Characterization of Intracellular Signaling Mediated by Human Somatostatin Receptor 5: Role of the DRY Motif and the Third Intracellular Loop

Abstract

Somatostatin (SST) exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5) linked to multiple cellular effectors. The receptor structural domains involved in these effects have been only partially elucidated. The aim of the study was to investigate the molecular determinants mediating the interaction of the human SST5 with intracellular signaling in the pituitary cell line GH3, focusing on the BBXXB domain in the third intracellular loop and the DRY motif in the second intracellular loop. We analyzed the effects of the SST5 agonist BIM23206 on cAMP accumulation, intracellular calcium, GH secretion, cell proliferation, and ERK1/2 phosphorylation in cells expressing either wild-type SST5 or mutant receptors, in particular the naturally occurring mutant R240W in the BBXXB domain and the D136A and R137A mutants in the DRY motif. We found that residues D136 and R137 were critical for SST5 signaling because their substitutions abolished all the intracellular responses. Conversely, third intracellular loop mutations resulted in receptor that inhibited intracellular cAMP levels similar to the wild-type (50 ± 9 vs. 53 ± 12% inhibition) but failed to mediate the other responses elicited by wild-type SST5, i.e. reduction of intracellular calcium levels as well as inhibition of ERK1/2. These events resulted in an absent inhibition of GH release and an impaired reduction of cell proliferation (38 ± 7 vs. 76 ± 6% inhibition in wild type, P < 0.05). These data indicate that different regions of SST5 are required for the activation of different signaling pathways.

Two regions are identified in the i2 and i3 loop of the human SST5 required for the generation of downstream effectors involved in the regulation of hormone secretion and cell proliferation.

Somatostatin (SST) is a widely distributed polypeptide that physiologically regulates several biological functions (1,2,3,4,5) that are mediated by five specific G protein-coupled receptors (GPCRs) termed SST1–5, coupled to multiple pertussis toxin-sensitive G-proteins (2,6,7). All five subtypes couple to adenylyl cyclase inhibition and some have also been found to reduce calcium entry by modulating L-type Ca²⁺ and K⁺ channels (8,9), all these events being involved in the inhibition of hormone release. The antimitotic effects of somatostatin are mediated mainly by SST2 and SST5 through partially different pathways. In fact, whereas the antimitotic effect of SST2 is mediated by tyrosine phosphatase activation (10), contradictory results were obtained for SST5 (11,12). Indeed, other signals, such as inhibition of ERK1/2, have been proposed to mediate the antiproliferative effect of SST5 (13,14).

The SST5 structural domains mediating these different effects are unknown. Although several structure-function studies indicate the second and third intracellular loop (i2 and i3) as the most critical regions for selectivity and efficiency of GPCR/G protein interactions, a consensus sequence of the binding interfaces between receptor and G proteins has not been defined (15). Because we recently demonstrated that the i3 loop of SST5 is an important mediator of β-arrestin/receptor interaction and receptor internalization by using the i3 loop mutants R240W, S242A, and T247A and the mutation of all these three residues (RST) (16), we decided to investigate the effects of i3 loop mutations on signal transduction. Indeed, these substitutions occur in the putative BBXXB consensus sequence (where B is a basic residue and X a nonbasic residue, Fig. 1A) in the C-terminal portion of the i3 loop, a domain that has been suggested to be involved in G protein activation (17,18,19,20,21).

Figure 1.

A, Schematic representation of wild-type and mutant SST5 receptors. The i2 and i3 loops are shown in detail. The localization of DRY motif and the BBXXB motif within SST5 sequence are indicated. Residues in the second intracellular loop mutated to Ala (Asp¹³⁶ and Arg¹³⁷) and residues of i3 loop mutated in the R240W and RST SST5 are indicated by arrows. B, Representative images of GH3 cells transiently transfected with SST5. Forty-eight hours after transfection, cells were fixed and examined by fluorescence microscopy. The figure shows wild-type (wt), D136A, and R137A SST5 intracellular distribution. Representative images from one of at least five individual experiments are shown. C, Representative Western blot of wt and mutated SST5 immunoprecipitated from cell membrane extracts of transiently transfected cells in basal conditions.

