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. 2009 Nov 12;24(1):240–249. doi: 10.1210/me.2009-0321

Agonist-Biased Signaling at the sst2A Receptor: The Multi-Somatostatin Analogs KE108 and SOM230 Activate and Antagonize Distinct Signaling Pathways

Renzo Cescato 1,a, Kimberly A Loesch 1,a, Beatrice Waser 1, Helmut R Mäcke 1, Jean E Rivier 1, Jean Claude Reubi 1, Agnes Schonbrunn 1
PMCID: PMC2802896  PMID: 19910453

Abstract

Somatostatin analogs that activate the somatostatin subtype 2A (sst2A) receptor are used to treat neuroendocrine cancers because they inhibit tumor secretion and growth. Recently, new analogs capable of activating multiple somatostatin receptor subtypes have been developed to increase tumor responsiveness. We tested two such multi-somatostatin analogs for functional selectivity at the sst2A receptor: SOM230, which activates sst1, sst2, sst3, and sst5 receptors, and KE108, which activates all sst receptor subtypes. Both compounds are reported to act as full agonists at their target sst receptors. In sst2A-expressing HEK293 cells, somatostatin inhibited cAMP production, stimulated intracellular calcium accumulation, and increased ERK phosphorylation. SOM230 and KE108 were also potent inhibitors of cAMP accumulation, as expected. However, they antagonized somatostatin stimulation of intracellular calcium and behaved as partial agonists/antagonists for ERK phosphorylation. In pancreatic AR42J cells, which express sst2A receptors endogenously, SOM230 and KE108 were both full agonists for cAMP inhibition. However, although somatostatin increased intracellular calcium and ERK phosphorylation, SOM230 and KE108 again antagonized these effects. Distinct mechanisms were involved in sst2A receptor signaling in AR42J cells; pertussis toxin pretreatment blocked somatostatin inhibition of cAMP accumulation but not the stimulation of intracellular calcium and ERK phosphorylation. Our results demonstrate that SOM230 and KE108 behave as agonists for inhibition of adenylyl cyclase but antagonize somatostatin’s actions on intracellular calcium and ERK phosphorylation. Thus, SOM230 and KE108 are not somatostatin mimics, and their functional selectivity at sst2A receptors must be considered in clinical applications where it may have important consequences for therapy.

SOM230 and KE108, two analogs purported to mimic somatostatin action at the sst2 receptor, unexpectedly block somatostatin signaling to calcium and ERK phosphorylation.

Somatostatins consist of two regulatory peptides, the 14-amino-acid form (SS-14) and the 28-amino-acid form (SS-28), that are widely distributed in the endocrine system, the nervous system, and the gastrointestinal tract (1,2). These peptides are physiologically important in the control of hormone and exocrine secretion, neurotransmission, and smooth muscle contraction. Somatostatins also inhibit the secretion of peptides and neuroregulators from a variety of neuroendocrine tumors and, in addition, often reduce tumor growth (1,3,4). These actions are mediated by a family of seven-transmembrane-domain receptors encoded by five genes (sst1 to sst5). Although human tissues express only somatostatin subtype 2A (sst2A) receptors, sst2 receptor mRNA is alternatively spliced in rodents to generate two splice variants, sst2A and sst2B, which differ in their carboxy termini. Other sst receptor mRNAs are not spliced.

The sst2A receptor has been targeted therapeutically because it is the most abundant and widely distributed somatostatin receptor subtype in both normal human tissues and neuroendocrine tumors (3,5). The first somatostatin analog introduced clinically was an octapeptide, octreotide, which exhibits markedly increased metabolic stability compared with natural somatostatins (6). Whereas the native peptides bind to all sst receptors with similar nanomolar affinities, octreotide is selective, potently activating the sst2 receptor, weakly activating the sst3 and sst5 receptors, and showing no activity at the sst1 and sst4 receptors. Both octreotide and lanreotide, another sst2-preferring somatostatin analog, are used in the standard long-term treatment of patients with GH-secreting pituitary and gastroenteropancreatic neuroendocrine tumors. [Tyr3]octreotide, which has the same receptor subtype specificity as octreotide, provides the backbone for the radioligand 90Yttrium-1,4,7,10-tetraazacyclododecane-1,4,7,10 tetraacetic acid-[Tyr3]octreotide, which has been used successfully for the radiotherapy of neuroendocrine tumors.

Despite this success, many neuroendocrine tumors are resistant to somatostatin analog therapy. Because such tumors often express several sst receptor subtypes, either instead of or in addition to the sst2A receptor, drug development has focused on stable somatostatin analogs that bind with high affinity to multiple somatostatin receptor subtypes to better mimic the broader actions of the native peptides. Two such somatostatin analogs are currently undergoing preclinical and clinical development: SOM230 binds with high affinity to the sst1, sst2, sst3, and sst5 receptor subtypes (7,8), whereas KE108 binds to all five sst receptors (9). Both compounds are reported to be full agonists at their targeted receptors based on their ability to inhibit adenylyl cyclase at nanomolar concentrations (9,10,11). Surprisingly, however, SOM230 and KE108 do not always elicit all the biological effects expected from the actions of native somatostatin. For example, unlike SS-14, neither KE108 nor SOM230 inhibit spontaneous epileptiform activity in mouse hippocampal slices even though all three ligands potently displace radiolabeled somatostatin binding in this tissue (12). Similarly, in acromegalic patients, high concentrations of SOM230 were less effective than octreotide at stimulating IGF-binding protein-1 levels (13). Such observations suggest that the binding of these two broad-spectrum somatostatin analogs may not couple sst receptors to all the same effector systems as somatostatin.

