Both Overlapping and Distinct Signaling Pathways for Somatostatin Receptor Subtypes SSTR1 and SSTR2 in Pituitary Cells*

(Received for publication, March 17, 1997, and in revised form, May 12, 1997)

Longchuan Chen Dagger , V. Danial Fitzpatrick §, Richard L. Vandlen § and Armen H. Tashjian Jr. Dagger par **

From the Dagger  Department of Molecular and Cellular Toxicology, Harvard School of Public Health, and the par  Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 and the § Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

To elucidate the signaling events mediated by specific somatostatin receptor (SSTR) subtypes, we expressed SSTR1 and SSTR2 individually in rat pituitary GH12C1 and F4C1 cells, which lack endogenous somatostatin receptors. In transfected GH12C1 cells, both SSTR1 and SSTR2 coupled to inhibition of Ca2+ influx and hyperpolarization of membrane potential via a pertussis toxin (PTx)-sensitive mechanism. These effects reflected modulation of ion channel activities which are important for regulation of hormone secretion. Somatostatin analogs MK678 and CH275 acted as subtype selective agonists as expected. In transfected F4C1 cells, both SSTR1 and SSTR2 mediated somatostatin-induced inhibition of adenylyl cyclase via a PTx-sensitive pathway. In addition, activation of SSTR2 in F4C1 cells, but not SSTR1, stimulated phospholipase C (PLC) activity and an increase in [Ca2+]i due to release of Ca2+ from intracellular stores. Unlike adenylyl cyclase inhibition, the PLC-mediated response was only partially sensitive to PTx. To determine the structural determinants in SSTR2 necessary for activation of PLC, we constructed chimeric receptors in which domains of SSTR2 were introduced into SSTR1. Chimeric receptors containing only the third intracellular loop, or all three intracellular loops from SSTR2, mediated inhibition of adenylyl cyclase, but failed to stimulate PLC activity as did wild-type SSTR2. Furthermore, the C-terminal tail of SSTR2 was not required for coupling to PLC. Thus, by expressing individual somatostatin receptor subtypes in pituitary cells, we have identified both overlapping and distinct signaling pathways for SSTR1 and SSTR2, and have shown that sequences other than simply the intracellular domains are required for SSTR2 to couple to the PLC signaling pathway.


INTRODUCTION

Somatostatin (SS)1 is a tetradecapeptide which has diverse physiological actions (1, 39). It was first isolated and identified as an inhibitor of growth hormone (GH) secretion from the anterior pituitary gland (2). It also inhibits secretion of insulin, glucagon, gastrin, and secretin (1, 39). In the central nervous system, SS acts as a neuromodulator, regulating neuronal firing and facilitating release of neurotransmitters such as dopamine, norepinephrine, and serotonin (3). SS acts by binding to specific membrane receptors (4). Five SS receptor (SSTR) subtypes have been cloned and they belong to a family of receptors which couple via G proteins to cellular effector systems (5-9). In the pituitary gland, mRNAs for all five SSTRs are present (10). In the past, rat pituitary GH3 and GH4C1 cells have been used extensively to characterize endogenously expressed somatostatin receptors (11, 12). Somatostatin was shown to activate K+ channels and inhibit voltage-dependent Ca2+ channels (VDCC) leading to inhibition of Ca2+ influx (12). The secretion of GH is regulated mainly by changes in the cytosolic Ca2+ level; thus, the action of SS on ion channel activity is critically relevant to the physiological role of SS as an inhibitor of secretion. Since both SSTR1 and SSTR2 are present in GH3 and GH4C1 cells (13), the functional importance of each subtype is not known. Expression of a single SSTR subtype in heterologous cells such as COS and CHO cells does not allow studies of coupling of receptors to those specific ion channels that are characteristic of pituitary cells. In the first part of this report, we describe the expression of either SSTR1 or SSTR2 individually in pituitary GH12C1 cells, which do not contain endogenous SSTRs, to study their independent roles in regulation of membrane ion channel activity.

