Homologous and Heterologous Regulation of Somatostatin Receptor 2
Gerard Elberg1,
R. William Hipkin2 and
Agnes Schonbrunn
Department of Integrative Biology and Pharmacology, University of Texas Health Sciences Center Houston, Houston, Texas 77225
Address all correspondence and requests for reprints to: Agnes Schonbrunn, Department of Integrative Biology and Pharmacology, University of Texas-Houston, P. O. Box 20708, Houston, Texas 77225. E-mail: Agnes.Schonbrunn{at}uth.tmc.edu.
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ABSTRACT
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We previously demonstrated that phosphorylation of somatostatin receptor 2A (sst2A) is rapidly increased in transfected cells both by agonist and by the protein kinase C (PKC) activator phorbol myristate acetate (PMA). Here, we investigate whether PKC-mediated receptor phosphorylation is involved in the homologous or heterologous regulation of endogenous sst2 receptors in AR42J pancreatic acinar cells upon stimulation by agonist or by cholecystokinin (CCK) or bombesin (BBS). Somatostatin, PMA, CCK, and BBS all increased sst2A receptor phosphorylation 5- to 10-fold within minutes. Somatostatin binding also caused rapid internalization of the ligand-receptor complex, and PMA, CCK, and BBS all stimulated this internalization further. Additionally, sst2 receptor-mediated inhibition of adenylyl cyclase was desensitized by all treatments. Somatostatin, as well as peptidic (SMS201995) and nonpeptidic (L-779,976) sst2 receptor agonists increased the EC50 for somatostatin inhibition 20-fold. In contrast, pretreatment with BBS, CCK, or PMA caused a modest 2-fold increase in the EC50 for cyclase inhibition. Whereas the PKC inhibitor GF109203X abolished sst2A receptor phosphorylation by CCK, BBS, and PMA, it did not alter the effect of somatostatin, demonstrating that these reactions were catalyzed by different kinases. Consistent with a functional role for PKC-mediated receptor phosphorylation, GF109203X prevented PMA stimulation of sst2 receptor internalization. Surprisingly, however, GF109203X did not inhibit BBS and CCK stimulation of sst2A receptor endocytosis. These results demonstrate that homologous and heterologous hormones induce sst2A receptor phosphorylation by PKC-independent and -dependent mechanisms, respectively, and produce distinct effects on receptor signaling and internalization. In addition, the heterologous hormones also modulate sst2 receptor internalization by a novel mechanism that is independent of receptor phosphorylation.
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INTRODUCTION
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SOMATOSTATIN OR SOMATOTROPIN release-inhibiting factor (SRIF) is a widely distributed peptide in both the central and peripheral nervous systems as well as in a variety of tissues including the pancreas, the pituitary, gastrointestinal tract, adrenals, kidneys, and immune system (1, 2, 3). Somatostatin modulates numerous functions in these targets, including exocrine and endocrine secretion, neurotransmission, motor, behavioral and cognitive operations, intestinal motility and nutrient absorption, vascular contraction, and cell proliferation (1, 2, 3). The cellular actions of somatostatin appear to be regulated via the expression of different somatostatin receptors in different cell types (1, 2, 3). The five known somatostatin receptor subtypes, named sst1 through sst5, share the predicted secondary structure of the G protein-coupled receptor (GPCR) family, namely seven-transmembrane domains connected by three intracellular and three extracellular loops (2, 3). In mice and rats, the sst2 receptor exits in two spliced isoforms, sst2A and sst2B, which differ in their C termini but the sst2B receptor variant remains to be demonstrated in humans (4). The sst2A receptor is the predominant isoform in all mouse tissues tested (5).
Somatostatin receptors are primarily coupled to pertussis toxin-sensitive, inhibitory G proteins (Gi/o). Although all the receptor subtypes regulate some common effectors, such as adenylyl cyclase, the receptors appear to also utilize distinct signaling pathways (2, 3, 6). In addition to adenylyl cyclase, sst receptors may modulate the activity of K+ and Ca2+ channels, phospholipase C (PLC), phosphoinositide-3 kinase, MAPKs, and protein phosphatases (2, 3).
As for other GPCRs, signaling by sst receptors can be diminished upon prolonged agonist stimulation in a process known as homologous desensitization (2, 3). A general model for GPCR regulation by agonist postulates that receptors, upon activation by ligand, undergo phosphorylation by GPCR kinases (GRKs) (7, 8). Receptor phosphorylation, in turn, promotes high- affinity binding of arrestins that sterically inhibit subsequent receptor-G protein interactions, thereby causing receptor desensitization. Arrestins also associate with clathrin heavy chains to mediate receptor internalization in clathrin-coated pits (8, 9, 10). Internalized receptors may be recycled to the cell surface or degraded intracellularly, thereby regulating cellular levels of receptor (11, 12).
Phosphorylation of receptors may also be catalyzed by second messenger-activated protein kinases, such as protein kinase C (PKC) or cAMP-dependent protein kinase (PKA) (7). In contrast to receptor phosphorylation by GRKs, phosphorylation by second messenger-activated kinases does not require receptor occupancy by agonist. Thus, second messenger-activated protein kinases may be involved in desensitization by ligands binding to heterologous receptors as well as by agonists for the homologous receptor (13, 14, 15).
Using cell lines transfected to express high receptor levels, we previously showed that sst1 and sst2A receptors are phosphorylated upon somatostatin binding and upon pharmacological activation of PKC with phorbol 12-myristate 13-acetate (PMA) (16, 17, 18). Both sst1 and sst2A receptors undergo rapid homologous desensitization although the sst2A receptor internalizes rapidly (t1/2 = 4 min) whereas the internalization of the sst1 receptor is slow (t1/2 > 180 min). Moreover, PMA activation of PKC stimulates sst2A receptor internalization (17). These and other similar studies, which used engineered cell lines overexpressing individual sst receptors, have provided important insights into the mechanisms regulating somatostatin receptor function (1, 2, 3, 16, 17, 18). However, it is not clear to what extent the regulation of exogenously expressed receptors mimics physiological events because variability in receptor desensitization and trafficking has been observed when somatostatin receptors are expressed in different cell lines (3). Further, there are no reports of the effect of physiological activation of PKC on somatostatin receptor phosphorylation, trafficking, and signal transduction. Thus, we do not know whether activation of heterologous receptors can regulate somatostatin function and whether such regulation is mediated by PKC-induced receptor phosphorylation or by other mechanisms. Our goal here was to investigate the manner in which endogenous somatostatin receptors are regulated by homologous and heterologous hormones in a more physiological context, using AR42J cells, a differentiated pancreatic acinar cell line widely used as a model for the exocrine pancreas (19, 20).
