37 °C for the times indicated. The cells were then scraped into cold
Hepes-buffered saline, pelleted, and solubilized in lysis buffer for 60 min at 4 °C. The detergent lysates were clarified by centrifugation
at 100,000 × g for 30 min, and the protein content of
the supernatants was assessed by the method of Bradford (31). Following
adsorption to WGA-agarose and deglycosylation by PNGase F as described
above, the supernatant was incubated for
90 min at 4 °C with
anti-sst1 receptor IgG covalently coupled to Protein A-Sepharose 4B.
The immunoprecipitated proteins were dissolved in sample buffer without reducing reagent for 60 min at 37 °C. After removal of the Sepharose beads and addition of 10% 2-mercaptoethanol (v/v) proteins were resolved on 12% SDS-polyacrylamide gels.
To investigate the subcellular distribution of phosphorylated
receptors, sst1 was also deglycosylated with
endo-N-acetylglucosaminidase H (Endo H, Rochem Molecular
Biochemicals), an enzyme which hydrolyzes high-mannose oligosaccharides
from glycoproteins (33). Following adsorption of glycoproteins to
WGA-agarose as described above, the washed beads were incubated with
lysis buffer (pH 5.8) containing 0.02% SDS (v/v) and 75 milliunits/ml
of Endo H at 37 °C overnight. Replicate aliquots were incubated with
PNGase F at the same time. Following deglycosylation, the WGA-agarose
beads were centrifuged and proteins released into the supernatant were
precipitated at 4 °C for 2 h by 12.5% (v/v) trichloroacetic
acid. Both the trichloroacetic acid-precipitated proteins and the
proteins remaining adsorbed to the WGA beads were solubilized in sample
buffer and subjected to SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) as described above.
Resolved proteins were transferred to PVDF membrane and immunoblotted
with 1:5000 dilution of anti-sst1 antibody R1-201 as described
previously (13). Immunoreactive proteins were detected with an ECL
detection system (Amersham Pharmacia Biotech).
For phosphatase treatment, anti-receptor IgG-Sepharose beads with
adsorbed receptor were washed once with phosphatase buffer (20 mM Hepes, pH 8.0, 25 mM KCl, 15 mM
MgCl2) and then incubated with 100 µl of buffer
containing 4 mg/ml dodecyl-
-D-maltoside, 0.1 SDS%
(v/v), and 5 units of bacterial alkaline phosphatase (Sigma) at
37 °C for 1 h (34). After centrifugation, the
immunoprecipitated receptor was eluted and analyzed as described above.
Phosphoamino Acid Analysis--
Following phosphorylation and
purification of the sst1 receptor as described above, SDS-PAGE-resolved
proteins were transferred to PVDF membrane, and the
32PO4 containing bands were localized by
autoradiography. Membrane pieces containing selected bands were excised
and incubated in 50 µl of 5.7 N HCl (Pierce) at 110 °C
for 30 min (26). Phosphoamino acids were resolved by two-dimensional
thin layer electrophoresis on cellulose plates (26).
Other Methods--
Protein A (Sigma) was covalently coupled to
CNBr-activated Sepharose 4B according to the manufacturer's
instructions (Amersham Pharmacia Biotech). Anti-receptor IgG was
covalently coupled to protein A-Sepharose as described previously (17).
Fitted values for the maximal effect and the EC50 were
obtained by least squares nonlinear regression analysis of
dose-response curves using the program D/R (Biomedical Computer, Inc.
Houston, TX). 32PO4-Labeled bands were
quantitated using a PhosphorImager and visualized by autoradiography on
Biomax MS film (Kodak, Rochester, NY). Unless otherwise indicated
results of a representative experiment are shown. All experiments were
repeated at least 2 times.
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RESULTS |
Desensitization of the Sst1 Receptor--
To investigate the
susceptibility of the rat sst1 receptor to desensitization, CHO-R1
cells were incubated in the absence or presence of 100 nM
SRIF for 30 min at 37 °C and the effect of pretreatment on hormonal
regulation of membrane adenylyl cyclase activity was determined (Fig.
1). SRIF pretreatment did not affect either basal or forskolin-stimulated adenylyl cyclase activity. Cyclase
activity was 6.0 ± 1.5 pmol/min/mg (mean ± S.E.,
n = 3) under basal conditions and 263 ± 43 pmol/min/mg (n = 5) in the presence of 10 µM forskolin. In membranes from untreated cells SRIF
inhibited forskolin-stimulated adenylyl cyclase activity with an
EC50 of 1.62 ± 0.75 nM (n = 5) (Fig. 1). Maximal inhibition was 42.0 ± 7.4%
(n = 5). Preincubation of cells with 100 nM
SRIF markedly attenuated the efficacy of SRIF inhibition (maximal
inhibition = 10.6 ± 2.2%, n = 5). Because
cyclase inhibition was so small in pretreated cells, it was not
possible to calculate the EC50 for SRIF.

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Fig. 1.