Another motif that plays a crucial role in regulating receptor conformation and G protein activation in a number of different GPCRs (22,23,24,25,26,27,28,29) is the highly conserved tripeptide Glu/Asp-Arg-Tyr (i.e. E/DRY motif) located between the third transmembrane domain and the i2 loop (Fig. 1A). In a subgroup of class A GPCRs, the DRY motif maintains the receptor in the ground state, and its mutations induce constitutive activity, whereas in other receptors, it is more directly involved in governing receptor conformation and G protein coupling/recognition, and its mutations impair agonist-induced receptor responses (reviewed in Ref. 30).

The aim of the study was to investigate the role of BBXXB and DRY motifs of human SST5 in the transduction of intracellular signals involved in the regulation of GH secretion and cell proliferation.

Materials and Methods

Materials

GH3 cells (ATCC CCL-82.1) and CHO-K1 cells were purchased from American Type Culture Collection (Manassas, VA). Ham F10 medium, MEM-α, fetal bovine serum (FBS), l-glutamine, penicillin, streptomycin, T₄ DNA ligase, AccuPrime proofreading polymerase were from Invitrogen (Carlsbad, CA). Restriction enzymes were from New England Biolabs (Beverly, MA). Transfection reagent Jet PEI was from Polyplus transfection (San Marcos, CA). cAMP Biotrack enzyme immunoassay and cell proliferation ELISA Biotrack system were from GE Healthcare (Buckinghamshire, UK), Rat GH enzyme immunoassay kit was from SPI bio (Montigny-le-Bretonneux, France). Fura-2 and FluoSave mounting medium were from Calbiochem (San Diego, CA). SSTR5(H-54) rabbit polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal antiphosho-p42/44 antibody and antitotal p42/44 antibody were from Cell Signaling Technologies (Danvers, MA). The superselective analog BIM23206, specific for SST5 subtype (31), was kindly provided by Biomeasure Inc./IPSEN (Milford, MA).

Plasmids and constructs

The wild-type or mutated SST5 receptors (R240W, RST) expression vector were obtained as previously reported (16). To generate the i2 loop mutants (Fig. 1A), point mutations were introduced into SST5 by PCR-based mutagenesis replacing D¹³⁶ and R¹³⁷ present in the highly conserved DRY motif with A and the cDNA was subcloned into the same expression vector. The primers used for the construction of mutant receptors are the following: D136A forward, 5′-CAGTCATGAGCGTGGCCCGCTACCTGGCAGT-3′; D136A reverse, 5′-ACTGCCAGGTAGCGGGCCACGCTCATGACTG-3′; R137A forward, 5′-TCATGAGCGTGGACGCCTACCTGGCAGTGGT-3′; R137A reverse, 5′-ACCACTGCCAGGTAGGCGTCCACGCTCATGA-3′.

The sequence of all constructs was verified by dideoxynucleotide sequencing.

To compare the results obtained with cells transfected with wild-type or mutant SST5, we used control cells transfected with the empty vector, which does not contain gene encoding proteins of interest.

Cell culture and transfection

Rat pituitary GH3 cells and CHO-K1 cells were grown in Ham F10 medium or MEM-α medium, respectively, supplemented with 10% FBS, 2 mm glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 C in a humidified atmosphere of 95% air-5% CO₂. Transient transfections of SST5 were performed using JetPEI according to the instruction of the manufacturer. Western blot analysis was performed in each experiment to control the expression level of SST5 in transiently transfected cells: SST5 was immunoprecipitated from cell membrane extracts of transiently transfected cells, as previously reported (16). Immunoblotting was performed using a 1:1000 dilution of the rabbit anti SST5 antibody and revealed by a chemiluminescent detection system.

cAMP measurement

To quantify the inhibition of forskolin-induced cAMP accumulation, GH3 and CHO-K1 cells transiently transfected for 48 h with wild-type or mutated SST5 were preincubated with 0.5 mm 3-isobutyl-1-methylxantine (IBMX) for 30 min and subsequently treated with 1 μm forskolin with or without increasing doses of BIM23206 for 60 min at 37 C. At the end of incubation, the medium was removed and intracellular cAMP was measured. Cells were lysed and assayed by enzymatic immunoassay according to the instruction of the manufacturer. Experiments were repeated at least three times and each determination was done in quintuple.