Recent studies have shown that drugs acting on a common G protein-coupled receptor (GPCR) can induce distinct and selective effects by stimulating some of the signaling pathways activated by the native ligand but antagonizing others (14,15). The effect of drugs acting at a single GPCR to induce a unique pattern of signaling events has been termed functional selectivity or biased agonism. This behavior is thought to result from the ability of different ligands to stabilize distinct active receptor conformations, each with its own signaling capacity (16). Previous evidence for functional selectivity at sst receptors has been scant (reviewed in Ref. 17). In the case of the sst2A receptor, functional selectivity was first demonstrated by the observations that 1) there was a large variation in the relative potencies of a series of somatostatin analogs to inhibit adenylyl cyclase and to stimulate receptor internalization (11), and 2) a nonpeptide full agonist had a reduced efficacy to induce receptor endocytosis and to bind arrestin (11). Nonetheless, there is no evidence that somatostatin analogs can activate distinct signaling pathways at sst receptor subtypes.

To better understand the pharmacological profile of SOM230 and KE108, we compared signaling by these two agonists with that of SS-14, octreotide, and [Tyr3]octreotide through the sst2A receptor. We chose two different cellular systems for these studies. HEK293 cells have been extensively used to elucidate signal transduction pathways by heterologously expressed GPCRs including those, like the sst2A receptor, that couple to Gi/Go proteins. AR42J cells are a differentiated pancreatic acinar cell line that has long served as a model for studies of the mechanisms by which somatostatin and other peptides regulate pancreatic exocrine secretion (18,19). We have shown that AR42J cells express only the sst2A receptor subtype using a combination of RT-PCR, sst receptor-specific antibodies, and receptor subtype-selective somatostatin analogs (20,21,22). Furthermore, these endogenous sst2A receptors are functionally coupled to multiple G proteins including Gαi1, Gαi3, and Gα14 that activate distinct signaling pathways (23,24). The results presented here demonstrate that in both HEK293-sst2 cells and AR42J cells KE108 and SOM230 are full agonists for adenylyl cyclase inhibition but antagonize the actions of SS-14 to stimulate calcium and ERK phosphorylation.

Results

Effect of KE108 and SOM230 on sst2A receptor signaling in HEK293 cells

To determine whether KE108 and SOM230 regulate the same signaling pathways as native SS-14, we first examined their ability to inhibit adenylyl cyclase and to stimulate intracellular calcium in HEK-sst2 cells (Fig. 1). SS-14, [Tyr3]octreotide, KE108, and SOM230 all inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner with the rank order of [Tyr3]octreotide more than KE108 more than SS-14 more than SOM230 (Fig. 1A). In addition, both [Tyr3]octreotide (Fig. 1B) and SS-14 (Fig. 2A) increased cytosolic calcium. However, even at a concentration of 10 μm, neither KE108 nor SOM230 altered cytosolic calcium (Fig. 1B). In fact, SOM230 and KE108 competitively antagonized SS-14 and [Tyr3]octreotide stimulation of calcium (Fig. 2, A and B). Antagonist affinities were calculated from the shift in the agonist dose-response curves for calcium stimulation and gave dissociation constant (KD) values of 22 nm for KE108 and 89 nm for SOM230. Because in the absence of biased agonism, full agonists will show identical relative potencies for different signaling pathways, these results demonstrate that SOM230 and KE108 are not full agonists at the sst2A receptor and suggest that they exhibit functional selectivity.

Figure 1.

Figure 1

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Agonist effect of somatostatin analogs on cAMP and cytosolic calcium in HEK-sst2 cells. A, HEK-sst2 cells were incubated for 30 min with 10 μm forskolin and different concentrations of [Tyr3]octreotide (•), KE108 (▪), SS-14 (▴), or SOM230 (▾), and then intracellular cAMP was measured as described in Materials and Methods. The EC50 values for the different analogs were as follows: [Tyr3]octreotide 0.012 nm, KE108 0.057 nm, SS-14 0.18 nm, and SOM230 0.86 nm. B, HEK-sst2 cells were loaded with Fluo-4NW dye and then treated with different concentrations of [Tyr3]octreotide (•), KE108 (▪), or SOM230 (▾). Intracellular calcium was measured for 60 sec immediately after the addition of the compounds as described in Materials and Methods and is expressed as a percentage of the calcium signal produced by 25 μm ionomycin. The EC50 value for [Tyr3]octreotide was 16 nm. In both panels, the data show the mean ± sem from at least three independent experiments.

Figure 2.