The effect of agonist pretreatment on the
subsequent inhibition of forskolin-stimulated adenylyl cyclase activity
by SRIF. CHO-R1 cells were incubated in the absence ( )
or presence of 100 nM SRIF ( ) for 30 min and then used
to prepare membranes. Adenylyl cyclase activity was subsequently
assayed in the presence of 10 µM forskolin in the absence
and presence of the indicated concentration of SRIF. Data represent the
mean ± range from two independent experiments each assayed in
triplicate and was expressed as a percentage of forskolin-stimulated
membrane adenylyl cyclase activity in the absence of SRIF. SRIF
pretreatment had no effect on either basal or forskolin-stimulated
adenylyl cyclase activity. However, the maximal inhibition of
forskolin-stimulated adenylyl cyclase by SRIF was reduced from
42.1 ± 1.5 to 10.0 ± 1.4% by the SRIF pretreatment. The
EC50 for SRIF inhibition of adenylyl cyclase in control
membranes was 1.16 ± 0.19 nM. The small SRIF
inhibition observed in pretreated membranes precluded an accurate
calculation of the EC50 after desensitization.
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To determine the time dependence of sst1 desensitization, cells were
incubated in the absence or presence of 100 nM SRIF for varying times and the effect of the different pretreatments on membrane
adenylyl cyclase activity was subsequently measured (Fig. 2). The sst1 receptor was maximally
desensitizated within 2 min and desensitization was maintained for at
least 30 min.

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Fig. 2.
Time course for SRIF-induced sst1 receptor
desensitization. Top panel, CHO-R1 cells were incubated
in the absence ( ) or presence of 100 nM SRIF for
2 min ( ), 5 or 30 min ( ) and then used to prepare membranes.
Adenylyl cyclase activity was subsequently assayed in the presence of
10 µM forskolin in the absence or presence of the
indicated concentrations of SRIF. Data represent the mean ± range
from two independent experiments each assayed in triplicate and are
expressed as a percentage of forskolin-stimulated adenylyl cyclase
activity in the absence of SRIF. Data for the 5-min pretreatment group
was similar to that for 2 min and was omitted for clarity. Bottom
panel shows the maximal inhibition by SRIF in control and
SRIF-treated membranes prepared after the indicated times of
pretreatment. Data represent mean ± range from two independent
experiments.
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The concentration dependence for sst1 desensitization was analyzed by
incubating cells for 5 min in the presence of varying amounts of SRIF
and then measuring the effect on membrane adenylyl cyclase activity.
Both the potency and the efficacy of SRIF inhibition of adenylyl
cyclase were diminished by pretreatment with low concentrations of
SRIF. The EC50 for SRIF inhibition of adenylyl cyclase was increased from 2.4 ± 1.0 nM in control membranes to
6.9 ± 2.5 nM after pretreatment with 1 nM
SRIF and the maximal inhibition was reduced from 42.6 ± 2.7 to
30.3 ± 2.4% (Fig. 3, top
panel). Desensitization was further increased at higher peptide
doses and pretreatment with 100 nM SRIF reduced maximal
cyclase inhibition to 8.3 ± 2.4% (Fig. 3, top panel).
The EC50 for SRIF-induced desensitization was 2 nM (Fig. 3, bottom panel). Together,
these studies demonstrate that exposure to agonist results in
homologous desensitization of the sst1 receptor in a time- and
concentration-dependent manner.

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Fig. 3.
Concentration dependence for SRIF induced
sst1 desensitization. Top panel, CHO-R1 cells were
incubated in the absence ( ) or presence of 1 ( ), 10 ( ), and
100 nM ( ) SRIF for 5 min and then used to prepare
membranes. Adenylyl cyclase activity was assayed in the presence of 10 µM forskolin in the absence or presence of the indicated
concentrations of SRIF. Data represent the mean ± range from two
independent experiments each assayed in triplicate and are expressed as
a percentage of forskolin-stimulated membrane adenylyl cyclase activity
in the absence of SRIF. Bottom panel shows maximal
inhibition by SRIF in control membranes and in membranes pretreated
with the indicated concentrations of SRIF. Data represent mean ± range from two independent experiments. Cyclase inhibition was reduced
from 40.5 to 8.7% by pretreatment with maximal concentrations of SRIF
and half-maximal desensitization was produced by 2 nM
peptide.
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Internalization of the Sst1 Receptor--
To ascertain whether
agonist binding induced the internalization of the ligand-receptor
complex, cells were incubated for 4 h at 4 °C with
[125I-Tyr11]SRIF to occupy cell surface
receptors, washed to remove unbound peptide, and then warmed to
37 °C for varying times (Fig. 4,
top panel). Internalization of the receptor-bound ligand
occurred slowly and only 30% of the complex was internalized even
after a 60-min incubation at 37 °C. The rate of internalization was fit to a first-order rate equation and the half-time for endocytosis of
the receptor-ligand complex was calculated to be 180 min.

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Fig. 4.