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RESULTS
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Identification of Functional sst2 Receptors in AR42J Cells
Previous studies have reported that AR42J cells express mRNA for sst1, sst2, and sst3, and not for sst5, but detection of different sst subtype mRNAs has been variable (21, 22, 23, 24). Further, the expression of sst4 mRNA has not been examined. Thus, we first determined which sst receptors were present in AR42J cells under our culture conditions using subtype-specific somatostatin analogs to detect functional receptor protein. We used two different radiolabeled agonists in competition binding experiments with AR42J membranes (Fig. 1A
): [125I-Tyr11]SRIF (somatostatin), which binds to all sst receptor subtypes, and [125I-Tyr3]SMS (octreotide), which binds primarily to sst2 and sst5 receptors (20). SMS inhibited the binding of both ligands by at least 95%, indicating that the receptors in AR42J cells were of the sst2 and/or sst5 subtypes. To distinguish between these receptors, the binding of [125I-Tyr3]SMS was competed with increasing concentrations of two nonpeptide sst receptor agonists: L-779,976, which is selective for the sst2 receptor; and L-817,818, which is selective for sst5 (25) (Fig. 1B
). Whereas L-817,818 was inactive, the EC50 values for somatostatin, SMS, and L-779,976 were 0.33, 0.28 and 1.87 nM, respectively, consistent with the reported affinities of these compounds for the sst2 receptor (25, 26, 27).

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Figure 1. Analysis of sst Receptor Subtypes in AR42J Cell Membranes Using Somatostatin Analogs
A, Competition for [125I-Tyr11]SRIF and [125I-Tyr3]SMS binding by 100 nM nonlabeled somatostatin or SMS. Specific binding for each labeled peptide, 13,921 ± 1,456 cpm for [125I-Tyr11]SRIF and 33,711 ± 2,210 cpm for [125I-Tyr3]SMS, are expressed as 100% ± SEM (n = 4). B, Competition of [125I-Tyr3]SMS binding by increasing concentrations of somatostatin, SMS, L-779,976, and L-817,818. Specific binding in the absence of competitor (8554 ± 324 cpm) is expressed as 100%. Error bars represent SEM of triplicate. C, Inhibition of adenylyl cyclase activity by increasing concentrations of somatostatin, SMS, L-779,976, and L-817,818. Adenylyl cyclase activity was assayed with 100 nM VIP in the presence of the indicated analog concentrations. Data represent the mean ± SEM (n = 3) as a percentage of the adenylyl cyclase activity in the absence of SMS in a representative experiment.
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We next addressed the signaling capability of somatostatin receptors in AR42J cells. As shown in Fig. 1C
, somatostatin, SMS, and L779976 inhibited vasoactive intestinal peptide (VIP)-stimulated adenylyl cyclase activity to the same extent, whereas L-817,818 was inactive. The EC50 values for somatostatin, SMS, and L-779,976 were 6.85 nM, 1.89 nM, and 11.38 nM, respectively. As expected, the EC50 values for cyclase inhibition were higher than those for membrane binding, since the former are determined in the presence of GTP, whereas the latter are measured in the absence of nucleotide. Overall, these results indicate that only sst2 receptors are functionally expressed in AR42J cells. In previous studies we had found that more than 70% of the [125I-Tyr11]SRIF-receptor complex solubilized from AR42J cells is immunoprecipitated with antibodies to the sst2A receptor subtype, an immunoprecipitation efficiency similar to that observed for receptors from CHO cells expressing only the sst2A receptor (28). Thus, most of the receptors in AR42J cells exist as the sst2A splice variant.
We previously reported that incubation of GH-R2 cells with somatostatin or with the PKC activator PMA markedly stimulates the phosphorylation of heterologously expressed sst2A receptors (16, 17). Therefore, we next determined whether the native sst2A receptor in AR42J cells was phosphorylated. As shown in Fig. 2
, the sst2A receptor from GH-R2 and AR42J cell membranes exhibited very similar apparent molecular weights, consistent with similar receptor glycosylation in the two cell lines. Exposure of AR42J cells for 15 min to 100 nM SMS or PMA increased sst2A receptor phosphorylation 10.5 ± 2.8 (n = 5)- and 4.8 ± 0.2 (n = 2)-fold, respectively. At the same time, cholecystokinin (CCK) and bombesin (BBS), which stimulate PKC via the production of diacylglycerol (23, 24, 25), increased receptor phosphorylation 7.4 ± 1.6 (n = 3)- and 7.8 ± 1.9 (n = 3)-fold, respectively. These results demonstrate that somatostatin agonists, pharmacological agents that directly activate PKC, and ligand activation of heterologous PLC-coupled receptors all markedly stimulate sst2A receptor phosphorylation.

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Figure 2. Western Blot and Phosphorylation Analysis of sst2A Receptor in AR42J Cells
Left, Detection of sst2A receptor. GH-R2 (20 µg) and AR42J (100 µg) cell membranes were subjected to immunoblot analysis using sst2A receptor-specific antibodies. Right, Phosphorylation of sst2A receptor. 32PO4-labeled AR42J cells were incubated in the absence or in the presence of 100 nM somatostatin, 200 nM PMA, 100 nM BBS, or 100 nM CCK for 15 min. Sst2 receptor was solubilized, WGA-agarose purified, and immunoprecipitated before analysis on SDS-PAGE and autoradiography. The left side of each panel shows molecular masses of standard proteins.
