Agonist-induced up-regulation of human somatostatin receptor type 1 is regulated by beta-arrestin-1 and requires an essential serine residue in the receptor C-tail

We have previously shown that the human somatostatin receptor type 1 (hSSTR1) does not undergo agonist-induced internalization, but is instead up-regulated at the membrane upon prolonged somatostatin (SST) exposure. The deletion of the carboxyterminal C-tail of the receptor completely abolishes up-regulation. To identify molecular signals that mediate hSSTR1 up-regulation, we created mutant receptors with progressive C-tail deletions. Up-regulation was found to be absent in mutants lacking residues Lys359-Ser360-Arg361. Moreover, point mutation of Ser360 to Ala completely abolished up-regulation. The coexpression of wild type hSSTR1 with V53D, a dominant negative mutant of beta-arrestin-1, completely blocked hSSTR1 up-regulation. Further analysis demonstrated that calcium-calmodulin (CaM) dependent kinases were essential for the SST-induced up-regulation response. Like wild type receptors, all mutants failed to internalize after agonist exposure and were able to inhibit forskolin-stimulated cAMP accumulation. Taking these data together, we suggest that SST-induced hSSTR1 up-regulation is critically dependent upon a specific Lys-Ser-Arg sequence in the C-tail of the receptor, with Ser360 being essential. Up-regulation also requires the participation of CaM protein kinases and interactions with beta-arrestins. In contrast, coupling to adenyl cyclase (AC) and internalization occur independently of molecular signals in the receptor's C-tail.     

Agonist-induced up-regulation of human somatostatin receptor type 1 is regulated by β-arrestin-1 and requires an essential serine residue in the receptor C-tail

We have previously shown that the human somatostatin receptor type 1 (hSSTR1) does not undergo agonist-induced internalization, but is instead up-regulated at the membrane upon prolonged somatostatin (SST) exposure. The deletion of the carboxyterminal C-tail of the receptor completely abolishes up-regulation. To identify molecular signals that mediate hSSTR1 up-regulation, we created mutant receptors with progressive C-tail deletions. Up-regulation was found to be absent in mutants lacking residues Lys³⁵⁹-Ser³⁶⁰-Arg³⁶¹. Moreover, point mutation of Ser³⁶⁰ to Ala completely abolished up-regulation. The coexpression of wild type hSSTR1 with V53D, a dominant negative mutant of β-arrestin-1, completely blocked hSSTR1 up-regulation. Further analysis demonstrated that calcium-calmodulin (CaM) dependent kinases were essential for the SST-induced up-regulation response. Like wild type receptors, all mutants failed to internalize after agonist exposure and were able to inhibit forskolin-stimulated cAMP accumulation. Taking these data together, we suggest that SST-induced hSSTR1 up-regulation is critically dependent upon a specific Lys-Ser-Arg sequence in the C-tail of the receptor, with Ser³⁶⁰ being essential. Up-regulation also requires the participation of CaM protein kinases and interactions with β-arrestins. In contrast, coupling to adenyl cyclase (AC) and internalization occur independently of molecular signals in the receptor's C-tail.     

C-tail mediated modulation of somatostatin receptor type-4 homo- and heterodimerizations and signaling

Somatostatin receptors show great diversity in response to agonist mediated receptor-specific homo- and heterodimerizations. Here, using photobleaching-fluorescence resonance energy transfer, immunocytochemistry, western blot and co-immunoprecipitation, we investigated dimerization, trafficking, coupling to adenylyl cyclase and signaling of human somatostatin receptor-4 (hSSTR4) in HEK-293 cells. We also determined the role of the C-tail of hSSTR4 on physiological responses of the cells. wt-hSSTR4 exogenously expressed in HEK-293 cells exhibits constitutive dimerization, inhibits forskolin-stimulated cAMP, and displays agonist dependent changes in pERK1/2 and pERK5 expressions. Upon C-tail deletion, the receptor loses membrane expression and ability to dimerize and inhibition of cAMP and pERK5 however, displays several-fold increases in the expression of pERK1/2. Chimeric hSSTR4 with the C-tail of hSSTR5 functions like wt-hSSTR4, in contrast, with the C-tail of hSSTR1 functions like C-tail deleted hSSTR4. hSSTR4 dimerization and signaling are associated with increased cyclin-dependent-kinase p27^kip1 expression and inhibition of the cell proliferation. We also report heterodimerization between hSSTR4/hSSTR5, but not between hSSTR4/hSSTR1, with significant changes in receptor functions. Taken together, these data define a novel mechanism for the role of hSSTR4 in cell proliferation and modulation of signaling pathways.     

Agonist-independent internalization and activity of a C-terminally truncated somatostatin receptor subtype 2 (delta349)

The rat somatostatin receptor subtype 2 (SSTR2) is rapidly internalized and phosphorylated in the presence of somatostatin 14 (SST14). Several C-terminal deletion constructs of SSTR2 have been investigated for their ability to undergo agonist-dependent internalization by using biochemical ligand binding assays and confocal microscopic analysis. Whereas mutant receptors lacking either 10 (delta359), 30 (delta339), or 44 (delta325) amino acid residues at the C terminus required SST14 for internalization, a construct lacking the last 20 amino acids (delta349) was detected mostly intracellularly and independently of the presence of the agonist. When internalization was blocked by sucrose, the delta349 receptor remained at the cell surface, strongly indicating that this mutant is internalized in an agonist-independent fashion. An increased affinity for agonists as measured in membrane binding assays and a reduced level of forskolin-stimulated cyclic AMP accumulation in human embryonic kidney cells expressing delta349 are properties that are characteristic of agonist-independent receptor activity. Delta349 is not phosphorylated detectably in the absence of agonist, demonstrating that phosphorylation per se is not a prerequisite for internalization of SSTR2. This observation is in line with data obtained for the delta325 mutant, which was internalized in an agonist-dependent manner, but not phosphorylated in either the presence or absence of SST14. We conclude that truncation of the SSTR2 C terminus at position 349 leads to agonist-independent, constitutive activity and internalization.     