Expression of SSTR1 and SSTR2 in heterologous systems such as COS and CHO cells has given inconsistent results as to whether both subtypes couple to inhibition of adenylyl cyclase (14-16). It is possible that coupling of SSTRs to a particular G protein is affected by the specific cellular environment in which the receptors are expressed. Therefore, it is essential to study SSTR signaling pathways in a cell type in which SSTRs are expressed physiologically. We describe in this report that SSTR1 and SSTR2 have both overlapping and distinct patterns of signaling in pituitary cells. In addition, we examined the structural basis for the difference in signaling using a chimeric receptor approach.


EXPERIMENTAL PROCEDURES

Materials

Pertussis toxin was purchased from List Biologicals (Campbell, CA). MK678 was obtained from Merck Laboratories (West Point, PA). Des-AA1,2,5-[D-Trp8,IAmp9]SS (CH275) was synthesized by Drs. Carl Heoger and Jean Rivier at Salk Institute. myo-[3H]Inositol (80-120 Ci/mmol) and [125I-Tyr11]SS (2000 Ci/mmol) were from Amersham Inc. (Arlington Heights, IL). Cyclic AMP radioimmunoassay kits were purchased from NEN Life Products Inc. (Boston, MA). Fura-2/AM and bisoxonol were purchased from Molecular Probes (Eugene, OR). Lipofectin was purchased from Life Technologies, Inc. (Grand Island, NY). Muta-gene kits and AG1-8X Dowex columns were from Bio-Rad. Somatostatin and other reagents were from Sigma.

Cell Culture

GH12C1 and F4C1 are clonal pituitary cell strains established from rat pituitary tumors (17). Both strains lack expression of endogenous SSTRs (4). Cells were grown in Ham's F-10 nutrient mixture supplemented with 15% horse serum and 2.5% fetal bovine serum (F10+) at 37 °C in a humidified atmosphere of 5% CO2, 95% air.

Transient and Stable Transfection

The cDNAs for SSTR1 from either the mouse (18) or rat (6) were used in this study, and no differences in SS binding and receptor-mediated signaling were detected. The cDNA for the mouse SSTR2A isoform was used throughout this study (referred to as SSTR2). SSTR1 and SSTR2 cDNAs cloned in the pcDNA3 vector (Invitrogen, Carlsbad, CA) were transfected into GH12C1 and F4C1 cells by the Lipofectin method according to the supplier's protocol, or by electroporation with a Bio-Rad electroporator. Briefly, 106 cells were suspended in 0.4 ml of F10+ medium containing 16 µg of plasmid DNA, and electroporation was performed at 260 V and 960 microfarads capacitance. In cotransfection experiments, 8 µg of human parathyroid hormone receptor cDNA and 8 µg of SSTR cDNA were used. For stable transfection, cells were selected in growth medium containing 500 µg/ml G418 beginning 48 h after transfection. After several weeks of culture in selection medium, single clones were isolated and screened using a radioligand binding assay for SSTRs (see below). Reverse transcriptase polymerase chain reaction using a pair of primers from the vector and coding region, and solution RNase protection assay using RNAs from different clones were used to confirm the expression of only the specific transfected SSTR isoform.2

Somatostatin Radioligand Binding Assay

Cells were plated and grown in F10+ medium in 24-well dishes for at least 24 h. Cells were incubated with minimal essential medium containing various concentrations (30 pM to 1 nM) of [125I-Tyr11]SS, 0.1% bovine serum albumin, 0.1% bacitracin, and 2 µg/ml aprotinin for 2 h at 37 °C. The dishes were then washed rapidly 3 times with ice-cold phosphate-buffered saline. Cells were lysed in 0.1 N NaOH and the lysates counted in a gamma -counter. Nonspecific binding was determined in the presence of 1 µM SS.