Internalization of receptor-bound
[125I-Tyr11]SRIF. Top panel,
CHO-R1 cells were incubated for 4 h at 4 °C with
[125I-Tyr11]SRIF (100,000 cpm/ml) in the
absence and presence of 100 nM SRIF, rapidly washed, and
then warmed to 37 °C. At the times shown replicate dishes were
chilled, rinsed, and then incubated for 5 min at 4 °C with acetic
acid-buffered saline to release surface bound ligand. Following removal
of the acid wash, the cells were dissolved in 0.1 N NaOH.
The radioactivity in both the acid wash ( ), representing
surface-bound ligand, and the cell lysates ( ), representing
internalized ligand, were then measured. Bottom panel,
CHO-R1 cells were incubated at 37 °C for the times shown with
[125I-Tyr11]SRIF (100,000 cpm/ml) in the
absence and presence of 100 nM SRIF. After the binding
incubation, the cells were rapidly washed to remove unbound ligand and
then subjected to the acetic acid buffer treatment described above. For
both panels, specific binding was calculated as the difference between
the amount of radioligand bound in the absence and presence of 100 nM SRIF and the data represent specifically bound
radioligand in triplicate wells of a representative experiment.
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Consistent with the slow rate of internalization of the sst1
receptor-ligand complex observed in temperature-jump experiments (Fig.
4, top panel), analysis of the distribution of
receptor-bound ligand during continuous incubation at 37 °C showed
that only 15% of the bound ligand was present intracellularly at
steady state (Fig. 4, bottom panel).
Agonist-stimulated Phosphorylation of the Sst1 Receptor--
We
previously showed that the R1-201 antibody specifically recognizes the
sst1 receptor (17). Furthermore, photoaffinity labeling and
immunoprecipitation experiments using CHO-R1 cells demonstrated that
the sst1 receptor migrates as a broad 60-kDa band on SDS-PAGE (17). We
next wanted to determine whether SRIF was able to stimulate the
incorporation of 32PO4 into the sst1 receptor
protein. To ensure that the R1-201 antibody was equally capable of
recognizing the sst1 receptor before and after SRIF stimulation, CHO-R1
cells were incubated with or without 100 nM SRIF for 15 min
and the solubilized receptors were subsequently immunoprecipitated with
antibody covalently coupled to Sepharose. Analysis of the
immunoprecipitated proteins by SDS-PAGE and immunoblotting with
receptor antibody showed that SRIF treatment did not alter the
immunoprecipitation efficiency of sst1 (data not shown).
To directly assess agonist-stimulated receptor phosphorylation, CHO-R1
cells were labeled with [32P]orthophosphate and incubated
in the absence or presence of 100 nM SRIF for 15 min. After
detergent solubilization, the receptor was purified by lectin affinity
chromatography followed by immunoprecipitation with receptor antiserum
and then analyzed by SDS-PAGE and autoradiography. A low level of basal
phosphorylation was observed in the 60-kDa broad receptor band as well
as in a narrower 45-kDa band (Fig. 5,
left panel). The 45-kDa band probably represents a
nonspecific contaminant because its intensity relative to the 60-kDa
band varied markedly between experiments and because SRIF treatment did
not affect 32P incorporation into the 45-kDa protein. In
contrast, treatment of cells with 100 nM SRIF increased the
amount of 32P incorporated the 60-kDa sst1 receptor protein
2.2 ± 0.4-fold over basal (n = 5). Therefore,
exposure to SRIF increases phosphorylation of the previously identified
60-kDa sst1 receptor protein within 15 min.

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Fig. 5.
The effect of SRIF on sst1 receptor
phosphorylation. Left panel,
32PO4-labeled CHO-R1 cells were incubated in
the absence or presence of 100 nM SRIF for 15 min.
Following detergent solubilization and incubation with WGA-agarose,
adsorbed glycoproteins were eluted at 37 °C for 30 min with 3 mM TACT and the receptor was then immunoprecipitated with
receptor antiserum (final dilution of 1:200) as described under
"Experimental Procedures." Right panel,
32PO4-labeled CHO-R1 cells were incubated in
the absence or presence of 100 nM SRIF for 15 min.
Following detergent solubilization and adsorption to WGA-agarose, the
adsorbed proteins were deglycosylated by incubating the beads overnight
at 37 °C with 10 units/ml PNGase F. The proteins released from
WGA-agarose by deglycosylation were subjected to immunoprecipitation
with receptor antiserum (1:200) in the absence or presence of 1 µM antigen peptide (Ag). In both panels,
immunoprecipitated proteins were analyzed by SDS-PAGE and
autoradiography.
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We next wanted to determine the time course and dose dependence of
receptor phosphorylation. Because receptor phosphorylation was
difficult to quantitate in the broad 60-kDa band we attempted to
sharpen the migration pattern of the receptor on SDS-PAGE by removing
the attached carbohydrate. 32PO4-Labeled CHO-R1
cells were incubated in the absence or presence of 100 nM
SRIF for 15 min. Following detergent solubilization and adsorption to
WGA-agarose, the adsorbed receptor was incubated with PNGase F. The
proteins released from the WGA-agarose by deglycosylation were then
immunoprecipitated with receptor antiserum and analyzed by SDS-PAGE and
autoradiography (Fig. 5, right panel). Two
32P-labeled bands were observed in control cells after
receptor deglycosylation, migrating at 34 and 38 kDa. SRIF treatment
led to the appearance of a third, strongly phosphorylated, band at 43 kDa (Fig. 5, right panel). None of the deglycosylated
32P-labeled proteins were immunoprecipitated by receptor
antibody in the presence of 1 µM antigen peptide,
indicating that they represented different forms of the sst1 receptor
(Fig. 5, right panel). In six independent experiments, a
15-min pretreatment with 100 nM SRIF led to a 4.7 ± 1.5-fold increase in the phosphorylation of the 43-kDa receptor band, a
sufficiently large increase to permit ready quantitation (see below).