Figure 2

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Antagonist effect of somatostatin analogs on cytosolic calcium in HEK-sst2 cells. A, HEK-sst2 cells were incubated with different concentrations of SS-14 in the absence (•) or presence (▴) of 10 μm SOM230. The EC50 values for SS-14 in the absence and presence of 10 μm SOM230 were 17 and 1920 nm, respectively B, HEK-sst2 cells were treated with different concentrations of [Tyr3]octreotide (TOC) in the absence (•) or presence (▴) of 1 μm KE108, and intracellular calcium was measured as described in Materials and Methods. The EC50 values for [Tyr3]octreotide in the absence and presence of 1 μm KE108 were 16 and 726 nm, respectively. C, HEK-sst2 cells were pretreated in the absence or presence of 100 ng/ml pertussis toxin (PTX) for 16 h and then treated with 1 μm [Tyr3]octreotide (TOC) in the continued absence or presence of PTX. Intracellular calcium was measured as described in Materials and Methods. Each panel shows the mean ± sem from at least three independent experiments.

Sst2A receptor-mediated inhibition of adenylyl cyclase in HEK293 cells is known to be pertussis toxin sensitive and thus to involve Gi/Go (25). Therefore, we next determined whether the increase in cytosolic calcium was also dependent on sst2A receptor coupling to pertussis toxin-sensitive G proteins. Pretreatment of HEK-sst2 cells with pertussis toxin abolished calcium mobilization by both [Tyr3]octreotide (Fig. 2C) and somatostatin (not shown). The observation that two pertussis toxin-sensitive signaling pathways, namely inhibition of adenylyl cyclase and stimulation of cytosolic calcium, were differentially regulated by KE108 and SOM230 indicates that the latter two analogs cannot couple the sst2A receptor to all the Gi/Go proteins activated by SS-14 and [Tyr3]octreotide.

Because GPCRs have previously been shown to stimulate ERK phosphorylation in HEK293 cells via both pertussis toxin-sensitive and -resistant G proteins (26,27), we next examined the effect of native SS-14 and the multi-sst receptor analogs on this signaling pathway. Figure 3, A and B, shows that SS-14, SOM230, and KE108 all induced a large increase in ERK phosphorylation within 2 min, although SOM230 and KE108 were less effective than SS-14. The stimulation was transient and returned to basal levels after about 10 min. Pretreating cells with pertussis toxin completely blocked the stimulatory effect of SOM230 and KE108 but only partially inhibited the effect of SS-14, indicating that SOM230 and KE108 were only able to stimulate ERK phosphorylation by a pertussis toxin-sensitive mechanism, whereas SS-14 was able to activate both pertussis toxin-sensitive and -resistant pathways. Consistent with this conclusion, SS-14 stimulation of ERK phosphorylation after pertussis toxin treatment was antagonized by SOM230 and KE108 (Fig. 3C). Together our data show that the sst2A receptor increases ERK phosphorylation in HEK-sst2 cells by two mechanisms: a Gi/Go-mediated process that is activated by SS-14 and the two multi-receptor analogs and a Gi/Go-independent process that is stimulated only by SS-14. These data support the conclusion that SOM230 and KE108 act as biased agonists at the sst2A receptor.

Figure 3.

Figure 3

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Effect of somatostatin analogs on ERK phosphorylation in HEK293 cells. A, HEK-sst2 cells were pretreated overnight in the absence or presence of pertussis toxin (PTX, 100 ng/ml) and then incubated with 100 nm SS-14, 1 μm SOM230, or 1 μm KE108 for the times shown. Cell proteins were extracted, resolved by SDS-PAGE, and then immunoblotted with both phospho-ERK (pERK) and total ERK-specific antibodies as described in Materials and Methods. B, Immunoblots were quantitated as described in Materials and Methods and expressed as the ratio of the phospho-ERK signal after 2 min peptide treatment compared with that of the vehicle control. The results show the mean ± sem of three independent experiments. C, HEK-sst2 cells were pretreated overnight in the presence of PTX (100 ng/ml) and then incubated for 2 min with 100 nm SS-14, 10 μm SOM230, or 10 μm KE108 individually or in combination as shown. Cell lysates were resolved by SDS-PAGE and immunoblotted sequentially with phospho-ERK and total ERK-specific antibodies.

Effect of KE108 and SOM230 on sst2A receptor signaling in AR42J cells

To determine whether functional selectivity also occurred with endogenous sst2A receptors in differentiated cells, we next examined somatostatin signaling in AR42J cells, a pancreatic cell line that has been used extensively to investigate somatostatin action (22,23,24,28). Using a combination of RT-PCR, sst receptor-specific antibodies, and receptor subtype-selective somatostatin analogs, we previously showed that AR42J cells express only the sst2A receptor subtype (20,21,22).