Spectrofluorometric Measurement of Cytosolic Free Calcium Concentration ([Ca2+]i) and Membrane Potential (Vm)

For [Ca2+]i measurement, cells were harvested using a HEPES-buffered salt solution (HBSS, 118 mM NaCl, 4.6 mM) KCl, 10 mM D-glucose, 20 mM HEPES, pH 7.4) containing 0.02% EDTA and then resuspended in HBSS containing 1 mM CaCl2. Cells were loaded using 1 µM fura-2/acetoxymethyl ester (fura-2/AM) for 30 min at 37 °C. Cells were then washed 3 times in HBSS containing 1 mM CaCl2. Fluorescence was measured using a Spex Fluorolog FIIIA spectrofluorometer at excitation wavelength of 342 nm and emission wavelength of 492 nm. The excitation and emission slit widths were 5 nm. [Ca2+]i was calibrated from the fluorescence signal as described previously (19). Changes in Vm were monitored using an anionic fluorescent dye bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (bisoxonol) (12). Cells were harvested as described above. About 5 × 106 cells were used for each run in the presence of 40 nM bisoxonol. Fluorescence was measured at excitation wavelength of 540 nm and emission wavelength of 580 nm. The excitation and emission slit widths were 5 and 10 nm, respectively.

Electrophysiological Measurements

The whole cell voltage clamp technique (20) was used to measure activity of VDCC in GH12C1 cells. The micropipet solution contained: 110 mM CsCl, 20 mM tetraethylammonium-Cl, 5 mM MgCl2, 5 mM Na2ATP, 4 mM BAPTA, 10 mM HEPES, pH 7.2, adjusted with Tris. The extracellular solution contained: 130 mM tetraethylammonium-Cl, 10 mM BaCl2, 10 mM D-glucose, 10 mM HEPES, and 100 nM tetrodotoxin (to block voltage-dependent Na+ channels). Ba2+ was used as the cation charge carrier because Ba2+ minimizes Ca2+ current rundown and increases the amplitude of the Ca2+ channel current. Only cells with total capacitance of 15-45 picofarads were used for recording. The chamber was perfused continuously during the experiment at a flow rate of 2 ml/min. All experiments were performed at room temperature.

Measurement of cAMP

F4C1 cells were plated in 24-well dishes after transient transfection. After 48 h, cells were incubated with test reagents in the presence of 0.2 mM isobutylmethylxanthine for 15 min at 37 °C. Intracellular cAMP was extracted with 50 mM HCl and quantitated by radioimmunoassay (21).

Measurement of Total Inositol Polyphosphates (IPs)

Twenty-four h after transfection, F4C1 cells were split into 24-well dishes at a density of 2 × 105 cells/well and incubated in F10+ medium containing [3H]inositol (2 µCi/ml) for 20 h. Cells were then washed with 0.5 ml of assay medium (20 mM HEPES-buffered minimal essential medium without sodium bicarbonate) with 5 mM LiCl for 10 min and then incubated with 0.5 ml of assay medium containing 5 mM LiCl and somatostatin for 1 h. Cold (4 °C) formic acid (0.75 ml, 20 mM) was added to each well to lyse the cells. IP fractions were separated by AG1-8X Dowex column chromatography (22). Inositol polyphosphates were eluted with 2 M ammonium formate, 20 mM formic acid and counted in a scintillation counter.

Construction of Chimeric SSTR1/SSTR2 Receptors

Chimeric receptors were constructed from parental mSSTR1 and mSSTR2 by the polymerase chain reaction and Kunkel's method (23). The junctions for IC3 in CH1 and CH2 (see "Results") were Leu233 and Val276. In CH2, the residues in IC1 and IC2 of SSTR1 were mutated one by one to the corresponding residues of SSTR2. In CH3, the junction is Val309. DNA sequencing was performed by the dideoxy method (24) to verify the constructs.


RESULTS

SSTR1 and SSTR2 Each Coupled to Inhibition of Ca2+ Influx through VDCC and Hyperpolarization of Membrane Potential in GH12C1 Cells

In control untransfected GH12C1 cells or GH12C1 cells transfected with the pCDNA3 vector alone, no specific high affinity receptors for SS were detected by radioligand binding assays, and SS had no effect on [Ca2+]i (Fig. 1, top panel) or membrane potential (see below). Several stable clones of GH12C1 expressing rat SSTR1 were obtained and a single clone expressing 4,600 receptors/cell was used for further study. To obtain stable clones expressing SSTR2, more than 200 clones were screened, and no clone with >1,000 receptors/cell was obtained. However, several clones expressed the transfected SSTR2 mRNA, and one of them was used in subsequent functional experiments. In those GH12C1 clones expressing either SSTR1 or SSTR2, SS caused a decrease in [Ca2+]i (Fig. 1, middle panels). Pretreatment of the cells with PTx completely abolished the decreases in [Ca2+]i mediated by either SSTR1 or SSTR2 (Fig. 1, bottom panels).