The multiple forms of the deglycosylated sst1 receptor could result
from different phosphorylation states of the receptor protein or from
other covalent modifications. Therefore we tested the effect of
alkaline phosphatase treatment on receptor mobility (Fig.
6, top panel). CHO-R1 cells
were incubated in the absence or presence of SRIF for 15 min. Following
detergent solubilization, partial purification by lectin chromatography
and deglycosylation by PNGase F, the sst1 receptor was
immunoprecipitated with receptor antiserum covalently coupled to
protein A-Sepharose. Replicate aliquots were then incubated in the
absence and presence of bacterial alkaline phosphatase (Fig. 6). As
observed in the 32PO4 incorporation experiment
shown in Fig. 5, immunoblots also showed that the deglycosylated sst1
receptor from nonpretreated cells migrates as a doublet at 34 and 38 kDa (Fig. 6, top panel). SRIF pretreatment led to the
appearance of a new immunoreactive band at 43 kDa. Moreover, alkaline
phosphatase digestion collapsed all receptor bands to a single species
at 34 kDa (Fig. 6, top panel). These results demonstrate
that both the 38- and 43-kDa bands represent phosphorylated forms of
the sst1 receptor protein, with the 43-kDa form being specifically
increased by SRIF treatment. Furthermore, the R1-201 antibody is able
to recognize the sst1 receptor in its different phosphorylated
states.

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Fig. 6.
Immunoblot analysis of phosphorylated and
dephosphorylated sst1 receptors. Panels A and
B, CHO-R1 cells were incubated in the absence or presence of
100 nM SRIF for 15 min. Following detergent solubilization,
partial purification by lectin chromatography, and deglycosylation by
PNGase F, the released receptor was immunoprecipitated with sst1
receptor antiserum covalently coupled to protein A-Sepharose. In
panel A, the immunoprecipitated proteins were solubilized in
sample buffer and directly subjected to SDS-PAGE. In panel
B, the immunoprecipitated proteins were incubated with bacterial
alkaline phosphatase for 60 min at 37 °C prior to solubilization in
sample buffer and SDS-PAGE. Panels C and D,
CHO-R1 cells were incubated in the absence or presence of 100 nM SRIF for 15 min as above. Following detergent
solubilization and adsorption to WGA-agarose, the adsorbed proteins
were incubated overnight at 37 °C with either PNGase F (Panel
C) or Endo H (Panel D). Proteins remaining adsorbed to
the WGA-agarose were then solubilized in sample buffer whereas the
proteins released from the agarose by deglycosylation were
trichloroacetic acid precipitated prior to solubilization in sample
buffer. Proteins were resolved by SDS-PAGE, electrophoretically
transferred to a PVDF membrane, and immunoblotted with sst1 receptor
antiserum (final dilution 1:5000).
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The results in Fig. 6, top panel, show that not all sst1
receptors are phosphorylated after SRIF treatment; a substantial fraction of the receptors from SRIF-treated cells continue to migrate
at 34 kDa. We therefore tested whether phosphorylation resistant sst1
receptors consisted of immature, intracellular proteins. Receptors in
the ER will contain core, high mannose carbohydrates that are sensitive
to digestion with the enzyme Endo H. In contrast, receptors that have
successfully traveled through the ER and cis-Golgi will possess mature,
complex carbohydrates that would be Endo H-resistant. Mature
carbohydrates would be expected either if the sst1 receptor was on the
cell surface or if it was sequestered in a post-Golgi intracellular
compartment such as endosomes.
CHO-R1 cells were incubated in the absence or presence of 100 nM SRIF for 15 min as before. Following detergent
solubilization and adsorption to WGA-agarose, the adsorbed proteins
were incubated with either PNGase F or Endo H (Fig. 6, bottom
panel). Most of the receptor was dissociated from the WGA-agarose
by the PNGase F incubation and the same pattern of receptor bands was
observed both without and with SRIF treatment as above (Fig. 6,
bottom left panel). In contrast, Endo H treatment released
only a small fraction of the sst1 receptors from WGA-agarose (Fig. 6,
bottom right panel) indicating that a relatively small
portion of the receptors are in the ER. However, those sst1 receptors
which were deglycosylated by Endo H were not affected by SRIF
pretreatment; Endo H-digested receptor from both control cells and from
SRIF-treated cells migrated at 34 kDa. Thus, the high mannose
containing precursor form of the sst1 receptor in the ER is resistant
to SRIF-stimulated phosphorylation, as would be expected from its
inaccessibility to extracellular ligand. These results indicate that
intracellular compartmentation contributes to the resistance of a
portion of the sst1 receptor population to SRIF-stimulated phosphorylation.