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Characterization of sst2A Receptor Phosphorylation
Figure 3
shows the time course of sst2A receptor phosphorylation after stimulation of AR42J cells by different agents. SMS, CCK, and BBS induced a maximal phosphorylation of 17.1-, 11.8-, and 12.7-fold over basal, respectively. Half-maximal phosphorylation was observed after about 5 min of SMS treatment. In contrast, CCK and BBS produced a maximal effect within 2 min. Thus, homologous and heterologous stimulation of sst2A receptor phosphorylation occurred at different rates.

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Figure 3. Time Course of sst2A Receptor Phosphorylation
32PO4-labeled AR42J cells were treated without or with 100 nM SMS, 100 nM BBS, or 100 nM CCK for the indicated times. Sst2A receptor was solubilized, WGA-agarose purified, and immunoprecipitated before analysis on SDS-PAGE and autoradiography. Sst2 receptor phospholabeling was quantitated by phosphoimaging and is expressed as fold over basal phosphorylation observed in nontreated cells.
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To identify the phosphorylated regions of the sst2A receptor, we carried out peptide mapping using N-chlorosuccinimide (NCS), which cleaves proteins at tryptophan residues. Cleavage of the receptor with NCS is predicted to generate nine peptides, five of which contain intracellular serines or threonines (see Ref. 17 for details). The predicted molecular masses of the third loop and the C-terminal tail of the sst2A receptor are 7 and 11 kDa, respectively. Using this method, we have shown that somatostatin and PMA stimulate phosphorylation on both the third loop and the C-terminal tail of the sst2A receptor in GH-R2 cells (17). Figure 4
shows that SMS, BBS, and CCK also stimulated receptor phosphorylation on both the third loop and C-terminal tail of the receptor in AR42J cells. With all three ligands the C tail was more intensely labeled than the third intracellular loop. Therefore, the same domains in the sst2A receptor are phosphorylated in response to SMS, BBS, or CCK treatment.

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Figure 4. Phosphopeptide Mapping of sst2A Receptor
32PO4-labeled AR42J cells were treated with 100 nM SMS, CCK, or BBS for the indicated times. Sst2AR was solubilized, WGA-agarose purified, and immunoprecipitated before analysis on SDS-PAGE and autoradiography. Sst2A receptor was eluted from the gel and cleaved with 25 mM NCS. The resulting phosphopeptides were subjected to tricine-SDS-PAGE, transferred to PVDF membrane, and analyzed by phosphoimaging. Based on predicted molecular mass and previous results, the two discernable phosphopeptides represent the third intracellular loop (3L; 7.5 kDa) and C-terminal tail (CT; 11 kDa).
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Stimulation of sst2 Receptor Internalization
We previously found that the internalization of receptor-bound somatostatin is increased in PMA-treated GH-R2 cells (17). Figure 5
shows the effect of BBS, CCK, and PMA on the subcellular distribution of bound [125I-Tyr3]SMS in AR42J cells. An extensive and time-dependent intracellular accumulation of [125I-Tyr3]SMS occurred at 37 C in both untreated and treated cells while surface binding was relatively low and essentially constant. None of the agents altered the amount of cell surface ligand. However, after a 60-min incubation with BBS, CCK, or PMA, internalized radioligand was increased 2.6 ± 0.2-, 2.3 ± 0.3-, and 1.8 ± 0.2-fold (n = 8), respectively. These values are significantly different from untreated cells (P < 0.01 in a paired t test). Further, stimulation by BBS and CCK was significantly higher than that by PMA (P < 0.01), whereas the effects of BBS and CCK were not significantly different from each other (P > 0.05). In contrast to BBS and CCK, which activate PLC- coupled receptors, VIP, which activates adenylyl cyclase via its own receptor in AR42J cells (Fig. 1
), did not affect [125I-Tyr3]SMS internalization (data not shown). This observation is consistent with our previous results showing that elevation of cAMP does not increase sst2A receptor phosphorylation (16). Thus, BBS, CCK, and PMA specifically stimulate the internalization of the ligand-sst2 receptor complex.

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Figure 5. Effect of BBS, CCK, and PMA on [125I-Tyr3]SMS Internalization
AR42J cells were preincubated in the absence or in the presence of 100 nM BBS or CCK for 2 min or PMA for 15 min before addition of [125I-Tyr3]SMS (55,000 cpm) at 37 C for the indicated times. Nonspecific binding determination was performed in the presence of 100 nM nonlabeled SMS. Cells were then washed with ice-cold PBS and incubated with acidic buffered-saline to remove surface-bound ligand. Then, cells were solubilized in 0.1 N NaOH to quantitate internalized ligand. The measured radioactivity was expressed as specific binding. Data represent the mean ± SEM (n = 4) of a representative experiment.
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Homologous Desensitization of the sst2 Receptor
To determine the analog specificity for homologous desensitization, AR42J cells were incubated in the presence of different sst2 receptor agonists, and then membranes were prepared and used for adenylyl cyclase measurements (Fig. 6A
). The EC50 for SMS inhibition of VIP-stimulated adenylyl cyclase was increased from 3.3 ± 0.1 nM in nontreated membranes to 122 ± 1.9, 74.6 ± 1.5, and 50.6 ± 0.8 nM in membranes treated for 30 min with 100 nM SMS, somatostatin, and L-779,976, respectively (Fig. 6A
).

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Figure 6. Homologous Desensitization of sst2 Receptor
AR42J cells, pretreated as indicated, were used to prepare membrane. Adenylyl cyclase activity was assayed with 100 nM VIP in the presence of the indicated SMS concentration. Data represent the mean ± SEM (n = 3) of a representative experiment and are expressed as a percentage of the adenylyl cyclase activity in the absence of SMS. A, Effect of pretreatment with different agonists on SMS inhibition of VIP-stimulated adenylyl cyclase. AR42J cells were incubated in the absence or in the presence of 100 nM somatostatin, SMS, or L-779,976 for 30 min. B, Time course for SMS-induced sst2 receptor desensitization. AR42J cells were incubated in the absence or in the presence of 100 nM SMS for 2, 30, or 60 min. C, Dose response for somatostatin-induced sst2 receptor desensitization. AR42J cells were incubated in the absence or in the presence of 2, 10, or 100 nM for 30 min.
