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Somatostatin receptor subtype 2A (sst2A) mediates many of the endocrine and neuronal actions of somatostatin and is the target of somatostatin analogs in cancer therapy. As with many G-protein-coupled receptors, agonist stimulation causes sst2A receptor desensitization and internalization, events that require receptor phosphorylation. Furthermore, heterologous receptor activation of protein kinase C (PKC) also increases sst2A receptor phosphorylation and internalization. Here we analyzed a series of sst2A receptor mutants biochemically to identify residues in the receptor carboxyl terminus that were phosphorylated upon agonist stimulation, and we then generated four phosphorylation-sensitive antibodies to those residues. Once the selectivity of each antibody for its phosphorylated and nonphosphorylated target sequence was determined, the phospho-site-specific antibodies were used to demonstrate that somatostatin treatment of Chinese hamster ovary (CHO) cells expressing the wild type sst2A receptor increased phosphorylation on five residues in the receptor C terminus: Ser341, Ser343, Ser348, Thr353, and Thr354. Phorbol 12-myristate 13-acetate (PMA) increased receptor phosphorylation only on Ser343. Inhibition of PKC blocked PMA but not somatostatin stimulation, showing that different kinases catalyzed Ser343 phosphorylation. In contrast, somatostatin-stimulated sst2A receptor phosphorylation was inhibited by knockdown of G-protein coupled receptor kinase-2 with siRNA. Somatostatin increased sst2A receptor phosphorylation on the same five residues in GH4C1 pituitary cells as in CHO cells. However, PMA stimulated sst2A receptor phosphorylation on both Ser343 and Ser348 in GH4C1 cells. These results characterize the complex pattern of sst2A receptor phosphorylation by agonist and second messenger-activated kinases for the first time and indicate that cell type-specific regulation of sst2A receptor phosphorylation occurs.

The somatostatin subtype 2A (sst2A) receptor, a member of the G protein-coupled receptor superfamily, mediates many of the neuroendocrine and neuromodulatory actions of somatostatin, and it represents the primary target for somatostatin analogs used in cancer therapy and tumor localization. Agonist stimulation leads to the rapid phosphorylation, endocytosis, and desensitization of the sst2A receptor; however, little is known about the role of phosphorylation in sst2A regulation. sst2A phosphorylation occurs on serine and threonine residues in the third intracellular loop and carboxyl terminus. Therefore, we generated mutant receptors in which serine (Ser-), threonine (Thr-), or both (Ser-/Thr-) residues in these regions were mutated to alanine. In contrast to the wild-type receptor, somatostatin treatment did not stimulate the phosphorylation of the Ser-/Thr- mutant, and it did not produce desensitization. Furthermore, internalization of the Ser-/Thr- mutant occurred 5 times more slowly than with the wild-type receptor. Mutating only the Ser residues did not inhibit either internalization or desensitization. In contrast, mutating only the Thr residues inhibited receptor endocytosis to the same extent as in the full mutant, but it did not affect receptor desensitization. In both the wild-type and Ser- receptors, agonist binding produced a stable arrestin-receptor complex that was maintained during receptor trafficking, whereas arrestin was not recruited to either the Thr- or the Ser-/Thr- receptors. These results demonstrate that agonist-stimulated receptor phosphorylation is necessary for both desensitization and rapid internalization of the sst2A receptor. However, sst2A receptor internalization and uncoupling can occur independently, involve different receptor phosphorylation sites, and exhibit different requirements for stable arrestin association.