Time Course and Dose Dependence of Sst1 Receptor
Phosphorylation--
If sst1 receptor phosphorylation plays a role in
agonist-stimulated desensitization or internalization, phosphorylation
should be increased within the time frame of one of these regulatory events. To test this hypothesis, 32PO4-labeled
CHO-R1 cells were incubated with 100 nM SRIF for various times. Following lectin chromatography and deglycosylation by PNGase F,
the sst1 receptor was immunoprecipitated with receptor antiserum and
analyzed by SDS-PAGE and autoradiography (Fig.
7). Stimulation of sst1 phosphorylation
reached 90% of maximum after a 2-min incubation, was complete by 5 min, and was then maintained for at least 15 min (Fig. 7).

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Fig. 7.
Time course for SRIF stimulation of sst1
receptor phosphorylation. 32PO4-Labeled
CHO-R1 cells were incubated with 100 nM SRIF for different
times. Following detergent solubilization, partial purification by
lectin chromatography, and deglycosylation by PNGase F, the sst1
receptor was immunoprecipitated with antiserum at a final dilution of
1:200. Immunoprecipitated proteins were subjected to SDS-PAGE and
analyzed by autoradiography (inset) and phosphorimaging
(graph). Plotted data represent the mean ± range for
the intensity of the 43-kDa band (arrow) from two
independent experiments
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Agonist stimulation of sst1 receptor phosphorylation was also
concentration dependent (Fig. 8). During
a 15-min incubation, phosphorylation of the sst1 receptor was
significantly elevated by 1 nM SRIF and was further
increased at higher doses up to a maximal effect with 100 nM peptide (Fig. 8). The EC50 for
SRIF-stimulated receptor phosphorylation was 1.3 ± 0.5 nM (n = 2). Interestingly, Fig. 8 also
shows that the 32P labeling of the 34-kDa band is reduced
at the same time as the incorporation into the 43-kDa band is
increased, indicating that increased phosphorylation of the 34-kDa band
gives rise to the 43-kDa form of the receptor. In six independent
experiments, a 15-min treatment with 100 nM SRIF decreased
32PO4 incorporation into the 34-kDa receptor
band by 36.7 ± 10.5% at the same time that it increased
32PO4 incorporation into the 43-kDa band
4.7 ± 1.5-fold. Overall, the results demonstrate that the
time course and concentration dependence of SRIF-stimulated sst1
receptor phosphorylation closely matches that for desensitization.

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Fig. 8.
Concentration dependence for SRIF stimulation
of sst1 receptor phosphorylation.
32PO4-Labeled CHO-R1 cells were incubated with
the indicated concentration of SRIF for 15 min. Following detergent
solubilization, partial purification by lectin chromatography, and
deglycosylation by PNGase F, the sst1 receptor was immunoprecipitated
with receptor antiserum. Immunoprecipitated proteins were subjected to
SDS-PAGE and analyzed by autoradiography (inset) and
phosphorimaging (graph). Plotted data represent mean ± range for the intensity of the 43-kDa band (arrow) from two
independent experiments. The fitted value for the EC50 was
1.3 ± 0.6 nM.
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Mechanisms Involved in Sst1 Receptor Phosphorylation--
To
assess the importance of sst1 receptor-Gi/o coupling for
SRIF-induced receptor phosphorylation, CHO-R1 cells were pretreated with 100 ng/ml pertussis toxin for 24 h. This treatment abolished SRIF inhibition of forskolin-stimulated adenylyl cyclase activity (data
not shown) as described previously (15). However, SRIF-induced phosphorylation of the sst1 receptor was unaffected by pertussis toxin
pretreatment (Fig. 9). Therefore,
functional interaction of the sst1 receptor with pertussis
toxin-sensitive G proteins is not necessary for agonist-induced
sst1 receptor phosphorylation.

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Fig. 9.
The effect of pertussis toxin on SRIF
stimulation of sst1 receptor phosphorylation. CHO-R1 cells were
incubated in the absence or presence of 100 ng/ml pertussis toxin
(PTX) for 24 h prior to and during cell labeling with
[32P]orthophosphate.
32PO4-Labeled cells were then incubated in the
absence or presence of 100 nM SRIF for 15 min. Following
detergent solubilization, partial purification by lectin
chromatography, and deglycosylation by PNGase F, the sst1 receptor
was immunoprecipitated and analyzed by SDS-PAGE and
autoradiography.
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We further evaluated the effect of second messenger-regulated protein
kinases on sst1 receptor phosphorylation. Either
32PO4-labeled CHO-R1 cells (Fig.
10, left panels) or
unlabeled cells (Fig. 10, right panel) were incubated for 15 min with either no addition (control), or the indicated agents followed
by sst1 receptor purification and analysis. Both SRIF and PMA increased
32P incorporation. However, whereas SRIF primarily
increased labeling of the 43-kDa receptor band, PMA stimulated
32P incorporation mainly into the 38-kDa band (Fig. 10,
left panel): the ratio of 32P in the 43:38-kDa
bands was 1.38 in SRIF-treated cells and 0.65 in PMA-treated cells.