[Tyr3]octreotide, SOM230, and KE108 all potently inhibited forskolin-stimulated cAMP accumulation in AR42J cells (Fig. 4A), as previously reported for SS-14 and octreotide (22). However, although [Tyr3]octreotide increased cytosolic calcium with an EC50 of 33 nm, neither KE108 nor SOM230 (Fig. 4B) had an effect, even at 10 μm. In fact, KE108 and SOM230 behaved as competitive antagonists for stimulation of intracellular calcium (Fig. 4C) with a calculated KD of 56 nm for KE108 and 50 nm for SOM230. In contrast to its inhibitory effect in HEK-sst2 cells, pertussis toxin pretreatment did not affect [Tyr3]octreotide stimulation of intracellular calcium in AR42J cells, although the same pretreatment blocked inhibition of cAMP accumulation by [Tyr3]octreotide (Fig. 4D). Together these results show that the sst2A receptor can regulate at least two signaling pathways in AR42J cells: a Gi/Go-mediated inhibition of adenylyl cyclase and a pertussis toxin-insensitive process that increases cytosolic calcium levels. Whereas SS-14 and [Tyr3]octreotide couple the receptor to both signaling pathways and behave as full agonists, KE108 and SOM230 regulate only adenylyl cyclase and antagonize signaling to intracellular calcium.

Figure 4.

Figure 4

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Effect of somatostatin analogs on intracellular cAMP and cytosolic calcium in AR42J cells. A, Cells were incubated for 30 min with 10 μm forskolin and different concentrations of [Tyr3]octreotide (•), KE108 (▪), or SOM230 (▴), and then intracellular cAMP was measured as described in Materials and Methods. The EC50 values were 0.073 nm for [Tyr3]octreotide, 0.31 nm for KE108, and 11.5 nm for SOM230. B, Cells were incubated with different concentrations of [Tyr3]octreotide (•), KE108 (▪), or SOM230 (▴), and cytosolic calcium was measured as described in Materials and Methods. The EC50 for [Tyr3]octreotide was 33 nm. C, Cells were incubated with different concentrations of [Tyr3]octreotide (TOC) in the absence (•) or presence of either 1 μm KE108 (▪) or 1 μm SOM230 (▴), and cytosolic calcium was measured after the addition of the compounds. The EC50 values for [Tyr3]octreotide when added alone was 33 nm, and this was increased to 630 nm in the presence of KE108 and 690 nm in the presence of SOM230. D, Cells were preincubated in the absence or presence of 100 ng/ml pertussis toxin (PTX) for 16 h. Left, Cells were subsequently incubated for 30 min with 10 μm forskolin and 1 μm [Tyr3]octreotide in the continued absence or presence of PTX, and intracellular cAMP was measured as described in Materials and Methods. The graph shows the percentage of forskolin-stimulated cAMP accumulated in the presence of [Tyr3]octreotide. Right, Cells were subsequently treated with 1 μm [Tyr3]octreotide in the continued absence or presence of PTX. The graph shows the stimulation of cytosolic calcium in the presence of [Tyr3]octreotide as a percentage of the ionomycin response. In each panel, the data show the mean ± sem in at least three independent experiments.

Liu et al. (24) previously identified a pertussis toxin insensitive mechanism for SS-14 signaling in AR42J cells: SS-14 was shown to increase ERK phosphorylation by coupling to G14. Therefore, we next determined the effect of SS-14 and its analogs on ERK phosphorylation in AR42J cells pretreated with pertussis toxin to block Gi/o-mediated signaling. Figure 5 shows that both SS-14 and octreotide caused a rapid increase in ERK phosphorylation that was evident within 1 min of stimulation and decayed to control levels after 5 min. In contrast, SOM230 and KE108 did not alter ERK phosphorylation under the same conditions. The effect of SS-14 was dose dependent with an EC50 of 9.4 nm (Fig. 6) and was antagonized by both KE108 and SOM230 (Fig. 7). Furthermore, in contrast to their effect in HEK-sst2 cells, KE108 and SOM230 did not increase ERK phosphorylation in AR42J cells not pretreated with pertussis toxin (not shown). Thus, in AR42J cells, KE108 and SOM230 behave as antagonists for two pertussis toxin-resistant signaling pathways activated by the sst2A receptor, namely stimulation of cytosolic calcium and ERK phosphorylation, even though they act as agonists for the Gi/Go-mediated inhibition of adenylyl cyclase. These results support the conclusion that KE108 and SOM230 act as biased agonists at the endogenous sst2A receptor present in AR42J cells.

Figure 5.

Figure 5

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Differential stimulation of ERK phosphorylation by somatostatin analogs in AR42J cells. A, Cells were pretreated overnight with 100 ng/ml pertussis toxin (PTX) and then incubated with 100 nm SS-14, 100 nm octreotide, 1 μm SOM230, or 1 μm KE108 for the times shown. Cell proteins were extracted, resolved by SDS-PAGE, and then immunoblotted with both phospho-ERK (pERK) and total ERK-specific antibodies as described in Materials and Methods. B, Immunoblots were quantitated as described in Materials and Methods and expressed as the ratio of the phospho-ERK signal at each time point to that of the vehicle control. •, SS-14; ▴, octreotide; ♦, SOM230; ▪, KE108. The results show the mean ± sem from multiple independent experiments.

Figure 6.