Measurement of the concentration of cytosolic free Ca²⁺

Cytosolic Ca²⁺ was measured by monitoring changes in the intensity of fura-2 fluorescence as previously described (32). Briefly, GH3 cells transiently transfected for 48 h with wild-type or mutated SST5 were resuspended at 1 × 10⁶ cells/ml in KRH incubation medium containing 125 mmol/liter NaCl, 5 mmol/liter KCl, 1.2 mmol/liter KH2PO4, 1.2 mmol/liter MgSO₄, 2 mmol/liter CaCl2, 25 mmol/liter HEPES-NaOH (pH 7.4), and 6 mmol/liter glucose. Cells were loaded with the fura-2 by incubation with 5 μmol/liter fura-2-acetoxymethylester for 30 min at 37 C. Cell suspension was inserted into a thermostatically controlled cuvette, maintained under continuous stirring, and stimulated with 10 nm TRH and 10 nm BIM23206. Fluorescence recordings were carried out in an LS5 spectrofluorimeter (PerkinElmer, Norwalk, CT) at 345 nm excitation and 490 nm emission, with slits of 5 and 10 nm, respectively. Intracellular free calcium concentrations ([Ca²⁺]i) was calculated according to the method of Grynkiewicz et al. (33). All values were corrected for changes in autofluorescence (32). Experiments were repeated at least five times.

GH secretion assay

GH3 cells were cultured in a 96-well plate (17,000 cells/well), transfected with wild-type or mutated receptors (R240W, RST, D136A, and R137A) for 48 h, and incubated with increasing concentrations of BIM23206 (0.1, 1, or 10 nm) for 3 h. The cells culture medium was used to measure GH by enzyme immunoassay according to the instruction of the manufacturer. All experiments were repeated at least three times and each determination was done in quintuple.

Proliferation assay

Cell proliferation was assessed by colorimetric measurement of 5-bromo-2′-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells according to the instruction of the manufacturer, as previously reported (34). Briefly, cells cultured in a 96-well plate (20,000 GH3 cells/well and 10,000 CHO-K1 cells/well) were transfected with the appropriate construct and starved for 24 h. Transfected cells were incubated with BIM23206 at 0.1, 1, or 10 nm for 48 h at 37 C and with BrdU for 2 h (CHO-K1 cells) or 24 h (GH3 cells) to allow BrdU incorporation in newly synthesized cellular DNA. Experiments were repeated at least three times and each determination was done in quintuple.

Immunoblotting analysis of p42/44

GH3 cells were transfected with wild-type or mutated SST5 for 48 h, serum starved for 24 h, and then stimulated with FBS 10% with or without BIM23206 for 10 min. The cells were then lysed in lysis buffer in the presence of protease inhibitors. Fifty micrograms of proteins from each sample were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose filter. To detect phosphorylated p42/44 proteins, 1:2000 dilution of antiphosho-p42/44 antibody and an antimouse horseradish peroxidase-linked antibody were used. The presence of total p42/44 was analyzed by stripping and reprobing with anti total p42/44 antibody (1:1000). Experiments were repeated at least five times. The resulting bands were evaluated with the image analysis program ImageJ (National Institutes of Health, Bethesda, MD).

Statistical analysis

The results are expressed as the mean ± se. A paired two-tailed Student’s t test was used to detect the significance between two series of data. P < 0.05 was accepted as statistically significant.

Results

Expression and cellular distribution of wild-type and mutant SST5 receptors

To investigate expression and subcellular localization of wild-type or mutated SST5, we analyzed transfected cells by fluorescence microscopy 48 h after transfection. As shown in Fig. 1B, wild-type SST5 appears to be localized at the plasma membrane of GH3 cells. To determine whether mutations of the DRY motif would reduce membrane expression, we evaluated the intracellular localization of the D136A and R137A mutant receptors. Fluorescence microscopy experiments showed that mutant receptors were correctly targeted to the plasma membrane (Fig. 1B). We also confirmed the expression and plasma membrane localization of R240W and RST mutants, as previously reported (16). In transiently transfected cells, the expression levels at the plasma membrane of the wild-type and mutated receptors in basal conditions were similar, as indicated by Western blot analysis of SST5 performed on plasma membrane cell extracts (Fig. 1C).