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The sst2A receptor was partially desensitized within 2 min of SMS treatment, maximal desensitization occurred by 30 min, and desensitization was sustained for at least 1 h (Fig. 6B
). The EC50 for SMS inhibition of VIP-stimulated adenylyl cyclase was increased from 3.1 ± 0.1 nM in nontreated membranes to 24.5 ± 0.2, 50.3 ± 0.9, and 50.0 ± 0.5 nM in membranes treated for 2, 30, and 60 min, respectively (Fig. 6B
). Somatostatin pretreatment produced a similar time course except that 1-h stimulation was slightly less effective than 30 min, perhaps due to the lower stability of somatostatin compared with SMS. Thus, homologous sst2A receptor desensitization and phosphorylation occur within the same time frame.
The sensitivity of sst2A receptor desensitization was analyzed by incubating AR42J cells for 30 min with different concentrations of somatostatin and measuring the effect on adenylyl cyclase activity (Fig. 6C
). The EC50 for SMS inhibition of adenylyl cyclase was increased from 2.2 ± 0.1 nM in untreated membranes to 3.7 ± 0.1, 9.1 ± 0.1, and 24.2 ± 0.5 nM after pretreatment with 2, 10, or 100 nM somatostatin, respectively. In a total of eight experiments, homologous desensitization with 100 nM somatostatin produced an average 20-fold shift in the EC50 for cyclase inhibition by SMS (Table 1
). Maximal inhibition of adenylyl cyclase in membranes from cells pretreated for 30 min with SRIF or SMS was not significantly different from that seen in control membranes.
Together, these results demonstrate that exposure to peptide and nonpeptide sst2 agonists results in a marked homologous desensitization of this receptor in a time- and concentration-dependent manner.
Heterologous Desensitization of the sst2 Receptor
To determine the effect of heterologous agents on sst2 receptor desensitization, AR42J cells were pretreated for 15 min at 37 C, and then the inhibitory effect of SMS on adenylyl cyclase activity was measured in membranes. Figure 7
shows that preincubation with BBS, CCK, or PMA reduced the potency of SMS to inhibit adenylyl cyclase, but this effect was much smaller than that produced by somatostatin treatment. Table 1
summarizes the data from multiple experiments. The EC50 value for SMS was about 20 times higher than control after somatostatin treatment, whereas the EC50 values after BBS, CCK, and PMA incubation for 15 min were only 2-fold higher (Table 1
). In addition, CCK and BBS slightly, but significantly, attenuated maximal inhibition of adenylyl cyclase whereas PMA and somatostatin did not (Table 1
). None of the agents affected VIP-stimulated adenylyl cyclase activity (data not shown). These results indicate that the sst2 receptor is subject to both homologous and heterologous desensitization in AR42J cells, although the magnitude of the effects differs markedly.

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Figure 7. Heterologous Desensitization of sst2 Receptor
AR42J cells, pretreated in the absence or in the presence of 100 nM BBS, CCK, PMA, or somatostatin for 15 min, were used for membrane preparation. Adenylyl cyclase activity was assayed with 100 nM VIP in the presence of the indicated SMS concentrations. Data represent the mean ± SEM (n = 3) of a representative experiment and are expressed as a percentage of the adenylyl cyclase activity in the absence of SMS.
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Role of PKC in the Regulation of the sst2A Receptor
We next examined the role of PKC in mediating the regulatory effects of different hormones and used GF109203X, a highly selective PKC inhibitor acting at the ATP binding site (29). AR42J cells were preincubated with GF109203X for 15 min, and then stimulated with either somatostatin or heterologous agents for 5 min. Figure 8
shows that GF109203X abolished the effect of BBS, CCK, and PMA on sst2A receptor phosphorylation but did not inhibit somatostatin receptor stimulation. Thus, the stimulatory effect of PMA, CCK, and BBS on sst2A receptor phosphorylation is mediated by activation of PKC, whereas agonist-induced phosphorylation is not.

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Figure 8. Effect of PKC Inhibition on the Phosphorylation of sst2A Receptor
32PO4-labeled AR42J cells were preincubated in the absence or in the presence of 4 µM GF109203X for 15 min and then incubated without or with 100 nM SMS, 200 nM PMA, 100 nM BBS, or 100 nM CCK for 5 min. Sst2A receptor was solubilized, WGA-agarose purified, and immunoprecipitated before analysis on SDS-PAGE and autoradiography.
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We next investigated the functional consequences of PKC inhibition (Fig. 9
). Although, in the experiments shown, GF109203X slightly reduced unstimulated [125I-Tyr3]SMS internalization, this effect was not significant when averaged over four independent experiments. However, GF109203X reduced 99 ± 15% of PMA-stimulated internalization of receptor-bound [125I-Tyr3]SMS (P < 0.05, paired t test, n = 4). In contrast, the PKC inhibitor did not significantly affect the BBS stimulation. Similarly, GF109203X did not reproducibly inhibit CCK stimulation (data not shown). These results indicate that PKC mediates the PMA stimulation of sst2 receptor internalization but not the effects of BBS and CCK.

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Figure 9. Effect of Various Inhibitors on [125I-Tyr3]SMS Internalization
A, AR42J cells were preincubated for 30 min in media supplemented with 5 mM EGTA in the absence or in the presence of 4 µM GF109203X, 2 µM thapsigargin, 1 µM K252a, or combinations of these agents, as indicated. Then, 100 nM BBS, CCK, or PMA was added to the cells, followed immediately by addition of [125I-Tyr3]SMS (100,000 cpm) with or without unlabeled SMS to allow the determination of specific binding. After a 60-min incubation at 37 C, internalized radioligand was measured as described in Fig. 5 . The bars show the mean ± SEM of internalized radioligand (specific binding) in quadruplicate samples. B, AR42J cells were preincubated in the absence or in the presence of 4 µM GF109203X or 200 µM genistein for 20 min. Then, 100 nM BBS, CCK, or PMA was added to the cells and followed immediately by the addition of [125I-Tyr3]SMS (100,000 cpm) with or without unlabeled SMS. After incubating at 37 C for 1 h, internalized radioligand was measured as described in Fig. 5 . The bars show the mean ± SEM of internalized radioligand (specific binding) in quadruplicate samples.