Comparable results were observed by immunoblotting the sst1 receptor
from unlabeled cells (Fig. 10, right panel). In two
independent experiments PMA increased 32P incorporation
into the combined 38- plus 43-kDa bands 2.63 ± 0.08-fold
(mean ± range). In contrast, forskolin did not affect sst1
receptor phosphorylation (Fig. 10, left panel). Following forskolin treatment 32PO4 incorporation into
the combined 38- plus 43-kDa bands was 1.18 ± 0.11 (mean ± range, n = 2) times control.

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Fig. 10.
The effect of heterologous agents on sst1
receptor phosphorylation. Left panels,
32PO4-labeled cells were incubated for 15 min
with either no addition (Control), 100 nM SRIF,
200 nM PMA, 10 µM forskolin (Fsk),
or both 100 nM SRIF and 200 nM PMA, as
indicated. Following detergent solubilization, partial purification by
lectin chromatography and deglycosylation by PNGase F, sst1 receptors
were immunoprecipitated and analyzed by SDS-PAGE and autoradiography.
Right panel, unlabeled CHO-R1 cells were treated with SRIF,
PMA, and forskolin and the receptor was solubilized, adsorbed to
WGA-agarose and deglycosylated with PNGase F as described above. The
sst1 receptor was then immunoprecipitated with receptor antiserum
covalently coupled to protein A-Sepharose. Immunoprecipitated proteins
were subjected to SDS-PAGE and then immunoblotted with the R1-201
antiserum (final dilution 1:5000) as described under "Experimental
Procedures."
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Since SRIF and PMA both increased sst1 receptor phosphorylation, we
next determined whether their effects were additive. Interestingly, simultaneous addition of agonist and PMA produced the same effect as
agonist alone (Fig. 10, left bottom panel). The
phosphorylation of the 43-kDa band was preferentially increased by the
combined treatment with 1.20 ± 0.13 times as much 32P
incorporated into the 43-kDa band as into the 38-kDa band
(n = 2). Furthermore, 32P incorporation
into the combined 38- plus 43-kDa bands was stimulated 2.6-fold in
cells treated with both SRIF plus PMA compared with a 2.8-fold
stimulation with SRIF alone and a 2.6-fold stimulation with PMA alone.
Together, these results demonstrate that protein kinase A activation
does not affect sst1 receptor phosphorylation. In contrast, protein
kinase C and SRIF both stimulate the phosphorylation of sst1 leading to
the formation of different phosphorylated products which can be
distinguished by their electrophoretic mobility. These results suggest
that stimulation of sst1 receptor phosphorylation by SRIF and PMA
involves different kinases and target different phosphorylation sites.
Since combined treatment with SRIF and PMA produced the same effect as
SRIF alone, hormone-stimulated phosphorylation appears to prevent the
protein kinase C-catalyzed reaction.
Phosphoamino Acid Analysis of Phosphorylated Sst1--
To identify
the phosphorylated residues in the sst1 receptor, phosphoamino acid
analysis was carried out with receptor from cells incubated with either
100 nM SRIF or 200 nM PMA for 15 min. The
43-kDa form of the receptor produced by SRIF treatment and the combined
43- and 38-kDa forms produced by PMA treatment were subjected to acid
hydrolysis for 30 min and analyzed for the presence of different
phosphoamino acids (Fig. 11). With both
treatments, the most heavily labeled residue was phosphoserine,
although a small amount of radioactivity was also incorporated into
phosphothreonine. Therefore, SRIF- and PMA-stimulated sst1 receptor
phosphorylation occur primarily on serine residues and secondarily on
threonine.

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Fig. 11.
Phosphoamino acid analysis of the
phosphorylated forms of sst1 receptor.
32PO4-Labeled cells were incubated for 15 min
with either 100 nM SRIF or 200 nM PMA.
Following detergent solubilization, partial purification by lectin
chromatography, and deglycosylation by PNGase F, sst1 receptors were
immunoprecipitated and subjected to SDS-PAGE. Following electrophoretic
transfer of the resolved proteins to a PVDF membrane, the
phosphorylated receptor bands were located by autoradiography. The
portions of the PVDF membrane containing the 43-kDa band after SRIF
treatment, and both the 43- and 38-kDa bands after PMA treatment were
excised and incubated at 110 °C in 6 N HCl for 30 min.
The hydrolyzed samples were analyzed by two-dimensional thin layer
chromatography in the presence of unlabeled phosphoserine
(PS), phosphothreonine (PT), and phosphotyrosine
(PY) standards as described under "Experimental
Procedures." The figure shows a phosphorimage of the TLC plate. The
migration of the unlabeled standards was determined by staining with
ninhydrin and is shown by the circles.