Upon hormone stimulation, the sst2 somatostatin receptor couples to adenylyl cyclase through G(i/o) proteins and undergoes rapid endocytosis via clathrin-coated pits. In this study, we determined the relationship between the ability of ligands to induce sst2 receptor internalization and inhibit adenylyl cyclase. Immunocytochemical studies demonstrated that peptide agonists [such as somatostatin-14, cortistatin-17, octreotide, vapreotide, KE108 (Tyr0-cyclo[d-diaminobutyric acid-Arg-Phe-Phe-d-Trp-Lys-Thr-Phe]), and SOM230 (cyclo[diaminoethylcarbamoyl-hydroxyproline-phenylglycine-d-Trp-Lys-(4-O-benzyl)-l-Tyr-Phe])] and nonpeptide agonists (such as L-779,976), stimulated the rapid endocytosis of sst2 receptors in human embryonic kidney 293 and CHO-K1 cells. In contrast, two antagonists did not induce receptor endocytosis by themselves and completely blocked agonist stimulation. Using a quantitative enzyme-linked immunosorbent assay to measure sst2 receptor sequestration, we found that peptide agonists varied by more than 100-fold in their potencies but exhibited the same efficacy as somatostatin14. In contrast, L-779,976 did not induce maximal receptor internalization. It is interesting that although betaarrestin-2 was recruited to cell surface sst2 receptors after stimulation with either somatostatin14 or L-779,976, the betaarrestin-receptor complex dissociated earlier in the endocytic pathway with the nonpeptide ligand. Although all agonists, including L-779,976, produced the same maximal inhibition of cyclic AMP, the potency ratio for inhibition of cyclic AMP and stimulation of receptor endocytosis varied by 15-fold. In general, native peptides showed similar potencies for cyclic AMP inhibition and receptor endocytosis, whereas short therapeutic analogs were substantially more potent at inhibiting cyclic AMP synthesis. These results demonstrate that the activity of somatostatin analogs to regulate receptor endocytosis and signaling are not tightly linked and provide compelling evidence for the induction of agonist specific states of the sst2 receptor.

The mechanisms regulating receptor internalization are not well understood and vary among different G protein-coupled receptors. The bombesin (Bn)/gastrin-releasing peptide receptor GRP-R, which is coupled to phospholipase C via the Gq family of transducing proteins, is internalized rapidly after Bn binding. Agonist stimulation leads to rapid receptor phosphorylation, as does activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA). However, agonist- and PMA-induced phosphorylation occur at different receptor sites. Here, we examined the role of PKC in GRP-R internalization after agonist and antagonist binding. We synthesized [D-Tyr6]Bn(6-13)propylamide ([D-Tyr6]Bn(6-13)PA) and found that it potently inhibited Bn-stimulated insulin release and [125I-Tyr4]Bn binding (Ki = 4.72 nM) in the HIT-T15 pancreatic cell line. The radiolabeled antagonist peptide, [125I-D-Tyr6]Bn(6-13)PA, bound with high affinity (KD = 0.29 nM at 4 degrees) to a single class of receptor sites, and competition binding studies exhibited the analog specificity expected for the GRP-R subtype. Although the agonist [125I-Tyr4]Bn was internalized rapidly at 37 degrees and subsequently degraded, [125I-D-Tyr6]Bn(6-13)PA was not internalized and was released into the medium mainly as intact peptide. The lysosomal inhibitor chloroquine (200 microM) increased the intracellular accumulation of [125I-Tyr4]Bn but had no effect on the subcellular distribution of [125I-D-Tyr6]Bn(6-13)PA. Consistent with these observations, the treatment of cells with 100 nM Bn at 37 degrees reduced cell surface receptors within minutes, whereas [D-Tyr6]Bn(6-13)PA had no effect. The addition of PMA did not induce the internalization of antagonist-occupied receptors, but pharmacological inhibition of PKC decreased the rate of agonist-induced receptor internalization. These results therefore demonstrate that although PKC contributes to agonist-induced internalization of the GRP-R, it does not elicit receptor internalization of the antagonist-occupied receptor.