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We also examined the effect of GF109203X on sst2A receptor desensitization. However, exposure of AR42J cells to 1 µM GF109203X alone decreased the potency of SMS to inhibit adenylyl cyclase from EC50 = 1.45 ± 0.02 to 2.67 ± 0.04 (n = 7). Although small, this effect was statistically significant in a paired t test (P < 0.01). Because GF109203X by itself increased the EC50 for SMS and because the effect of BBS, CCK, and PMA on sst2 receptor desensitization also produced only a 2-fold increase in the EC50, we were unable to determine whether GF109203X prevented heterologous desensitization of sst2A receptor. However, this inhibitor did not affect somatostatin-induced desensitization (data not shown).
Taken together, our results demonstrate that PKC is involved in heterologous, but not homologous, regulation of sst2A receptor phosphorylation and internalization in AR42J cells. Additionally, they suggest that PKC-independent pathways play a role in the regulation of sst2 receptor internalization by Gq-coupled receptors.
PKC-Independent Mechanisms Stimulating sst2A Receptor Internalization
To further examine the mechanism by which BBS and CCK stimulated sst2 internalization, we used several known inhibitors of BBS and CCK signaling. Figure 9A
shows the effect of thapsigargin, a microsomal ATPase inhibitor that causes depletion of intracellular calcium stores and has been shown to inhibit intracellular Ca2+ release in pancreatic acinar cells as well as in AR42J cells (30, 31) and K252a, a potent inhibitor of Ca2+/calmodulin kinase, which also inhibits PKA, PKC, and cGMP-dependent protein kinase at higher doses (32, 33). These inhibitors were used, in the presence of 5 mM EGTA, to chelate extracellular Ca2+ (Fig. 9A
). Neither thapsigargin nor K252a affected BBS-stimulated internalization either alone or in combination with GF109203X, whereas both GF109203X and K252a abolished the PMA effect in the same experiment. These results indicate that neither BBS receptor-induced increases in intracellular Ca2+ nor the subsequent activation of calmodulin kinase are involved in BBS stimulation of sst2 receptor internalization.
We also assessed the effect of genistein, a tyrosine kinase inhibitor that was previously shown to block BBS stimulation of tyrosine phosphorylation of p125FAK and paxillin in AR42J cells (34). Although genistein inhibited basal [125I-Tyr3]SMS internalization, it did not prevent the stimulatory effect of either BBS or PMA (Fig. 9B
). In contrast, in the same experiment, GF109203X blocked PMA stimulation both by itself and in the presence of genistein. These results indicate that the effect of BBS on sst2A receptor internalization is not related to its previously reported stimulation of tyrosine kinase activity. Similar results were observed for CCK (data not shown). Thus, BBS and CCK stimulate sst2A internalization independently of Ca2+ mobilization as well as activation of PKC, Ca2+/calmodulin kinase II, and tyrosine kinases.
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DISCUSSION
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Although the regulation of somatostatin receptors has been extensively investigated in a variety of differentiated cells, most previous studies either did not identify the particular receptor subtype being examined or did not distinguish between multiple somatostatin receptor types present. As a result, little is known about the molecular mechanisms involved in the control of individual sst receptor subtypes in somatostatin targets. Using a pancreatic acinar cell line that expresses sufficient receptor for both biochemical and functional analysis, we show for the first time that rapid agonist-stimulated phosphorylation of the sst2A receptor occurs concomitantly with receptor desensitization. Homologous sst2A receptor desensitization is induced by a novel nonpeptidic agonist (L-779,976), by the clinically used somatostatin analog, octreotide, as well as by the native peptide. Further, heterologous activation of PLC-coupled receptors endogenously expressed in AR42J cells also stimulates the rapid phosphorylation, desensitization, and internalization of the sst2A receptor, but the rate and extent of these effects differ from those induced by agonists. Whereas protein kinase C is involved in sst2A receptor phosphorylation by PLC-coupled receptors, it does not play in a role in homologous receptor phosphorylation. Unexpectedly, PKC-dependent sst2A receptor phosphorylation cannot account for the increased internalization produced by PLC-coupled receptors, as this stimulatory effect was not blocked by PKC inhibitors. Together, our studies indicate that the sst2A receptor subtype is subject to complex modulation by both homologous and heterologous mechanisms that produce different functional outcomes.
Somatostatin action is thought to depend both on the nature of the receptor subtypes in a cell and cell-specific regulatory functions. For example, in the GH4C1 and AtT20 pituitary cell lines, the magnitude of the somatostatin inhibition of intracellular calcium concentrations is diminished within a few minutes of continuous somatostatin exposure (35, 36). In contrast to this transient effect, inhibition of intracellular calcium levels is sustained for at least 20 min in the GH3 pituitary cell line (35). GH3 and GH4C1 cells express sst1 and sst2 receptors (23, 28, 37), but AtT20 cells have been reported to contain five different sst receptor subtypes (sst1, sst2A, sst2B, sst4, and sst5) (38, 39). Unfortunately, the multiplicity of sst receptors expressed complicate mechanistic analysis of sst receptor regulation in these models.