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DISCUSSION |
We show for the first time that the sst1 somatostatin receptor
undergoes rapid, agonist-stimulated phosphorylation and that receptor
phosphorylation correlates with homologous desensitization but not with
receptor internalization. Our results indicate that receptor uncoupling
rather than receptor endocytosis is responsible for sst1
desensitization and suggest that receptor phosphorylation is involved
in the desensitization process.
Previous studies had suggested that the internalization of the sst1
receptor was species specific. Thus, hormone treatment produced
endocytosis of the rat sst1 receptor into intracellular vesicles within
minutes (35). In contrast, somatostatin did not stimulate the
internalization of human sst1 (36, 37). These results were surprising
in view of the few differences between the rat and human sst1 receptor
sequences. In fact, the intracellular domains of the rat (X61630) and
human (M81829) sst1 differ by only two amino acids: at the COOH
terminus the rat sst1 receptor contains the sequence ASRISTL whereas
the human receptor contains the sequence TSRITTL. Our observation (Fig.
4) that the rat sst1 receptor internalized as slowly in CHO-K1 cells as
did the human receptor (36) shows that the small sequence divergence
between these species does not significantly affect the rate of
receptor internalization when measured in the same host. The study of
rat sst1 by Roosterman et al. (35) differed experimentally
from ours in two ways. First, we used different parental cells for expression of rat sst1: Roosterman et al. (35) transfected
RIN1046-38 cells, a differentiated insulinoma cell line, whereas we
used CHO-K1 cells, an established model for studies of G
protein-coupled receptor regulation. Second, we studied the native
receptor whereas Roosterman et al. (35) used a receptor
containing an epitope tag on the C terminus (rat sst1-tag). At present,
we do not know whether the observed differences in the rate and extent
of hormone-induced internalization of rat sst1 in the two studies
result from differences in the internalization machinery between the
two host cells or from the presence of an epitope tag on the receptor
used by Roosterman et al. (35). Perhaps it is pertinent in
this regard that the endogenous, but unidentified, SRIF receptors in
another rat insulinoma cell line, RINm5F cells, were not rapidly
internalized following hormone binding (38). These studies emphasize
that it will be important to examine receptor processing in cells
expressing sst1 endogenously.
Pretreatment of cells with SRIF leads to desensitization of both the
rat and human sst1 receptor (35, 39). In HEK cells expressing rat
sst1-tag no effect was observed after a 10-min pretreatment with 1 µM SRIF but significant desensitization occurred after
preincubation for 30 and 120 min (35). However, the relationship between desensitization and receptor internalization was not examined in this cell line (35). In another study, the human sst1 receptor expressed in CHO-K1 cells was shown to desensitize after treatment with
SRIF for 60 min when little receptor internalization had occurred (36,
39). We show here that desensitization of the native rat sst1 receptor
proceeds extremely rapidly in CHO-K1 cells, with a half-time less than
2 min. This is well before significant receptor internalization occurs.
Thus, desensitization of sst1 results from receptor uncoupling rather
than receptor sequestration. This contrasts with the behavior of the
sst2A receptor, which undergoes desensitization and internalization at
similar rates (26)
The mechanisms mediating sst1 receptor desensitization are not known
but, as has been postulated for other G protein-coupled receptors
(23-25), may involve receptor phosphorylation. We show for the first
time that hormone binding stimulates the phosphorylation of the sst1
receptor. Moreover, we demonstrate that (a) agonist stimulation of sst1 receptor phosphorylation occurs concurrently with
receptor desensitization, and (b) the dose-response for
SRIF-stimulated receptor phosphorylation and SRIF-induced receptor
desensitization are indistinguishable. By analogy to results with other
members of the G protein-coupled receptor family, these observations
indicate that receptor phosphorylation is important in sst1 desensitization.
Our first evidence that hormone binding led to phosphorylation of
the sst1 receptor was based on increased incorporation of 32PO4 into immunoprecipitated receptor protein
(Fig. 5). However, in subsequent studies we observed that
phosphorylation correlated with a decrease in the mobility of the sst1
receptor on SDS-PAGE, and that this change in mobility was reversed by
alkaline phosphatase digestion, as would be expected for a
phosphorylation dependent effect. Although such mobility shifts are
often exploited in studies of kinase signaling cascades, they have been
used rarely in the study of G protein-coupled receptor phosphorylation
(40, 41). The ability to distinguish the phosphorylated receptor from
the unphosphorylated form on Western blots enabled us to determine the
fraction of receptors phosphorylated. Given the close correlation between receptor phosphorylation and desensitization, we were surprised
to find that less than half of the sst1 receptors were phosphorylated
following agonist stimulation. Since this partial phosphorylation was
associated with essentially complete desensitization, we surmised that
the unphosphorylated receptors were present in an inactive receptor
pool, i.e. not coupled to cyclase inhibition.