Gastrin-releasing peptide and other bombesin-like peptides stimulate secretion, cell proliferation, and smooth muscle contraction via a family of G protein-coupled receptors that activate phospholipase C. Second messenger formation by one of these receptors, called BR1, is rapidly desensitized after treatment of cells with either agonists or the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether receptor phosphorylation was involved in BR1 desensitization, we generated antibodies to a peptide corresponding to a unique sequence within the COOH terminus of this receptor. One antibody (BR1-517) immunoprecipitated 60% of the solubilized [125I-Tyr4]bombesin/receptor complex prepared from either Swiss 3T3 fibroblasts or CHO-K1 cells transfected to express high levels of mouse BR1 (CHO-mBR1). Furthermore, immunoprecipitation of photoaffinity-labeled receptors yielded the expected 87-kDa radiolabeled band on gel electrophoresis. Phosphorylation of this immunoprecipitated receptor protein was markedly stimulated when [32P]orthophosphate-labeled Swiss 3T3 cells or CHO-mBR1 cells were treated with 100 nM bombesin for 5 min. 32PO4 incorporation into immunoprecipitated receptor was detectable after 2 min and maximal after 15 min of bombesin treatment. Phosphoamino acid analysis showed 32P labeling of serine and theonine but not tyrosine residues. Pretreatment of CHO-mBR1 cells with 100 nM TPA for 30 min also desensitized bombesin stimulation of inositol-1,4,5-trisphosphate formation. However, TPA did not increase 32PO4 incorporation into the immunoprecipitated receptor, although protein kinase C inhibition potentiated bombesin-induced receptor phosphorylation. Subsequent studies showed that TPA did stimulate receptor phosphorylation, but the antibody did not recognize this phosphorylated state of the receptor. Thus, TPA decreased the efficiency of receptor immunoprecipitation, and subsequent incubation of receptor with alkaline phosphatase reversed this TPA inhibition. The differential specificity of the antibody for various phosphorylated forms of BR1 demonstrates that agonist-induced and TPA-induced phosphorylations of the receptor occur at distinct sites.

The signal transduction pathways regulated by somatostatin receptor subtype 1 (sst1) have been difficult to define because of the variability observed when this receptor is expressed in different cell types by transfection and because pharmacological approaches are inadequate to distinguish sst1 receptor subtypes. To study the sst1 receptor in its endogenous environment, we developed a polyclonal antibody to a 15-amino acid peptide corresponding to a unique sequence in the receptor carboxyl terminus. The peptide antibody routinely precipitated 70% of the soluble [125I-Tyr11]somatostatin/receptor complex prepared from Chinese hamster ovary-K1 cells expressing the sst1 receptor but precipitated1% of the complex from cells expressing other sst receptor subtypes. Photoaffinity-labeled sst1 receptor was also specially immunoprecipitated and migrated as a broad 60-kDa band on sodium dodecyl sulfate polyacrylamide gels. The observation that sst receptors from GH4C1 pituitary cells were immunoprecipitated by the antibody and that receptors from AR4-2J pancreatic acinar cells were not indicated that only the former expressed sst1 receptor protein. Because reverse transcription-polymerase chain reaction showed that GH4C1 cells contained both sst1 and sst2 receptor mRNA, immunoprecipitation permitted the sst1 receptor to be separated from the other receptors present. Two observations showed that G proteins were coprecipitated with sst1 receptors from GH4C1 cells. First, pertussis toxin pretreatment markedly decreased hormone binding in the immunoprecipitate. Second, the addition of 20 microM guanosine-5'-(gamma-thio)triphosphate to the immunoprecipitated [125I-Tyr11]somatostatin/receptor complex stimulated the rate of dissociation of bound ligand by 10-fold. Interestingly, however, the dissociation rate of approximately 30% of the ligand/receptor complex was unaffected by guanosine-5'-(gamma-thio)triphosphate. In summary, we have developed an sst1 receptor-specific antibody and used it to show that sst1 receptors endogenously expressed in GH4C1 pituitary cells couple primarily to pertussis toxin-sensitive G proteins. Furthermore, these receptors exist in two distinct high affinity states distinguished by their GTP sensitivity.