Effects of SST5 mutations on intracellular cAMP levels

Native GH3 cells did not express SST5, as previously demonstrated (16), and no changes in forskolin-induced cAMP accumulation were observed on BIM23206 stimulation in cells transfected with empty vector (GH3; Fig. 2A). On the contrary in cells transiently transfected with wild-type SST5, we found that BIM23206 dose-dependently inhibited forskolin-induced cAMP accumulation, with a maximal inhibition (53 ± 12%) at 1 nm BIM23206. This effect was biphasic, as already reported in human somatotropes (35). To investigate the effects of i2 and i3 loop mutations on SST5 functionality, we evaluated the inhibition of cAMP accumulation mediated by different mutant receptors. As shown in Fig. 2A, BIM23206 caused an inhibition of forskolin-stimulated cAMP accumulation in GH3 cells transfected with R240W or RST mutants similar to that found in cells with wild-type SST5 (50 ± 9 and 48 ± 8% inhibition, respectively). In contrast, the expression of D136A or R137A mutants resulted in complete loss of cAMP inhibition. These results suggest that an intact DRY motif, but not i3 loop, is essential for coupling with Gαi proteins and adenylyl cyclase inhibition.

Figure 2.

Effects of SST5 mutations on intracellular cAMP levels. A, GH3 cells transiently transfected with wild-type (WT) or mutated SST5 were tested with increasing concentration of BIM23206 for their ability to inhibit forskolin-stimulated cAMP. Cells were preincubated with 0.5 mm IBMX for 30 min and subsequently treated with 1 μm forskolin with or without increasing doses of BIM23206 for 60 min at 37 C. At the end of incubation, the medium was removed and intracellular cAMP was measured. No changes in forskolin-induced cAMP accumulation were observed on BIM23206 stimulation in cells transfected with empty vector (GH3). Wild-type receptor showed a maximum 53% inhibition of forskolin-stimulated cAMP accumulation, and R240W and RST mutants induced a similar inhibition (50 and 48% respectively), whereas the i2 loop mutants showed a complete loss of coupling to adenylyl cyclase. Experiments were repeated at least three times and each determination was done in quintuple. The mean value (±se) was used for the graph. *, P < 0.05 vs. corresponding basal. B, To quantify the inhibition of forskolin-induced cAMP accumulation, CHO-K1 cells transiently transfected for 48 h with wild-type or mutated SST5 were preincubated with 0.5 mm IBMX for 30 min and subsequently treated with 1 μm forskolin with or without increasing doses of BIM23206 for 60 min at 37 C. At the end of incubation, cells were lysed and assayed by enzymatic immunoassay. Experiments were repeated at least three times and each determination was done in quintuple. The mean value (±se) was used for the graph. *, P < 0.05 vs. corresponding basal.

The same effects of SST5 mutations on intracellular cAMP inhibition were observed in CHO-K1 cells (Fig. 2B). One nanomole BIM23206 induced 78 ± 3% inhibition of forskolin-stimulated cAMP accumulation in CHO-K1 cells transfected with wild-type SST5, and similar results were obtained with R240W or RST mutants (76 ± 6 and 72 ± 5% inhibition, respectively). No intracellular cAMP inhibition was mediated by BIM23206 in D136A and R137A SST5-expressing cells or in empty vector transfected cells.

Effects of SST5 mutations on intracellular calcium inhibition

We investigated the effects of i2 and i3 loop mutations on the SST5 ability of decreasing [Ca²⁺]i. In cells expressing the wild-type SST5, the addition of 10 nm BIM23206 did not affect resting [Ca²⁺]i (Fig. 3). Conversely, this agent was effective in dramatically reducing the [Ca²⁺]i rise induced by TRH. In fact, the addition of TRH caused a rapid and transient [Ca²⁺]i rise, followed by a plateaux phase that was maintained for many minutes. This biphasic effect was due to both Ca²⁺ mobilization from intracellular stores because it was maintained in Ca²⁺ free medium (containing 3 nm EGTA) and Ca²⁺ influx from extracellular spaces because in Ca²⁺ free medium, the [Ca²⁺]i rise was transient (Fig. 3). BIM23206 added 2 min after TRH rapidly interrupted the plateaux phase and reduced [Ca²⁺]i to basal values, with an effect similar to those brought about by EGTA. By contrast, no reduction in the TRH-induced [Ca²⁺]i rise was observed in R240W, RST, D136A, and R137A SST5-expressing cells or in cells transfected with empty vector after BIM23206 treatment, suggesting that both the DRY motif and the i3 loop are crucial for the inhibition of Ca²⁺ influx.

Figure 3.