Fig. 1. Effect of somatostatin on [Ca2+]i in control GH12C1 and transfected GH12C1 cells stably expressing either SSTR1 or SSTR2. Cytosolic free Ca2+ concentration ([Ca2+]i) was measured using the calcium probe fura-2/AM as described under "Experimental Procedures." After washing, the cells were suspended in HBSS buffer containing 1 mM CaCl2 during the measurement. The traces are representative results of at least six independent experiments for each type. Top, control GH12C1 cells transfected with vector alone; middle left, SSTR1-expressing clone; middle right, SSTR2-expressing clone; bottom, cells were preincubated with pertussis toxin (180 ng/ml) for 18 h. Arrows mark the addition of 1 µM SS; however, the effect of SS can be observed at concentrations as low as 1 nM (data not shown).
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The decreases in [Ca2+]i mediated by SSTR1 and SSTR2 (Fig. 2, top panel) were abolished by either chelating extracellular Ca2+ with EGTA (Fig. 2, middle panels) or by incubating the cells with the L-type Ca2+ channel blocker nifedipine (Fig. 2, bottom panels). Thus, the decreases in [Ca2+]i induced by SS acting via SSTR1 and SSTR2 were mediated by inhibition of Ca2+ influx through VDCC. To verify this conclusion, the action of SS on Ca2+ channel activity was studied directly using the whole cell voltage clamp technique. Consistent with the effects of SS on [Ca2+]i, the inward Ca2+ currents were inhibited acutely by SS in cells expressing either SSTR1 (Fig. 3B) or SSTR2 (Fig. 3C). The inhibition was reversed by washing out SS. No inhibition by SS was observed in GH12C1 cells transfected with vector alone (Fig. 3A).


Fig. 2. In GH12C1 cells stably expressing either SSTR1 or SSTR2, SS caused inhibition of Ca2+ influx through L-type voltage dependent Ca2+ channels. Left panels, SSTR1; right panels, SSTR2. Top, addition of 1 µM SS alone; middle, addition of 1.5 mM EGTA before 1 µM SS; bottom, addition of 10 µM nifedipine before 1 µM SS. The SS-induced decrease in [Ca2+]i was completely abolished by either EGTA or nifedipine. The traces are representative of at least three independent experiments.
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Fig. 3. Effects of SS on Ca2+ currents in GH12C1 cells. Whole cell Ca2+ currents were recorded under voltage-clamp conditions by depolarizing pulses from -80 mV to -10 mV. Representative traces are shown. A, lack of effect of SS in control GH12C1 cells transfected with vector alone; B, effect of SS in GH12C1 cells expressing SSTR1; C, effect of SS in GH12C1 cells expressing SSTR2. Control indicates current before addition of SS; SS indicates currents during superfusion of the cells with 1 µM SS; and Washout indicates currents after removal of SS.
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We next examined the ligand specificity of the action of SS on [Ca2+]i using receptor subtype selective SS analogs. MK678, which binds preferably to SSTR2 (18), had no effect on [Ca2+]i in cells expressing only SSTR1, but it induced a decrease in [Ca2+]i in cells expressing SSTR2 (Fig. 4). A new SS analog CH275, reported to bind with high affinity only to SSTR1 (25), induced a decrease in [Ca2+]i in cells expressing SSTR1 but not in cells expressing SSTR2 (Fig. 4). These results are the first demonstration that CH275 acts as an agonist to activate SSTR1 selectively.