Figure 6

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Dose dependence for SS-14 stimulation of ERK phosphorylation in AR42J cells. A, Cells were pretreated overnight with 100 ng/ml pertussis toxin and then incubated with various concentrations of SS-14 for 2 min. Cell proteins were extracted, resolved by SDS-PAGE and then immunoblotted with phospho-ERK (pERK) and total ERK-specific antibodies as described in Materials and Methods. B, Immunoblots were quantitated as described in Materials and Methods. The data are expressed as a percentage of the signal obtained with 1 μm SS-14 and show the mean ± sem of three independent experiments. An EC50 of 9.4 nm was calculated by nonlinear regression curve fitting using GraphPad Prism.

Figure 7.

Figure 7

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SOM230 and KE108 antagonize somatostatin-stimulated ERK phosphorylation in AR42J cells. A, Cells were pretreated overnight with 100 ng/ml pertussis toxin and then incubated for 2 min with either 10 nm SS-14, 1 μm SOM230, or 1 μm KE108 either alone or in combination. Cell proteins were extracted, resolved by SDS-PAGE, and immunoblotted with both phospho-ERK (pERK) and total ERK-specific antibodies as described in Materials and Methods. B, Immunoblots were quantitated as described in Materials and Methods, and the data are expressed as the ratio of the phospho-ERK signal obtained in peptide-stimulated compared with unstimulated cells. The results show the mean ± sem of three independent experiments.

Effect of somatostatin analogs on [35S]GTPγS binding in AR42J cells

To directly assay for the ability of different somatostatin analogs to couple the sst2A receptor to G proteins, we determined their effect on [35S]GTPγS binding to AR42J membranes. Figure 8 shows that KE108 and SOM230 were both less efficacious at increasing [35S]GTPγS binding than SS-14, indicating that the native peptide is better able to couple the sst2A receptor to G proteins than either KE108 or SOM230. This result is consistent with the data showing that the binding of SS-14 to the sst2A receptor results in the activation of both Gi/Go and the pertussis toxin-insensitive G protein G14, whereas KE108 and SOM230 couple the sst2A receptor only to pertussis toxin-sensitive G proteins.

Figure 8.

Figure 8

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Stimulation of [35S]GTPγS binding by somatostatin analogs in AR42J cells. A, Cell membranes were incubated for 2 h with [35S]GTPγS and various concentrations of SS-14 (•), KE108 (▪), or SOM230 (▴), and the amount of membrane-associated radioactivity was then determined as described in Materials and Methods. The results represent the mean ± sem for [35S]GTPγS binding above the basal levels in at least three independent experiments. B, AR42J cell membranes were incubated with [35S]GTPγS for 2 h in the presence of 2 μm of the indicated peptides either alone or in the presence of 10 μm GTPγS. The results show an autoradiogram of the [35S]GTPγS bound to representative membrane aliquots.

Discussion

The results presented here indicate that the sst2A somatostatin receptor exhibits ligand-directed signaling both when exogenously expressed in HEK293 cells and when endogenously present in AR42J pancreatic acinar cells. In fact, this functional selectivity is evident even though the signaling events activated by the sst2A receptor in the two cell lines are distinct. Specifically, we show that KE108 and SOM230, two somatostatin analogs of great current clinical interest, produce a different pattern of signaling at the sst2A receptor than either the native ligand SS-14 or the U.S. Food and Drug Administration-approved drug octreotide. All four compounds potently inhibit adenylyl cyclase via Gi/Go, but only SS-14 and octreotide increase cytosolic calcium, whereas KE108 and SOM230 act as antagonists. In addition, KE108 and SOM230 antagonize the pertussis toxin-insensitive pathway by which SS-14 stimulates ERK phosphorylation. Thus, we can no longer assume, as has been done in the past, that the biological activity of somatostatin analogs can be generalized from their inhibitory effect on adenylyl cyclase. Most importantly, the functional selectivity exhibited by the sst2A receptor in signaling may well affect the therapeutic actions of somatostatin analogs used clinically, and therefore it will be important to determine how the signaling differences reported here modify tumor responsiveness.

Our previous studies demonstrated functional selectivity at the sst2A receptor by comparing the activities of a group of somatostatin agonists to stimulate sst2A receptor internalization and to inhibit adenylyl cyclase (11). Strikingly, these studies showed that the nonpeptide agonist L-779,976 was less effective than the native hormone at inducing receptor internalization and binding arrestin, although it behaved as a potent and full agonist for inhibition of adenylyl cyclase. To extend these observations, we here examined the effect of two therapeutically promising multi-somatostatin agonists on signaling at the sst2A receptor. We initially investigated sst2A action in HEK293 cells because this cell line has been used extensively to study GPCR signaling including GPCRs that, like the sst2A receptor, are preferentially coupled to pertussis toxin-sensitive G proteins. The cell line selected for these studies expressed high levels of the sst2A receptor (8.9 pmol/mg protein) (11) to ensure that even weak signaling by partial agonists would be readily detected.