Representative traces of the intracellular calcium concentration changes induced by BIM23206 treatment. GH3 cells were transiently transfected with wild-type or mutated SST5, loaded with fura-2 as described and stimulated with 10 nm TRH and 10 nm BIM23206 at the time points indicated by arrows. Left panel, The addition of 10 nm BIM23206 did not affect resting [Ca²⁺]i. The addition of TRH caused a rapid [Ca²⁺]i rise, due to Ca²⁺ mobilization, followed by a plateau phase that was maintained for many minutes. The [Ca²⁺]i rise was transient in Ca²⁺ free medium (3 nm EGTA). Right panel, In wild-type SST5-expressing cells, BIM23206 reduced the [Ca²⁺]i rise induced by TRH to basal values. By contrast, no reduction in the THR-induced [Ca²⁺]i rise was observed in mutant SST5 expressing cells. The figure shows representative traces of at least five experiments with wild-type, R240W, and D136A SST5-expressing cells. Similar results were obtained for the other mutants, R137A and RST.

Effects of R240W mutation on GH secretion

To analyze the effects of SST5 agonist in inhibiting GH secretion, GH3 cells transiently transfected with wild-type or mutated SST5 were incubated for 3 h with or without BIM23206. As shown in Fig. 4, wild-type SST5 mediated a dose-dependent reduction in GH secretion, and 10 nm BIM23206 treatment resulted in a 21 ± 3% reduction of GH secretion with respect to basal. Conversely, this analog did not affect GH release from GH3 cells expressing R240W, RST, D136A, or R137A mutants. No alterations of GH secretion from cells transfected with empty vector were induced by BIM23206 treatment. These data suggest that the loss of mutant receptors ability to inhibit calcium levels has direct consequences on the regulation of GH release.

Figure 4.

GH secretion of GH3 cells stimulated with increasing concentrations of BIM23206 (0.1, 1, or 10 nm). The cells were transiently transfected with empty vector (GH3) wild-type (WT), R240W, RST, D136A, or R137A SST5 and incubated 3 h with or without BIM23206. Culture medium was used to measure GH by enzyme immunoassay. Experiments were repeated at least three times and each determination was done in quintuple. The mean value (±se) was used for the graph. *, P < 0.05 vs. corresponding basal.

Effects of SST5 mutations on cell proliferation and ERK1/2

To analyze the SST5 inhibitory effect on GH3 cell proliferation, we performed proliferation assay by measuring incorporation of BrdU after 48 h incubation with or without BIM23206. As shown in Fig. 5A, BIM23206 induced a dose-dependent reduction in cell proliferation (76 ± 6% inhibition at 10 nm) in GH3 cells transfected with wild-type SST5, whereas no effect was observed in cells transfected with empty vector. When GH3 cells were transfected with R240W or RST SST5, the inhibitory effect on cell proliferation was significantly reduced (38 ± 7 and 31 ± 8% at 10 nm, respectively, P < 0.05 vs. corresponding wild type), suggesting that these mutations impair the ability of SST5 receptor to inhibit proliferation. An altered DRY motif completely abolished the effect of SST5 agonist on cell proliferation, as demonstrated by transfections with D136A and R137A mutated receptors.

Figure 5.

A, Effect of BIM23206 treatment (0.1, 1, or 10 nm) on proliferation of GH3 cells expressing wild-type (WT) or mutated SST5. No changes in cell proliferation was observed in cells transfected with empty vector (GH3). In cells transfected with wild-type SST5, BIM23206 caused a dose-dependent inhibition of GH3 proliferation, whereas a slight reduction of cell proliferation was observed in R240W and RST SST5 expressing cells. No effect on cell proliferation was mediated by receptors with mutations in the DRY motif (D136A and R137A). Experiments were repeated at least three times and each determination was done in quintuple. bas, Basal. *, P < 0.05 vs. corresponding basal; °, P < 0.05 vs. corresponding wt. B, Effect of 10 nm BIM23206 incubation on proliferation of CHO-K1 cells expressing wild-type (wt) or mutated SST5. In cells transfected with wild-type SST5, BIM23206 caused a significant inhibition of CHO-K1 proliferation, whereas no changes in cell proliferation were observed in cells transfected with empty vector (CHO) and R240W, RST, D136A, and R137A SST5-expressing cells. Experiments were repeated at least three times and each determination was done in quintuple. *, P < 0.05 vs. corresponding basal (bas).