Fig. 4. Subtype selective SS analogs induced [Ca2+]i responses in appropriate GH12C1 cell clones. [Ca2+]i was measured in GH12C1 cells stably expressing SSTR1 (left) or SSTR2 (right). Arrows indicate application of 100 nM MK678 or CH275. MK678 or CH275 caused no changes in [Ca2+]i in control GH12C1 cells lacking SSTRs (data not shown). Each trace is representative of at least three independent experiments.
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Bisoxonol fluorescence has been used to measure changes in membrane potential in GH4C1 cells, and hyperpolarization of Vm induced by SS in GH4C1 cells was caused by activation of K+ channels (12). Using this technique, SS caused a decrease in bisoxonol fluorescence in GH12C1 cells expressing either SSTR1 or SSTR2 (Fig. 5, middle panels), indicating that activation of each receptor subtype caused hyperpolarization of membrane potential. These responses were inhibited completely by preincubation with PTx (Fig. 5, bottom panels). Thus, both SSTR1 and SSTR2 mediate modulation of ion channel activity in GH12C1 cells, via a PTx-sensitive G protein, probably Go or Gi.


Fig. 5. Both SSTR1 and SSTR2 mediated membrane hyperpolarization in GH12C1 cells. Changes in membrane potential in GH12C1 cells were monitored using bisoxonol as described under "Experimental Procedures." Delta F is the change in fluorescence in arbitrary units. Top, lack of effect of SS in control GH12C1 cells transfected with vector alone; middle left, SSTR1-expressing clone; middle right, SSTR2-expressing clone; bottom, cells were preincubated with pertussis toxin (180 ng/ml) for 18 h. Arrows mark the addition of 1 µM SS.
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Both SSTR1 and SSTR2 Mediated Inhibition of Adenylyl Cyclase Activity in F4C1 Cells via a Pertussis Toxin-sensitive Mechanism

We next examined the coupling of SSTR1 and SSTR2 to adenylyl cyclase activity in rat pituitary F4C1 cells, which also lack high affinity binding sites for SS. Stable clones of F4C1 cells expressing 17,200 rat SSTR1/cell or 5,400 SSTR2/cell (determined by saturation binding assays) were used for the study of signaling properties. Inhibition of adenylyl cyclase activity by SS was determined by measuring the decrease in forskolin (Fsk)-stimulated cAMP accumulation. SS had no effect on basal or forskolin-stimulated cAMP levels in wild-type F4C1 cells not transfected with SSTRs (Fig. 6, left panel). However, SS acting via either SSTR1 or SSTR2 inhibited forskolin-stimulated cAMP accumulation, and this process was reversed completely by pretreatment with pertussis toxin (Fig. 6, middle and right panels). These results demonstrate that both SSTR1 and SSTR2 can mediate inhibition of adenylyl cyclase activity via a PTx-sensitive G protein (possibly Gi/Go) in F4C1 cells. In GH12C1 clones expressing either SSTR1 or SSTR2, SS did not inhibit vasoactive intestinal peptide-stimulated cAMP accumulation, while in the same cells carbachol, acting via endogenous acetylcholine receptors, did inhibit the vasoactive intestinal peptide effect (data not shown).


Fig. 6. Effects of SS on forskolin-stimulated cAMP accumulation in control F4C1 cells transfected with vector alone and in clones stably expressing SSTR1 or SSTR2. C, basal cAMP level; F, 10 µM forskolin; F+S, 10 µM forskolin + 1 µM SS; PTx, cells were preincubated with pertussis toxin (180 ng/ml, 18 h). Bars and brackets represent the mean and S.D., respectively, of triplicate measurements in a single experiment. Two additional experiments yielded similar results.
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Only SSTR2 Coupled to the Inositol Lipid/[Ca2+]i Pathway in F4C1 Cells

Because several Gi-coupled receptors can activate more than one intracellular signaling pathway (26, 27), we examined the coupling of SSTR1 and SSTR2 to the inositol lipid pathway. In F4C1 cells stably expressing SSTR2, SS stimulated total inositol polyphosphate production about 5-fold over the basal level (Fig. 7). Stimulation was reduced about 35% by preincubation with PTx. In F4C1 cells expressing an even higher level of SSTR1, no stimulation of IP production was induced by SS (Fig. 7).