The neuropeptide bombesin has a powerful but transient stimulatory effect on insulin secretion in the pancreatic islet cell line HIT-T15. We have previously shown that pretreatment of HIT-T15 cells with a saturating concentration of bombesin (100 nM) for 1.5-2 hr abolishes their secretory response to a second challenge with peptide and decreases [125I-Tyr4]bombesin binding by over 90%. In this study we examined the mechanisms involved in desensitization to bombesin. To determine whether receptor modulation was responsible, we compared the effect of bombesin pretreatment on [125I-Tyr4]bombesin binding and on the ability of bombesin to stimulate insulin release. Both effects occurred very rapidly and were maximal by 10 min. Although pretreatment of cells for 90 min with a subsaturating concentration of bombesin did not affect either the ED50 for bombesin-stimulated secretion or the apparent Kd for bombesin binding, it decreased both the maximum secretory response to a subsequent challenge with the peptide and bombesin receptor number. However, the extent of desensitization was greater than the extent of receptor down-regulation at all times examined during pretreatment and recovery. Furthermore, bombesin was 3 times more potent at inducing desensitization (ED50 = 0.35 +/- 0.08 nM) than down-regulation (ED50 = 1.1 +/- 0.4 nM). These results suggest that desensitization was not due solely to a reduction in receptor number. Because bombesin stimulates diacylglycerol production in HIT-T15 cells, we used the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to determine whether protein kinase C also played a role in desensitization to the peptide. Pretreatment of cells with TPA did not affect either [125I-Tyr4]bombesin binding or the dose dependence for bombesin-stimulated hormone release. However, TPA pretreatment did decrease the maximum secretory response to bombesin by 40% and caused a 50% reduction in bombesin-induced accumulation of inositol triphosphates and elevation of intracellular free calcium. Conversely, bombesin pretreatment reduced the secretory response to TPA by 40%. These studies indicate that the mechanism for desensitization to bombesin is a complex process that involves down-regulation of the bombesin receptor, inhibition of intracellular second messenger production, and reduction of protein kinase C-stimulated secretion.

GH4C1 cells are a clonal strain of rat pituitary tumor cells which contain high affinity receptors for the inhibitory neuropeptide somatostatin (SRIF). In contrast to other peptides that bind to specific receptors on these cells, receptor-bound [125I-Tyr1]SRIF does not undergo rapid endocytosis. Rather, partial degradation to 125I-tyrosine occurs concomitantly with the dissociation of [125I-Tyr1]SRIF from cell surface receptors. In this study we characterize the binding, biological activity and receptor-mediated degradation of [125I-Tyr11]SRIF, a SRIF analog that is radiolabeled in the center of the molecule. The binding of trace concentrations of [125I-Tyr11]SRIF (less than 50 pM) required 6 hr to reach equilibrium at 37 degrees compared with the 60 min required for [125I-Tyr1]SRIF. Analysis of the kinetics of [125I- Tyr11]SRIF binding showed that the rate constant for association (kon = 1.7 x 10(8) M-8min-1) was similar to that for [125I-Tyr1]SRIF (0.8 x 10(8) M-1min-1). However, the two radioligands exhibited markedly different dissociation kinetics; the koff for [125I-Tyr11]SRIF was 0.002 min-1 compared with the value of 0.02 min-1 for [125I-Tyr1] SRIF. In agreement with its much slower rate of dissociation, [125I-Tyr11]SRIF bound to the SRIF receptor with higher affinity (Kd = 70 pM) than did [125I-Tyr1]SRIF (Kd = 350 pM). However, the apparent ED50 for [I-Tyr11]SRIF to inhibit cAMP accumulation (1.9 +/- 0.4 nM) was greater than the ED50 for SRIF (0.19 +/- 0.04 nM). The low potency of [I-Tyr11]SRIF probably resulted from the fact that subsaturating concentrations of this peptide did not achieve equilibrium binding during the 30-min incubation used to assay biological activity. As previously reported for [125I-Tyr1]SRIF, receptor-bound [125I-Tyr11]SRIF was not internalized and was released from the cells as a mixture of intact [125I-Tyr11]SRIF (30%) and the degradation product 125I-tyrosine (65%). Only approximately 5% of receptor-bound [125I-Tyr11]SRIF was released as a different degradation product. Our data demonstrate that [125I-Tyr11]SRIF is a better radioanalog than [125I-Tyr1]SRIF for binding studies with intact cells because of its higher affinity for the SRIF receptor. In addition, inasmuch as receptor-mediated degradation of bound ligand releases iodotyrosine from both position 1 and position 11 substituted analogs, aminopeptidases are unlikely to be entirely responsible for SRIF degradation. The superior binding properties of [125I-Tyr11]SRIF should facilitate the detection of SRIF receptors in other cell types.