To test the effects of SST5 mutations on cell proliferation in a different cell line, we performed proliferation assay on CHO-K1 cells. The results are shown in Fig. 5B. The antiproliferative effect of wild-type SST5 (48 ± 5% at 10 nm BIM23206, P < 0.05) was completely abolished by the mutations in the i3 loop and in the DRY motif, confirming a crucial role of these regions in mediating the antiproliferative signaling of SST5.

To elucidate the mechanism involved in the antimitotic effect of wild-type SST5, we analyzed the effects of BIM23206 on ERK1/2 phosphorylation by Western blotting. In GH3 cells transfected with wild-type SST5, we found a 66 ± 6% reduction of serum-induced ERK1/2 phosphorylation after short-term incubation (10 min), which was similar to that obtained after more prolonged agonist stimulation (30 and 60 min). Conversely, no change was observed in cells transfected with i3 loop (R240W and RST) and DRY motif (D136A and R137A) mutant receptors or empty vector (Fig. 6, A and B, and data not shown).

Figure 6.

A, The figure shows a representative immunoblot of ERK1/2 phosphorylation analysis. Cells were transfected with empty vector, wild-type (WT), or mutated SST5 for 48 h; serum starved for 24 h; and then stimulated with 10% FBS with or without BIM23206 for 10 min. We observed a reduction in FBS-induced ERK1/2 phosphorylation in cells expressing wild-type SST5, whereas no inhibition of ERK1/2 activation was observed with R240W receptor or empty vector. The presence of total p42/44 was analyzed by stripping and reprobing with anti-total p42/44 antibody. B, The graph shows the quantification of phospho-ERK1/2 normalized to total ERK1/2 (mean value ± se from five independent experiments). The analysis of the data obtained was performed with the image analysis program ImageJ (National Institutes of Health). *, P < 0.05 vs. corresponding basal (bas).

Discussion

In the present study, we identified two regions in the i2 and i3 loop of the human SST5 required for the generation of downstream effectors involved in the regulation of hormone secretion and cell proliferation. Somatostatin is the hypothalamic peptide that physiologically inhibits GH secretion and cell growth. The high density of its receptors, in particular SST2 and SST5, on GH-secreting adenomas is used clinically to treat acromegalic patients with specific somatostatin analogs that have been developed in recent years. However, the structural domains mediating the interaction of these receptors with G proteins and subsequent signal transduction are largely unknown.

Because all five SST subtypes are functionally coupled to the inhibition of adenylyl cyclase and cAMP accumulation via Gαi protein, we first investigated the ability of wild-type or mutated SST5 to mediate this pathway. In GH3 and CHO-K1 cells expressing wild-type SST5, the SST5 superselective analog BIM23206 caused the expected inhibition of cAMP accumulation, which reflects the reduced activity of adenylyl cyclase due to the presence of phosphodiesterase inhibitors. When expressed in GH3 or CHO-K1 cells, R240W or RST mutant SST5 appeared to be effective in inhibiting cAMP accumulation, the extent of this inhibition being similar to that induced by the wild-type receptor. We previously demonstrated that the i3 loop of SST5 is crucial for arrestin binding and receptor internalization (16) and that these processes are altered in the presence of the R240W mutation or other mutations also involving phosphorylated residues within the i3 loop (RST mutant). The basic residue R²⁴⁰ represents the first residue of the conserved BBXXB motif located within the i3 loop (Fig. 1A), a consensus sequence identifying a potential site for coupling a number of GPCRs to G proteins (17,18,19,20,21). Although a putative BBXXB sequence exists in the i3 loop of SST2, -3, -4, and -5 (1), the role of this region in G protein coupling has not been investigated. Our data suggest that the BBXXB motif of SST5 does not engage in direct interactions with Gαi protein, and the substitution of a basic residue within this region does not affect adenylyl cyclase inhibition.