HEK293 cells express the α-subunits of all three Gi proteins, as well as Go, Gz, Gq/11, and G12 but not G14 or G16 (24,29). Other Gi-coupled receptors expressed in HEK293 cells act by pertussis toxin-sensitive mechanisms to both inhibit adenylyl cyclase and to increase cytosolic calcium by stimulating calcium release from intracellular stores (30). Thus, the ability of SS-14 to produce both of these pertussis toxin-sensitive effects upon activation of the sst2A receptor was not surprising (Fig. 9). However, the observation that SOM230 and KE108 behaved as agonists for adenylyl cyclase inhibition but antagonized somatostatin stimulation of cytosolic calcium was unexpected and indicated that these two signaling events were mediated by different pertussis toxin-sensitive G proteins. In fact, the sst2A receptor was previously shown to couple to multiple Gi proteins. Agonist activation produces a stable complex between the sst2A receptor and the α-subunits of Gi-1, Gi-2, and Gi-3 in CHO cells (21). The results described here indicate that coupling of the sst2A receptor to different pertussis toxin-sensitive G proteins results in the regulation of distinct signaling pathways that are differentially activated by somatostatin analogs.

Figure 9.

Figure 9

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Signaling by the sst2A receptor upon activation by different somatostatin analogs. Top, In HEK293 cells, SS-14 and octreotide (SS/Oct) inhibit adenylyl cyclase (AC) and stimulate cytosolic calcium via Gi/Go, whereas they increase ERK phosphorylation by two distinct mechanisms: a Gi/Go-mediated pathway, which is inhibited by pertussis toxin, as well as a pertussis toxin-insensitive process, shown by X. Although SOM230 and KE108 (Som/KE) behave as full agonists to inhibit adenylyl cyclase, they antagonize the pertussis toxin-sensitive process that leads to an increase in intracellular calcium. Moreover, because the effect of SOM230 and KE108 to stimulate ERK phosphorylation is completely blocked by pertussis toxin, this action occurs solely via Gi/Go, and these analogs antagonize SS-14 stimulation of ERK phosphorylation by this pertussis toxin-insensitive pathway. Bottom, In AR42J cells, SS-14 and octreotide (SS/Oct) inhibit adenylyl cyclase (AC) via Gi/Go but act by the pertussis toxin-insensitive G protein G14 to stimulate ERK phosphorylation and increase cytosolic calcium. SOM230 and KE108 (Som/KE) are only able to activate Gi/Go signaling and act as antagonists for sst2A receptor signaling via G14.

In addition to inhibiting cAMP and stimulating cytosolic calcium, SS-14 produced a rapid and transient increase in ERK phosphorylation in HEK293 cells. Although SOM230 and KE108 also stimulated ERK phosphorylation, the magnitude of their effect was less than that with SS-14. Pertussis toxin completely blocked the stimulatory action of SOM230 and KE108 on ERK phosphorylation, indicating that it was entirely mediated by Gi/Go. Interestingly, however, pertussis toxin only partially inhibited SS-14-induced ERK phosphorylation, suggesting that both pertussis toxin-sensitive and -resistant pathways were stimulated by SS-14 to activate ERK. This conclusion was further supported by the observation that SOM230 and KE108 antagonized only the pertussis toxin-resistant mechanism by which SS-14 stimulated ERK phosphorylation. Together, these results demonstrate that SS-14 activates ERK by two pathways: a pertussis toxin-sensitive process that is also stimulated by SOM230 and KE108 and a pertussis toxin-resistant mechanism that is antagonized by SOM230 and KE108 (Fig. 9).

Although HEK293 cells provide a well characterized model system in which to examine receptor signaling, the sst2A receptor was highly overexpressed in these cells, and thus its signaling may not reflect its normal behavior in a native environment. To ensure that the functional selectivity observed with the sst2A receptor in HEK293 cells also occurred with endogenous receptors, we examined the effect of SOM230 and KE108 on sst2A receptor function in AR42J pancreatic acinar cells, which contain a much lower receptor density (1.1 pmol/mg protein) (28) than found in the transfected HEK293 cell line (8.9 pmol/mg protein) (11). We have shown that Gαi1 and Gαi3 but not Gαi2 or Gαo are associated with the agonist-occupied sst2A receptor in AR42J cells (23). Furthermore, SS-14 and octreotide both inhibit adenylyl cyclase (22) and stimulate cytosolic calcium (31) in this cell line. In addition, SS-14 and octreotide increase ERK phosphorylation via the pertussis toxin-insensitive G protein G14 (24), which couples the sst2A receptor to phospholipase C (32). As in HEK293 cells, SS-14, octreotide, SOM230, and KE108 all behaved as full agonists for adenylyl cyclase inhibition. However, although SS-14 and octreotide both increased cytosolic calcium, SOM230 and KE108 again antagonized this effect. The observation that only SS-14 and octreotide stimulated intracellular calcium whereas SOM230 and KE108 behaved as antagonists was particularly interesting because calcium stimulation occurred by a pertussis toxin-sensitive mechanism in HEK293 cells and by a pertussis toxin-resistant mechanism in AR42J cells. Thus, the binding of SOM230 and KE108 to the sst2A receptor has distinct effects on its signaling through different pathways. In addition, SS-14 and octreotide rapidly increased ERK phosphorylation in AR42J cells by a pertussis toxin-insensitive mechanism, as previously reported (24). However, SOM230 and KE108 were unable to activate this signaling pathway and instead behaved as antagonists. Together these results demonstrate that in a differentiated cell line in which it is endogenously expressed, the sst2A receptor exhibits functional selectivity with SOM230 and KE108 acting as full agonists for inhibition of adenylyl cyclase and as antagonists for stimulation of cytosolic calcium and ERK phosphorylation (Fig. 9).