Fig. 7. Effects of SS on total inositol polyphosphate production in F4C1 cells expressing either SSTR1 or SSTR2. SS had no effect on IP production in cells transfected with vector alone (left panel) or cells expressing SSTR1 (middle panel). SS caused stimulation of IP production in cells expressing SSTR2 (right panel). C, basal level; S, in the presence of 1 µM SS; PTx, pretreated with pertussis toxin (180 ng/ml) for 18 h. Bars and brackets represent the mean and S.D., respectively, of triplicate measurements in a single experiment. Two additional experiments yielded similar results.
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The functional consequences of IP production were determined by measuring changes in [Ca2+]i. In control F4C1 cells or F4C1 cells expressing only SSTR1, SS had no effect on [Ca2+]i, whereas in cells expressing SSTR2, SS induced an acute increase in [Ca2+]i (Fig. 8, top). The increase in [Ca2+]i in cells expressing SSTR2 was not abolished by chelation of extracellular Ca2+ with EGTA (Fig. 8, middle panel). Preincubation of cells with 1 µM thapsigargin for 10 min, to deplete intracellular Ca2+ pools (28), abolished the increase of [Ca2+]i induced by SS (Fig. 8, bottom panel), supporting the conclusion that the increase in [Ca2+]i was due to release of Ca2+ from IP3-sensitive intracellular Ca2+ stores. These results demonstrate that SSTR1 and SSTR2 differ in their ability to couple to the inositol lipid/[Ca2+]i pathway.


Fig. 8. Effects of SS on [Ca2+]i in control F4C1 cells (top left), F4C1 cells expressing SSTR1 (top middle), or SSTR2 (top right). Effect of adding 1.5 mM EGTA (middle trace) and of pretreating the cells with 1 µM thapsigargin (Tg) for 10 min (bottom trace) on the subsequent SS response in cells expressing SSTR2 are shown. The SS concentration was 1 µM. Traces shown are representatives of at least three independent experiments.
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Chimeric SSTR1/SSTR2 Receptors

To identify the structural determinants for SSTR2-mediated inositol lipid signaling, we constructed a series of SSTR1 and SSTR2 chimeras in which specific domains of SSTR2 were introduced into the SSTR1 backbone. From sequence comparison, SSTR1 and SSTR2 differ in the intracellular domains, especially the third intracellular loop (IC3) and C-terminal tail. Because the intracellular loops, especially IC3, have been identified as sites of interaction with G proteins for several heptahelical receptors (29, 30), we replaced IC3 of SSTR1 with IC3 of SSTR2 in chimeric construct CH1 (Fig. 9). In CH2, the first, second, and third intracellular loops of SSTR1 were replaced with those of SSTR2. In another construct (CH3), the 7th transmembrane domain and the C-terminal tail of SSTR2 were replaced by the corresponding sequence from SSTR1 (Fig. 9). Each chimeric receptor was tested for its ability to express on the cell surface, to stimulate PLC activity, and to inhibit adenylyl cyclase activity in transient transfection assays. When transiently transfected into F4C1 cells, CH1, CH2, and CH3 expressed at levels comparable to wild-type SSTR1 and SSTR2 as determined by ligand binding experiments (Fig. 10). However, CH1 and CH2 did not couple to the inositol lipid pathway in response to SS as determined by the lack of rise in IP production. In F4C1 cells expressing CH3, SS induced formation of IPs as it did in cells expressing the intact SSTR2 (Fig. 11). Therefore, the three intracellular loops of SSTR2 are not sufficient for coupling to the inositol lipid pathway, and the C-terminal domain of SSTR2 is not required for this pathway. To exclude the possibility that CH1 and CH2 are functionally impaired to couple to any G protein, we studied their ability to inhibit adenylyl cyclase activity. To evaluate the action of SS on ligand-stimulated cAMP production, the human PTH receptor, a receptor known to couple to Gs (21), was transiently co-expressed with SSTR constructs in F4C1 cells. F4C1 cells lack functional PTH receptors, and PTH does not stimulate cAMP accumulation in untransfected F4C1 cells (data not shown). SS had no effect on PTH-stimulated cAMP accumulation in F4C1 cells transfected with only the human parathyroid hormone receptor. All three chimeric receptors as well as wild-type SSTR1 and SSTR2 mediated inhibition of PTH-stimulated cAMP accumulation by SS (Fig. 12). Thus, the lack of coupling to PLC in CH1 and CH2 was not due to global nonfunctionality of these constructs. The finding that replacement of all three intracellular loops of SSTR1 with those of SSTR2 was not sufficient to confer SSTR2-specific signaling suggests that additional domains of SSTR2, possibly the transmembrane helices, are involved in determining the specificity of coupling of SSTR2 to the inositol lipid signaling pathway.