On the contrary, we found that residues D¹³⁶ and R¹³⁷ within the DRY motif in the i2 loop of SST5 were important for coupling the receptor to Gαi protein. Indeed, GH3 or CHO-K1 cells expressing SST5 mutants with substitution at D¹³⁶ and R¹³⁷ failed to mediate adenylyl cyclase inhibition. The importance of the i2 loop for G protein interaction, receptor activation, and signal transduction has been documented for a number of GPCRs (30). The tripeptide Glu/Asp-Arg-Tyr (i.e. E/DRY motif, permutated to ERW in the TSH, LH, and FSH receptors), forming part of the amino-terminal subregion of i2, is specific to class-A receptors and highly conserved in all five somatostatin receptor subtypes, but its role in signal transduction for SST1–5 has not been investigated. Mutational analysis of several GPCRs has demonstrated that replacement of Arg residue by various other amino acids abolishes or drastically reduces agonist-stimulated signal transduction (22,36,37,38,39,40) or results in constitutive activation of the altered receptor (25,41). This study first demonstrates that SST5 belongs to the subgroup of GPCRs in which DRY motif is directly involved in governing receptor conformation and G protein coupling and recognition. A recent study demonstrated that dopamine type 2 and type 3 receptors fit in the same group, given that mutation of the Arg residue in the DRY motif abolishes their signaling (42).

Because SSTs regulates Ca²⁺ and K⁺ channels on somatotrope to reduce Ca²⁺ influx and [Ca²⁺]i, to further investigate signal transduction mediated by wild-type or mutant SST5, we measured intracellular calcium in transfected GH3 cells. Our data showed that the activation of the wild-type SST5 resulted in a strong inhibition of the [Ca²⁺]i, as previously shown (43). Conversely, in the same experimental conditions, R240W, RST, D136A, and R137A mutants failed to mediate this effect, suggesting that both the BBXXB sequence and the DRY motif are crucial for coupling SST5 with the blockade of Ca²⁺ entry.

We then investigated the ability of mutant receptors to mediate the most relevant biological effect of somatostatin, that is the inhibition of hormone release. In GH3 cells, a model of rat somatotropic cells, which synthesizes and secretes GH, we demonstrated that wild-type SST5 caused a reduction of GH release that, although less pronounced than that reported for SST2 (44), is in agreement with previous observations (44). Conversely, the i3 loop (R240W and RST) and DRY motif (D136A and R137A) mutants were not able to induce any inhibitory effect on GH release. Taking into account that R240W and RST mutants were effective in inhibiting cAMP accumulation, it is tempting to speculate that the absent inhibition of GH release may be attributable to the inability of these receptors to reduce Ca²⁺ influx. Moreover, it is worth noting that R240W mutation was originally identified in one acromegalic patient in whom octreotide treatment did not inhibit GH hypersecretion, a clinical phenotype that is consistent with the results obtained in GH3 cells expressing the same SST5 mutation (45).

Finally, we demonstrated that SST5 mutations in i3 and i2 loop affected the antiproliferative action of SST5, in either GH3 or CHO-K1 cells. In particular, the activation of the wild-type SST5 caused a dramatic reduction of both cell growth and ERK1/2 phosphorylation. Mutants in the BBXXB motif caused a marked reduction of the antiproliferative action of BIM23206 and a nearly total abrogation of negative effect on ERK1/2 phosphorylation, whereas DRY mutations were ineffective on both parameters. Although it is well known that SST5 mediates antiproliferative signals, the intracellular pathway is still unclear. The involvement of specific phosphotyrosine phosphatases was suggested for SST2 (10), whereas contradictory data are available for SST5. The antiproliferative effect mediated by SST5 has been reported to be independent from cAMP inhibition and associated with an inhibition of ERK activity (11,13,14), as also supported by the results of the present study. Furthermore, taking into account that R240W and RST mutants failed to interact with β-arrestin-2 (16) and that this protein may act as scaffold protein to activate additional signaling pathways (46,47), it is tempting to speculate that i3 loop mutations may affect not only SST5 internalization but also potential arrestin-mediated signal transduction.

In conclusion, in the present study, we characterized the structural domains of human SST5 involved in signal transduction in a pituitary cell model and CHO-K1 cells. Our data indicate that mutants in the BBXXB motif within i3 loop maintain coupling with cAMP inhibition but not other signaling pathways involved in the inhibition of GH secretion and cell growth, suggesting that different regions of SST5 are required for the activation of different signaling pathways. On the contrary, DRY motif plays a crucial role in coupling SST5 with its downstream effectors and alterations in this region completely abolished agonist-induced receptor responses, suggesting its direct involvement in the interaction with all G protein family members required for somatostatin signaling or in governing receptor conformation. Finally, understanding of the molecular determinants mediating the interaction of the human SST5 with intracellular signaling pathways might provide the basis for new molecular studies on possible association of single nucleotide polymorphisms in the SST5 gene with susceptibility to diseases and/or resistance to drugs in acromegalic patients (48).