To examine the behavior of individual sst receptor subtypes in a common cellular background, a number of investigators have analyzed sst receptor function in transfected cell lines and provided clear evidence for subtype-specific desensitization and endocytosis behavior (2, 3, 40). For example, sst3 receptor inhibition of adenylyl cyclase is rapidly desensitized in HEK embryonic kidney cells (41), whereas sst4 receptor inhibition is not (42). Similarly, sst receptor subtypes, like other GPCRs, show strikingly different internalization kinetics when expressed in different cell types (2, 3, 43). However, there are two major disadvantages of transfected cell systems. First, specific desensitization and internalization components may be limiting or overabundant in host cells that do not naturally express a particular receptor (44, 45). Second, high levels of expressed receptors, often 10100 times greater than those found in cells expressing sst receptors endogenously, may induce molecular events that do not occur at physiological receptor levels or may overwhelm particular cellular constituents present in limiting amounts. In fact, there are now numerous examples of the same sst receptor subtype showing different signaling or regulatory properties when expressed in different host cell types (3). Therefore, in this study we examined the regulation of endogenous somatostatin receptors in AR42J cells, which are a well established cell model that mimics normal exocrine pancreatic cell function (19, 20). These cells express about 10% the amount of sst2A receptor protein found in GH-R2 cells (Fig. 2
). This result is consistent with previous measurements using radioligand binding, which reported that AR42J cells contain 1 pmol of somatostatin receptor per mg membrane protein (46) compared with 10 pmol/mg membrane protein in GH-R2 cells (16).
Somatostatin agonists inhibit the proliferation of AR42J cells and counteract the mitogenic action of a variety of growth factors, including epidermal growth factor, basic fibroblast growth factor, and gastrin (26, 46, 47). Somatostatin-induced growth inhibition is proposed to involve activation of tyrosine phosphatases, including SH2 domain containing phosphatase 1 (commonly called SHP-1), which associates with the sst2 receptor in rat pancreatic membranes (48, 49). Inhibition of the phosphatidylinositol 3-kinase signaling pathway may also be involved, leading to inhibition of Akt phosphorylation (50). Both stimulation of SHP-1 phosphatase and inhibition of Akt kinase contribute to the up-regulation of the cyclin kinase inhibitor p27(kip). As expected, somatostatin agonists suppress mitogen-induced p27(kip) down-regulation (50, 51). Somatostatin also increases intracellular Ca2+ in AR42J cells, in contrast to the inhibition observed in most cell lines (35, 36). This increase has been proposed to occur by activation of a cell-surface calcium channel without stimulation of PLC (52). By characterizing the binding and cyclase-inhibitory effects of selective somatostatin analogs, we found that only the sst2 receptor is functionally expressed in AR42J cells (Fig. 1
). Since the sst2A receptor is also present in normal acinar cells (53) and pancreatic tumors (54), where somatostatin also regulates cell proliferation (54), the AR42J cell line provides a particularly suitable and important model to analyze sst2A receptor regulation.
Our results indicate that agonist and PKC activation both induce sst2A receptor phosphorylation in AR42J cells. Interestingly, the rate of homologous receptor phosphorylation is somewhat slower than the rate of heterologous receptor phosphorylation (Fig. 2
), suggesting that the concentrations and activities of the kinases may determine the relative importance of different modulatory effects. In contrast to the marginal effect of BBS on sst2A receptor phosphorylation in GH-R2 cells (17), the stimulation by BBS and CCK is pronounced in AR42J cells, possibly due to higher BBS and CCK receptor levels in the latter. The phosphorylation induced by agonist occurs at the third intracellular loop and C tail of the sst2A receptor in both cell lines. The same receptor domains are also phosphorylated when AR42J cells are stimulated by heterologous ligands. Together, our results demonstrate that phosphorylation of the endogenous sst2A receptor is markedly stimulated by agonist as well as by heterologous hormones that activate PLC, such that the functional consequences of homologous and heterologous receptor phosphorylation could be assessed in AR42J cells.
We previously showed that sst2A inhibition of adenylyl cyclase was desensitized after a 30-min exposure to SMS in GHR2 cells (16) but the kinetic and the dose-response characteristics for desensitization of this receptor have not been reported. We show here that homologous desensitization of the sst2A receptor occurs at low, physiological concentrations of agonist (nanomolar) and proceeds extremely rapidly. Desensitization is detectable within 2 min and is sustained for more than 1 h. In fact, desensitization appears to occur somewhat more rapidly than receptor phosphorylation, which takes longer to reach a maximum. This difference may reflect the fact that different residues on the sst2A receptor are phosphorylated at different rates by either one or multiple protein kinases and that only a subset of these phosphorylations lead to desensitization. Alternatively, rapid desensitization may result from molecular events that are independent of sst2A phosphorylation. Numerous examples now exist to indicate that the phosphorylation, desensitization, and internalization of various GPCRs are not necessarily linked (55, 56, 57, 58). Therefore, to understand how phosphorylation is involved in sst2A receptor desensitization, clearly it will be necessary to identify and eliminate the different phosphorylation sites on the receptor.
Although numerous examples now exist for differential regulation of GPCRs by structurally distinct agonists (59, 60, 61, 62), the extent to which various analogs produce somatostatin receptor desensitization is unknown despite its therapeutic importance. Our studies demonstrate that desensitization of the sst2A receptor occurs with a recently developed nonpeptidic agonist highly selective for the sst2 receptor subtype (25) as well as with the clinically used peptide analog octreotide (SMS201995), in the same way as it does with the endogenous hormone, somatostatin. These observations suggest that these structurally diverse compounds induce similar active receptor conformations.
Little is known about the mechanisms involved in heterologous regulation of somatostatin receptors. Several previous studies reported the effects of long-term treatment (hours to days) by different hormones on sst2 receptor function. For example, a 48-h exposure to gastrin, which binds to the CCK receptor, or to epidermal growth factor, was shown to inhibit the effect of somatostatin on VIP-stimulated cAMP accumulation in AR42J cells (21). We report here for the first time that heterologous hormone receptors are involved in the acute regulation of the sst2A receptor. In contrast to the extensive homologous desensitization, heterologous desensitization was modest although significant, suggesting that distinct mechanisms mediate these effects. This conclusion is supported by the fact that inhibition of PKC by GF109303X blocks the stimulation of sst2A receptor phosphorylation by BBS, CCK, and PMA but not by somatostatin. Thus, PKC is implicated in the heterologous, but not homologous, phosphorylation of the sst2A receptor.