Previous studies showed that some somatostatin analogs exhibit different potency ratios and efficacies at the sst2A receptor for signaling to adenylyl cyclase and for stimulating receptor internalization, providing the first clear evidence for functional selectivity at somatostatin receptors (11,33). Here we have extended these observations to show that SOM230 and KE108, two multi-sst receptor targeting ligands that have been described as full agonists at the sst2A receptor, exhibit a striking functional selectivity for different signaling pathways activated by sst2A; they behave as agonists for some signaling processes and as antagonists for others. Our studies highlight the importance of testing the actions of somatostatin analogs on multiple signaling pathways known to be involved in the regulation of secretion, cell proliferation, and apoptosis. In the case of SOM230 and KE108, which are currently being investigated for a variety of clinical applications, our results show that these analogs cannot be considered as simple mimics of the native peptide.

Materials and Methods

Reagents

Peptides were obtained as follows: SS-28, SS-14, and KE108 (9) were synthesized at the Salk Institute and provided by Jean E. Rivier or purchased from Bachem (Torrance, CA); [Tyr3]octreotide and octreotide (SMS201-995) were from Novartis Inc. (Basel, Switzerland); SOM230 (7) was provided by either Novartis or Dr. J. E. Rivier. [35S]GTPγS (1250 Ci/mmol) was from PerkinElmer (Waltham, MA). Phospho-p44/42 ERK (Thr202/Tyr204) and total ERK-specific antibodies were from Cell Signaling Technologies (Danvers, MA). Goat antirabbit IgG (heavy and light chain) conjugated to horseradish peroxidase was from Kirkegaard & Perry Laboratories (Gaithersburg, MD). All other reagents were of the best grade available and were purchased from common suppliers.

Cell culture

HEK293 cells stably expressing either a T7-epitope-tagged sst2A receptor or a triple hemagglutinin-tagged sst2A receptor were grown as described previously (11,33). The rat pancreatic tumor cell line AR42J (CRL-1492) was obtained from American Type Culture Collection (LGC Standards, Teddington, Middlesex, UK) and cultured at 37 C and 5% CO2 in Ham’s F12K medium containing 2 mm l-glutamine and supplemented with 20% (vol/vol) fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin or as previously described (22).

Measurement of cAMP formation

HEK-sst2 or AR42J cells were seeded in poly-d-lysine (20 mg/ml) coated 96-well plates at 20,000 cells per well and grown for 24 h at 37 C in 5% CO2. Culture medium was then removed, and fresh medium containing 0.5 mm 3-isobutyl-1-methylxanthine was added to each well. After 30 min incubation at 37 C in 5% CO2, fresh medium was added containing 0.5 mm 3-isobutyl-1-methylxanthine, with or without 10 μm forskolin and various concentrations of somatostatin analogs. Cells were incubated for 30 min at 37 C in 5% CO2 and then lysed as recommended by the manufacturer. cAMP accumulation was measured using either the cAMP scintillation proximity assay system (RPA 538) from GE Healthcare (Little Chalfont, UK) or the adenylate cyclase activation flash-plate assay (SMP004) from PerkinElmer according to the manufacturers’ instructions. When indicated, cells were pretreated in the absence or presence of 100 ng/ml pertussis toxin for 16 h, and cAMP accumulation was measured as described above in the continued presence or absence of the toxin. All experiments were repeated at least three times.

Measurement of intracellular calcium

Cytosolic calcium was measured in HEK-sst2 and AR42J cells using the Fluo-4NW calcium assay kit (Molecular Probes Inc., Eugene, OR) as described previously (34). In brief, HEK-sst2 or AR42J cells were seeded (25,000–50,000 cells per well) in poly-d-lysine-coated (20 μg/ml) 96-well plates and cultured at 37 C in 5% CO2. On the day of the experiment, the cells were loaded with Fluo-4NW dye in assay buffer (Hanks’ balanced salt solution; and 20 mm HEPES, pH 7.4) containing 2.5 mm probenecid for 30 min at 37 C in 5% CO2 and then for an additional 30 min at room temperature. The dye-loaded cells were transferred to a SpectraMax M2e microplate reader (Molecular Devices, Sunnyvale, CA), and fluorescence emission was recorded at room temperature with λem = 520 nm and λex = 485 nm. Baseline (Fbaseline) measurements were taken for dye-loaded, untreated cells, and then fluorescence was measured for 60 sec in a kinetic experiment after the addition of somatostatin analogs. Maximum fluorescence (Fmax) was measured after the addition of 25 μm ionomycin. Analog effects are presented as a percentage of the maximal calcium response obtained with ionomycin (Fmax− Fbaseline = 100% of maximal calcium response). When indicated, cells were pretreated in the absence or presence of 100 ng/ml pertussis toxin for 16 h, and calcium measurements were then performed as described above in the continued presence or absence of the toxin. All experiments were repeated at least three times in triplicate.