A previous report showing that a substantial portion of sst1 receptors
are localized intracellularly in unstimulated CHO cells (39) suggested
that the fraction of receptors resistant to phosphorylation may derive
from this cytoplasmic pool. To determine whether the phosphorylated and
phosphorylation resistant forms of sst1 were present in different
intracellular compartments, we exploited the specificity of the
carbohydrate selective endoglycosidase, Endo H. This enzyme can
differentiate between immature receptors in the ER which contain high
mannose carbohydrates and are sensitive to Endo H digestion and fully
processed receptors containing complex N-linked
oligosaccharides which are Endo H resistant (42). Our observation that
Endo H-sensitive receptors were not phosphorylated after SRIF exposure
demonstrated that only fully processed, mature receptors were
substrates for hormone-stimulated phosphorylation. Thus, different sst1
receptor pools were differentially sensitive to agonist-stimulated
phosphorylation. Unfortunately, because fully processed endosomal
receptor pools cannot be distinguished from plasma membrane receptors
by endoglycosidase digestion we do not yet know whether plasma membrane
sst1 represents the receptor population which is susceptible to
hormone-stimulated phosphorylation. Nonetheless, our results clearly
show that phosphorylation of a receptor subpopulation is sufficient to
cause complete desensitization of SRIF inhibition of adenylyl cyclase.
If this is true for other G protein-coupled receptors, it may help
explain why receptor phosphorylation has been so difficult to detect in
some instances. Our results suggest that the development of a mobility
shift assay with deglycosylated receptors may help overcome such
sensitivity problems.
Two types of kinases are known to phosphorylate G protein-coupled
receptors: second messenger-activated kinases and G protein-coupled receptor kinases. The latter preferentially phosphorylate
agonist-occupied, activated receptors and are independent of second
messenger formation. Forskolin did not affect sst1 receptor
phosphorylation despite the presence of protein kinase A consensus
sites in the intracellular regions of the receptor (43). However, both
agonist and the protein kinase C activator, PMA, stimulated sst1
receptor phosphorylation. Two observations indicated that these
phosphorylation reactions are catalyzed by different kinases. First,
the mobility of the phosphorylated sst1 receptor is different following
SRIF and PMA stimulation: SRIF increases 32PO4
incorporation into a 43-kDa band whereas PMA primarily stimulates labeling of a 38-kDa band (Fig. 10). This difference in mobility must
result from phosphorylation of the receptor at different sites, since
it disappears after alkaline phosphatase treatment, and therefore
indicates that SRIF and PMA stimulate kinases with different substrate
specificities. Second, SRIF-stimulated phosphorylation does not require
signal transduction. Although sst1 activation can weakly stimulate
PIP2 hydrolysis in CHO-K1 cells and thereby could
potentially increase PKC activity, this effect is prevented by
pertussis toxin treatment (44). However, since pertussis toxin did not
affect SRIF-induced sst1 phosphorylation, PKC cannot be involved in the
stimulation of receptor phosphorylation by agonist. Based on these
results, we conclude that the sst1 phosphorylation produced by SRIF and
PMA must involve different kinases and must generate different
products. Interestingly, simultaneous treatment with both SRIF and PMA
produced the same effect as SRIF alone, indicating that agonist may
prevent receptor phosphorylation by heterologous kinases.
The inability of pertussis toxin to block SRIF-stimulated sst1
phosphorylation also suggests that agonist-stimulated phosphorylation is independent of receptor-G protein coupling, a characteristic of G
protein-coupled receptor kinase-catalyzed receptor phosphorylation. Although it remains possible that a second messenger cascade which is
activated by pertussis toxin-insensitive G proteins mediates SRIF
stimulation of sst1 receptor phosphorylation, this seems unlikely for
two reasons. First, agonist binding to the sst1 receptor is completely
resistant to GTP inhibition after pertussis toxin treatment of CHO-K1
cells (17). Second, all sst1-mediated signaling in CHO-K1 cells,
including cyclase inhibition, inositol 1,4,5-trisphosphate formation,
tyrosine phosphatase stimulation, and mitogen-activated protein
kinase activation are blocked by pertussis toxin (15, 20, 44).
Together, these observations provide strong support for the conclusion
that the sst1 receptor is coupled exclusively to pertussis
toxin-sensitive G proteins in this cell line. Therefore, the inability
of pertussis toxin to affect SRIF-stimulated sst1 receptor
phosphorylation demonstrates that this phosphorylation is independent
of receptor-G protein coupling and rules out the involvement of second
messenger-activated kinases.
As described here for the sst1 receptor, we had previously shown that
the sst2A receptor subtype is also phosphorylated independently by PMA
and agonist stimulation but is unaffected by protein kinase A
activation (26, 27). Recent studies demonstrated that PKC stimulation
increases the rate of sst2A receptor internalization (27). In contrast,
PMA did not induce or inhibit the endocytosis of the rat sst1-tag
receptor in RIN1046-38 (35). Thus, the biological significance of the
PKC-mediated sst1 receptor phosphorylation remains to be elucidated.
As a first step toward identifying the sites of sst1 receptor
phosphorylation, we determined which amino acids were phosphorylated following agonist and PMA stimulation. Although a small amount of
labeled phosphothreonine was observed, phosphoserine was the predominantly labeled residue found under both conditions. Clearly identification of the sites phosphorylated upon hormone and PMA stimulation will be crucial for determining the functional consequences of sst1 receptor phosphorylation and for elucidating the relationship between receptor phosphorylation and desensitization.