The sst2 receptor is rapidly internalized in AR42J cells (Fig. 5
), consistent with previous data in some other experimental systems (17, 63). The present study shows that PLC-coupled receptors, such as the ones for BBS and CCK, stimulate the internalization of the sst2 receptor at least as effectively as pharmacological activation of PKC by PMA. PMA stimulation of sst2 receptor internalization was completely blocked by PKC inhibition with either GF109203X or K252a, demonstrating that PKC activation is sufficient to mediate this effect. Surprisingly, however, BBS and CCK stimulation of sst2 receptor internalization was not suppressed by PKC inhibition. Since the PKC inhibitor GF109203X completely blocked the stimulation of sst2A receptor phosphorylation by BBS and CCK (Fig. 8
), these peptides must be able to stimulate receptor internalization by a mechanism independent of receptor phosphorylation.
Both PKC-dependent and -independent signaling pathways are known to be activated by BBS and CCK receptors (64, 65, 66, 67). To identify the PKC-independent pathway by which BBS and CCK stimulate sst2 internalization, we examined the effects of several inhibitors. BBS and CCK receptor stimulation of PLC leads to rapid production of inositol 1,4,5-trisphosphate followed by Ca2+ release from intracellular stores, as well as the production of the PKC activator, diacylglycerol. Simultaneous inhibition of both intracellular calcium mobilization by thapsigargin and of PKC by GF109203X are necessary to abolish the stimulatory effect of CCK on PYK2/CAKß tyrosine phosphorylation in pancreatic acini (30). However, in our experiments thapsigargin combined with GF109203X caused no additional inhibition of CCK- and BBS-stimulated sst2 receptor internalization over that seen with the PKC inhibitor alone (Fig. 9
). Therefore, the effect of BBS and CCK must be independent of PLC activation. We also found that neither Ca2+-activated calmodulin-kinase nor PKC, both of which are inhibited by K252a (32, 33), are involved in BBS stimulation of sst2A receptor internalization.
BBS also stimulates the tyrosine phosphorylation of p125FAK and paxillin in AR42J cells (34). Interestingly, this stimulation is completely inhibited by genistein, a tyrosine protein-kinase inhibitor (34). However, in our experiments genistein or genistein combined with GF109203X did not inhibit BBS-stimulated internalization of the sst2A receptor (Fig. 9
), suggesting that BBS stimulation of tyrosine kinase activity is not involved.
Recently, Pfeiffer et al. (68) showed that agonist and PMA treatment both lead to the internalization of the sst2A receptor transfected in HEK293. Thus, PKC activation alone is able to induce sst2A receptor internalization in the absence of receptor occupancy. In the same study, the authors proposed that heterodimerization between sst2A receptor and opioid receptors cross- modulate receptor phosphorylation, internalization, and desensitization. Whether or not a similar mechanism occurs with sst2A and BBS or CCK receptors remains to be determined, although we did not see any evidence for such dimers on SDS-PAGE gels (Fig. 2
).
In summary, our study demonstrates that the sst2A receptor is rapidly regulated by agonist as well as by heterologous GPCRs. Because SMS is used clinically to inhibit a variety of neuroendocrine tumors and radiolabeled SMS analogs are used for tumor diagnosis (69), modulation of sst2A receptor function is likely to have important therapeutic consequences and possibly may be exploited to improve the sensitivity of diagnostic procedures involving sst2 receptor-mediated radioligand internalization. Therefore, elucidation of early events, which occur after exposure to agonist as well as heterologous hormones, may provide new strategies to enhance the clinical utility of sst2 receptor-targeted drugs.
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MATERIALS AND METHODS
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Hormones and Supplies
SRIF and [Tyr11]SRIF were purchased from Bachem (Torrance, CA). [Tyr3]SMS201995 and SMS201995 (SMS) were a gift from Sandoz Pharmaceuticals Corp. (Basel, Switzerland). L-779,976 and L-817,818 were generously provided by Merck \|[amp ]\| Co., Inc. (Rahway, NJ). Cell culture media and fetal bovine serum were purchased from Life Technologies, Inc. (Gaithersburg, MD) and Atlanta Biologicals (Norcross, GA), respectively. The generation and specificity of sst2A receptor antiserum (R288) have been described previously (18). Leupeptin, pepstatin A, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, bacitracin, PMA, NCS, protein A, and p-nitrophenyl phosphate were obtained from Sigma (St Louis, MO). N-Dodecyl-ß-D-maltoside was purchased from Calbiochem (San Diego, CA). Okadaic acid, thapsigargin, and GF109203X-HCl were purchased from Alexis Biochemicals (San Diego, CA). K252a was purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). Dowex AG 1-X8 anion exchange resin (200400 mesh, chloride form), Bradford reagent, electrophoresis reagents, and polyvinylidene difluoride (PVDF) membrane were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Enhanced chemiluminescence Western blotting detection reagents, CNBr-activated Sepharose 4B, and carrier-free Na125I were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). [
-32P]ATP was from Perkin-Elmer Life Sciences Inc. (Boston, MA). [3H]cAMP was purchased from Moravek Biochemicals, Inc. (Brea, CA). Phosphate-free DMEM and [32P]orthophosphate were purchased from ICN Biomedicals, Inc. (Irvine, CA). Wheat germ agglutinin (WGA) agarose was purchase from Vector Laboratories, Inc. (Burlingame, CA). All other reagents were of analytical grade and purchased from common suppliers.
Cell Culture
The rat pancreatic acinar cell line AR42J was grown in F12/DMEM supplemented with 10% (vol/vol) fetal bovine serum, 20 U/ml penicillin, and 20 µg/ml streptomycin. Cell cultures, plated at density of 107 per 150-mm plates for membrane preparation and 2 x 105 per 35-mm dish for internalization experiments, were grown for 7 d with a medium change every 3 d. The GH-R2 cell line (GH4C1 cells stably transfected with sst2A receptor) was generated and grown as reported previously (16).