Measurement of ERK phosphorylation

HEK-sst2 or AR42J cells were plated in six-well plates at a confluency of approximately 30% and grown for 3–5 d to 80% confluency. Cells were then incubated in the absence or presence of 100 ng/ml pertussis toxin (List Biological Labs, Campbell, CA) for 18–24 h in DMEM/F12 containing 0.5% (wt/vol) BSA (Sigma-Aldrich, St. Louis, MO). The next day, cells were treated at 37 C with somatostatin analogs in fresh serum-free medium for the times specified. After peptide stimulation, cells were washed four times with ice-cold PBS and then solubilized on ice for 20 min in lysis buffer containing 1% Triton X-100 (wt/vol), 150 mm NaCl, 10% glycerol (vol/vol), 50 mm HEPES (pH 7.4), 1 mm EGTA, 10 mm sodium pyrophosphate, 100 mm sodium fluoride, 1.5 mm MgCl2, 1:500 dilution of protease inhibitor cocktail (Sigma-Aldrich), and 1:100 dilution of phosphatase inhibitor cocktail 2 (Sigma-Aldrich). Detergent extracts were centrifuged at 20,000 × g for 15 min at 4 C, diluted into Laemmli SDS sample buffer and then resolved by SDS-PAGE under reducing conditions. Proteins were transferred to polyvinylidene difluoride membranes using the Bio-Rad semi-dry blotter. Membranes were blocked with 2% (wt/vol) BSA in Tris-buffered saline/Tween 20 for at least 30 min at room temperature and then immunoblotted with antibodies to phospho-ERK. Proteins were detected with secondary antibody horseradish peroxidase-conjugated antirabbit and enhanced chemiluminescence detection reagents (Pierce Chemical Co., Rockford, IL). Blots were then stripped and reprobed with total ERK antibodies. Each immunoblot shown is representative of at least two independent experiments. The intensity of receptor bands was quantitated using Scion Image version 1.63 (Scion Corp., Frederick, MD) after scanning autoradiograms with an Epson Expression 1680 scanner. Band intensity with phosphospecific antibodies was normalized for total ERK levels in the same experiment and is expressed as mean ± sem of values from at least three independent experiments. Differences between treatment groups were determined by two-way analysis of variance and a Bonferroni post test.

[35S]GTPγS binding

AR42J cell membrane pellets were prepared as previously described and stored at −80 C (35). The [35S]GTPγS binding assay was performed on 20-μm-thick cryostat (Microm HM 500, Walldorf, Germany) sections of the membrane pellets and mounted on microscope slides. The sections were first preincubated for 10 min at room temperature in assay buffer [100 mm Tris-HCl buffer (pH 8.2), 1% BSA, 40 mg/liter bacitracin, and 10 mm MgCl2) and then for 30 min in assay buffer containing 50 μm GDP. Sections were subsequently incubated for 2 h at room temperature in assay buffer containing 50 μm GDP and approximately 40 pm [35S]GTPγS (100,000 cpm/ml), in the absence (basal level) or presence (stimulated) of test compounds. At the end of the incubation, sections were washed on ice with assay buffer. After a brief dip in assay buffer without BSA and bacitracin to remove excess salts, the sections were dried and exposed to Kodak BioMax MR film. OD were determined using the MCID Basic 7.0 program (Inter Focus GmbH, Meiring, Germany) and were expressed as percentage above basal level.

Data analysis and curve fitting

EC50 values were calculated by least-squares nonlinear regression analysis of dose-response curves using GraphPad Prism (GraphPad Software, San Diego, CA). Antagonist KD values were calculated from the shift of the agonist dose-response curves in the presence of a fixed concentration of antagonist using the following equation: dose ratio = 1 + [Ant]/KD, where the dose ratio is the ratio of the agonist concentration required to produce an identical response in the presence and absence of a fixed concentration of antagonist ([Ant]).

Acknowledgments

We thank Dr. Terry Kenakin for helpful discussions.

Footnotes

This work was supported by National Institutes of Health (NIH) Grants DK032234 to A.S. and DK059953 to J.R. and J.C.R. K.L. was supported by a fellowship from the Pharmacoinformatics Training Program of the Keck Center of the Gulf Coast Consortia (NIH Grant T90 DK070109).

Disclosure Summary: R.C., K.A.L., B.W., J.E.R., and A.S. have nothing to disclose. H.R.M. and J.C.R. are inventors on a patent by the University of Berne on KE108 but do not have a financial relationship with any commercial entity that has an interest in the subject of this manuscript.

First Published Online November 12, 2009

Abbreviations: GPCR, G protein-coupled receptor; SS-14, the 14-amino-acid form of somatostatin; sst2A, somatostatin receptor subtype 2A.

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