Fig. 9. Diagram of chimeric SSTR1/SSTR2 receptors. The vertical bars represent putative transmembrane domains. The top and bottom portions represent the extra- and intracellular domains of each receptor. In chimeric receptors, the dark or light bands indicate the contribution by SSTR1 (dark) or SSTR2 (light). The junctions for IC3 in CH1 and CH2 were Leu233 and Val276, respectively. In CH2, the distinctive residues of SSTR1 in IC1 and IC2 were mutated to the corresponding residues of SSTR2. In CH3, the junction was at Val309.
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Fig. 10. Expression level of SSTR1/SSTR2 chimeras in F4C1 cells determined by ligand binding assay. Forty-eight hours after transfection, cells were incubated with minimal essential medium containing 30 pM [125I-Tyr11]SS, 0.1% bovine serum albumin, 0.1% bacitracin, 2 µg/ml aprotinin for 2 h at 37 °C. Each well was then rinsed rapidly three times with ice-cold phosphate-buffered saline. T, total binding; or N, nonspecific binding was determined in the absence or presence of 1 µM SS. Each bar represents the mean and the brackets represent the S.D. of triplicate measurements in a single experiment.
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Fig. 11. Effect of SS on IP formation in F4C1 cells transfected with various SSTR constructs. IP formation was determined as described under "Experimental Procedures." B, basal IP level; S, IP level in the presence of 1 µM SS. Each bar represents the mean and the brackets the S.D. of triplicate measurements in a single experiment. Two additional experiments yielded similar results.
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Fig. 12. Effect of SS on PTH-stimulated cAMP accumulation in F4C1 cells co-transfected with various SSTR constructs and human parathyroid hormone receptor cDNA. Intracellular cAMP levels were determined as described under "Experimental Procedures." B, basal cAMP level; P, cAMP level in the presence of 100 nM PTH; P+S, cAMP level in the presence of 100 nM PTH and 1 µM SS. Data are expressed as percentage of the PTH-stimulated cAMP level. Each bar gives the mean value and the brackets give the S.D. of three samples.
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DISCUSSION

SS exerts its physiological and pharmacological actions by binding to one or more subtypes of cell surface receptors. Different SSTR isoforms have overlapping but distinct expression patterns (16), raising the possibility that some of the diverse actions of SS are mediated by different receptor subtypes. Alternatively, it is possible that a single receptor subtype can couple to more than one effector system and that such coupling may vary among cell types. It has been reported that SSTR1 and SSTR2 do not couple to inhibition of adenylyl cyclase in CHO cells (14); however, it has also been shown that SSTR1 and SSTR2 can couple, via Gi, to inhibition of adenylyl cyclase in other cell types (15). One possible explanation for these differences is that the G protein and effector components differ between the cell types. In the present study, SS did not inhibit adenylyl cyclase activity stimulated by vasoactive intestinal peptide in GH12C1 clones expressing either SSTR1 or SSTR2, while SS modulated ion channel activity in these cells. Both SSTR1 and SSTR2 mediated inhibition of adenylyl cyclase activity in F4C1 cells. Although we cannot rule out the possibility that the lack of effect of SS on adenylyl cyclase in GH12C1 cells was due to a lower level of receptor expression, it is likely that SSTR coupling patterns to adenylyl cyclase depend on the specific cellular environment.

Fujii et al. (31) reported, that in rat insulinoma RINm5F cells, SSTR2, but not SSTR1, coupled to inhibition of VDCC, suggesting that there are subtype specific differences in regulation of ion channel activity. On the other hand, we have demonstrated that in GH12C1 cells, both SSTR1 and SSTR2 mediate inhibition of voltage-gated Ca2+ channels. These results again demonstrate that SSTR isoforms couple to specific signal transduction pathways depending on the specific type of cells in which they are expressed.