Membrane Preparation
Cells were pretreated at 37 C as indicated in each figure legend either in growth medium or in serum-free F12 medium containing 5 mg/ml lactalbumin hydrolysate (LH) and 20 mM HEPES, pH 7.4 (F12/LH). After pretreatment, cells were washed with ice-cold PBS, scraped into PBS, and centrifuged. The cell pellet was frozen on dry ice and stored until use. Cells were homogenized with a Dounce homogenizer pestle B in ice-cold 20 mM HEPES buffer (pH 7.6), supplemented with 1 mM EDTA, 2 mM tetrasodium pyrophosphate, 50 µg/ml bacitracin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium metavanadate, and 100 nM okadaic acid. The homogenates were centrifuged on a step gradient of 2343% (wt/vol) sucrose in 20 mM HEPES (pH 7.6), supplemented with 1 mM EDTA, 100 µM benzamidine, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 100 µM sodium metavanadate, and 100 nM okadaic acid. The fraction at the 23:43% (wt/vol) sucrose interface was collected and stored at -80 C.
Membrane preparation for Western blot analysis of GH-R2 and AR42J cells for the experiment shown in Fig. 2
were carried out as previously described (16).
Adenylyl Cyclase Assay
Membranes (5 µg protein/tube) were assayed at 30 C for 10 min in triplicate as described previously (16, 18) with modifications. The reaction was performed in the presence of 100 nM VIP and the indicated SMS concentrations in the presence of 1.2 mM MgCl2 and a cocktail of 100 nM okadaic acid, 1 mM p-nitrophenyl phosphate, and 200 µM sodium metavanadate to prevent sst2 receptor dephosphorylation during the assay.
Radioligand Binding and Internalization in Intact Cells
[Tyr3]SMS and [Tyr11]SRIF were radioiodinated using chloramine T and subsequently purified by reverse-phase HPLC as described previously (19). For internalization experiments, AR42J cells were incubated at 37 C in F12/LH media in the absence or presence of 100 nM BBS, CCK, or PMA and [125I-Tyr3]SMS (
50,000100,000 cpm/ml) without or with 100 nM unlabeled SMS. The experiment in Fig. 9A
was performed in F12/LH media supplemented with 5 mM EGTA to chelate medium calcium. After a wash with ice-cold PBS to remove unbound trace, cells were incubated on ice with acidic buffered saline (0.5 N acetic acid, pH 2.5; 200 mM NaCl) to dissociate surface-bound ligand. After collection of the acidic buffer, the cells were dissolved in 0.1 N NaOH to quantitate internalized ligand.
Radioligand Binding to Membranes
Membrane binding assays were performed in 35 mM Tris buffer, pH 7.6, supplemented with 5 mM MgCl2, 1.5 mM EDTA, 1.4 U/ml bacitracin, and 0.07% (wt/vol) BSA in 100 µl final volume. The binding reaction contained sucrose gradient-purified AR42J cell membranes (
2 µg protein), approximately 100,000 cpm [125I-Tyr3]SMS, or [125I-Tyr11]SRIF in the absence or the presence of 100 nM somatostatin or SMS. Reaction was carried out at 30 C for 2 h in multiscreen-FC, opaque plates (1.2-µm glass fiber type C filter, Millipore Corp., Molheim, France). After vacuum filtration of the binding reaction, the filters were washed with cold membrane binding buffer and counted for radioactivity. Specific binding was calculated as the difference between the amount of radioligand in the absence and presence of unlabeled SMS or somatostatin.
Phosphorylation of sst2A Receptor
Metabolic labeling of cells with 32PO4 and subsequent purification of the sst2 receptor have been described previously (15, 16, 17). In brief, after incubation for 3 h in phosphate-free DMEM supplemented with 1 mCi 32PO4/3.5 ml, AR42J cells were treated with different hormones or pharmacological agents as indicated in the figure legends. Cells were scraped into cold HEPES-buffered saline (20 mM HEPES, pH 7.4; 150 mM NaCl supplemented with 5 mM EDTA; 3 mM EGTA; 10 mM tetrasodium pyrophosphate; 50 µg/ml bacitracin; 10 µg/ml soybean trypsin inhibitors; 10 µg/ml leupeptin; 0.1 mM sodium orthovanadate; and 100 nM okadaic acid) and centrifuged. The cell pellet was solubilized in HEPES-buffered saline with 4 mg/ml dodecyl-ß-D-maltoside, after which the sst2 receptor was purified by WGA agarose chromatography and immunoprecipitation with R288 antibody. Sst2A receptor was solubilized in sample buffer [62.5 mM Tris-HCl, 2% (wt/vol) sodium dodecyl sulfate, 10% (vol/vol) 2-mercaptoethanol, 6 M urea, 20% (vol/vol) glycerol (pH 6.8)] for SDS-PAGE. Receptor phosphorylation was quantitated using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). For peptide mapping sst2A receptor was cleaved at tryptophan residues with NCS, and the resulting phosphopeptides were analyzed on tricine-urea SDS-PAGE as described previously (17).
Immunoblotting
Membrane proteins were subjected to SDS-PAGE and transferred to PVDF membrane (28). Immunoblot analysis was performed using R288 antibody as previously described (28) and detected using the enhanced chemiluminescence detection system from Amersham Pharmacia Biotech.
Statistical Analysis
Curve fitting and statistical analyses were performed using Prism software (GraphPad Software, Inc., San Diego, CA).
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FOOTNOTES
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This work was supported by research grants (to A.S.) from the NIH (DK-32234) and from the Welch foundation (AU1410).
1 Current address: Department of Pediatrics, The University of Oklahoma, Health Sciences Center, Oklahoma City, Oklahoma 73104. 
2 Current address: Department of Immunology, Schering-Plough Corp. Research Institute, Kenilworth, New Jersey 07033-0539. 
Abbreviations: BBS, Bombesin; CCK, cholecystokinin; Gi, G-inhibitory; GPCR, G protein-coupled receptor; LH, lactalbumin hydrolysate; NCS, N-chlorosuccinimide; PKC, protein kinase C; PMA, phorbol myristate acetate; SMS, SMS201995 or octreotide that has the structure FCFWKTCT(ol) with a disulfide bond between the two cysteine residues; PVDF, polyvinylidene difluoride; SRIF, somatotropin release-inhibiting factor or somatostatin; sst, somatostatin receptor; VIP, vasoactive intestinal peptide; WGA, wheat germ agglutinin.
Received for publication June 5, 2002.
Accepted for publication August 7, 2002.
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