Different signaling through the same receptor can be transduced by different G proteins (27). Specifically, the alpha 1, beta 1, and gamma 3 subunits of Go have been shown to mediate inhibition of Ca2+ channels by SSTRs in GH3 cells (32). In a GH4C1 cell strain in which Go was knocked out by overexpressing antisense mRNA (33), the effect of SS on Ca2+ influx was no longer observed; however, the effect of SS on membrane potential was not altered in these knockout cells.3 Thus, membrane hyperpolarization which is due to activation of K+ channels is not dependent on Go and may be mediated by Gi subunits. It is likely that in GH12C1 cells, SSTR1 and SSTR2 couple to their cognate G proteins with similar specificity.

In human embryonic kidney 293 cells expressing SSTR2, we have reported that SS activates PLC and increases [Ca2+]i (38). Tomura et al. (34) reported, that in COS cells expressing SSTR2, SS stimulated PLC/[Ca2+]i, while in cells expressing SSTR1, SS had little or no effect. In the present report, we have shown that in pituitary F4C1 cells expressing about 5,400 SSTR2 receptors/cell, that SS clearly activates the PLC/[Ca2+]i pathway. Because this response was only partially blocked by PTx, it was mediated, at least in part, by a PTx-insensitive G protein, possibly Gq/11. SS induced increases in [Ca2+]i have also been observed in normal cells, including astrocytes (35) and intestinal smooth muscle cells (36). Therefore, coupling to multiple pathways by SSTR2 cannot be attributed to an artifact of the transfected cell systems.

The intracellular domains of a receptor serve as contact sites for interacting with G proteins and, in many cases, determine the specificity of coupling (30). Therefore, it was somewhat unexpected that the introduction of the putative intracellular domains of SSTR2 into SSTR1 did not confer SSTR2-like PLC activation in response to SS under conditions in which the chimeric receptors were expressed as well as wild-type receptors. Thus, the specificity of G protein coupling by SSTR2 is not determined solely by its intracellular domains. Additional structural determinants, probably from the transmembrane domains, are needed to enable coupling to the inositol lipid signaling pathway. These findings are consistent with the concept that the transmembrane domains of a G protein-coupled receptor act in concert with the intracellular loops to affect the conformation of the contact regions for the G protein (37). In an attempt to define the transmembrane sequences required, we tested a series of SSTR1/SSTR2 chimeras in which both transmembrane and intracellular domains were swapped. Unfortunately, these chimeras all expressed poorly on the cell surface, making it impossible to study their signaling properties. Further studies, using chimeras with more subtle exchanges and adequate receptor expression will be necessary to identify the critical transmembrane and intracellular sequences needed to confer SSTR2-specific signaling.


FOOTNOTES

*   This work was supported in part by a research grant from the National Institute of Diabetes, Digestive and Kidney Diseases (DK11011), National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
**   To whom correspondence should be addressed: Dept. of Molecular and Cellular Toxicology, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. Tel.: 617-432-1177; Fax: 617-432-1780; E-mail: tashjian{at}hsph.harvard.edu.
   Current address: Dept. of Biology, Pharmacopeia, Inc., 3000 East Park Blvd., Cranbury, NJ 08512.
1   The abbreviations used are: SS, somatostatin; SSTR, somatostatin receptor; Vm, membrane potential; CH275, des-AA1,2,5-[D-Trp8,IAmp9] somatostatin; [Ca2+]i, cytosolic Ca2+ concentration; IP, inositol polyphosphate; PTx, pertussis toxin; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; VDCC, voltage-dependent Ca2+ channel; GH, growth hormone; CHO, Chinese hamster ovary; PTH, parathyroid hormone; IC3, intracellular loop 3.
2   J. Bruno and L. Chen, unpublished results.
3   L. Chen, unpublished data.

ACKNOWLEDGEMENTS

We thank Drs. Zhiling Xiong and Dr. Gary Strichartz (Harvard Medical School) for their help with the electrophysiological studies, Drs. Yun Xu and John Bruno (SUNY, Stony Brook) for RNA solution hybridization assays of various cell clones. We also thank Dr. Xiao-Jiang Li for rSSTR1 cDNA, Dr. Larry Suva for human parathyroid hormone receptor cDNA, Drs. Carl Hoeger and Jean Rivier (Salk Institute) for the SS analog CH275, and Jean Foley for help with the preparation of this manuscript.


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