The role of subtype-specific ligand binding and the C-tail domain in dimer formation of human somatostatin receptors

G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors. Several GPCRs have been documented to dimerize with resulting changes in pharmacology. We have previously reported by means of photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence correlation spectroscopic (FCS) analysis in live cells, that human somatostatin receptor (hSSTR) 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14. In contrast, hSSTR1 remained monomeric when expressed alone regardless of agonist exposure in live cells. In an effort to elucidate the role of ligand and receptor subtypes in heterodimerization, we have employed both pb-FRET microscopy and Western blot on cells stably co-expressing hSSTR1 and hSSTR5 treated with subtype-specific agonists. Here we provide evidence that activation of hSSTR5 but not hSSTR1 is necessary for heterodimeric assembly. This property was also reflected in signaling as shown by increases in adenylyl cyclase coupling efficiencies. Furthermore, receptor C-tail chimeras allowed for the identification of the C-tail as a determinant for dimerization. Finally, we demonstrate that heterodimerization is subtype-selective involving ligand-induced conformational changes in hSSTR5 but not hSSTR1 and could be attributed to molecular events occurring at the C-tail. Understanding the mechanisms by which GPCRs dimerize holds promise for improvements in drug design and efficacy.     

The role of G-proteins in the dimerisation of human somatostatin receptor types 2 and 5

Somatostatin (SST) is a peptide hormone that acts through a family of heptahelical receptors belonging to the G-protein coupled receptor (GPCR) superfamily. There are five known SST receptor subtypes termed SSTR1–5 and all couple to Gα_i/o G-proteins. It has been previously demonstrated that these receptors can form both homo- and heterodimers within their family or with other GPCR family members. Although agonist was demonstrated as a factor in modulating certain dimeric pairs, the molecular mechanism(s) underlying this regulation remains undetermined. Here, we demonstrate the coupling of G-protein as a contributing factor in the homo- and heterodimerisation of human (h) SSTR2 and SSTR5. When cells stably expressing hSSTR2 are pretreated with pertussis toxin (PTX), dissociation of hSSTR2 dimers occurs. Interestingly, although dimerisation of hSSTR5 was unaffected following PTX treatment, heterodimerisation between hSSTR2 and hSSTR5 is potentiated in the absence of receptor-stimulation. These results demonstrate the importance of G-protein in the maintenance and regulation of hSSTR dimers.     

Somatostatin receptor type 5 modulates somatostatin receptor type 2 regulation of adrenocorticotropin secretion

Somatostatin inhibits adrenocorticotropin (ACTH) secretion from pituitary tumor cells. To assess the contribution of somatostatin receptor subtype 5 (SST5) to somatostatin receptor subtype 2 (SST2) action in these cells, we assessed multipathway responses to novel highly monoreceptor-selective peptide agonists and multireceptor agonists, including octreotide and somatostatin-28. Octreotide and somatostatin-28 cell membrane binding affinities correlated with their respective SST2-selective peptide ligand. Although octreotide had similar inhibiting potency (picomolar) for cAMP accumulation and ACTH secretion as an SST2-selective agonist, somatostatin-28 exhibited a higher potency (femtomolar). Baseline spontaneous calcium oscillations assessed by fluorescent confocal microscopy revealed two distinct effects: SST2 activation reduced oscillations at femtomolar concentrations reflected by high inhibiting potency of averaged normalized oscillation amplitude, whereas SST5 activation induces brief oscillation pauses and increased oscillation amplitude. Octreotide exhibits an integrated effect of both receptors; however, somatostatin-28 exhibited a complex response with two separate inhibitory potencies. SST2 internalization was visualized with SST2-selective agonist at lower concentrations than for octreotide or somatostatin-28, whereas SST5 did not internalize. Using monoreceptor-selective peptide agonists, the results indicate that, in AtT-20 cells, SST5 regulates the dominant SST2 action, attenuating SST2 effects on intracellular calcium oscillation and internalization. This may explain superior somatostatin-28 potency and provides a rationale for somatostatin ligand design to treat ACTH-secreting pituitary tumors.     

Interactions with PDZ domain proteins PIST/GOPC and PDZK1 regulate intracellular sorting of the somatostatin receptor subtype 5

By yeast two-hybrid screening we have identified interaction partners for the intracellular C-terminal tail of the human and rodent somatostatin receptor subtype 5 (SSTR5). Interactions with the PDZ domain-containing proteins PIST and PDZK1 are mediated by the PDZ ligand motif at the C terminus of the receptor; in case of the human and mouse (but not the rat) receptors, a slight sequence variation of this motif also allows for binding of the peroxisomal receptor PEX5. PIST is Golgi-associated and retains SSTR5 in the Golgi apparatus when coexpressed with the receptor; PDZK1 on the other hand associates with the SSTR5 at the plasma membrane. Endogenous SSTR5 in the neuroendocrine AtT-20 tumor cell line is colocalized with PIST in the Golgi apparatus. On a functional level, removal of the PDZ ligand motif of the receptor does not interfere with agonist-dependent internalization of the receptor or its targeting to a Golgi-associated compartment; however, recycling of the receptor to the plasma membrane after washout of the agonist is inhibited, suggesting that the PDZ-mediated interaction of SSTR5 is required for postendocytic sorting.     

The PDZ/coiled-coil domain containing protein PIST modulates insulin secretion in MIN6 insulinoma cells by interacting with somatostatin receptor subtype 5

The multi-domain protein PIST (protein interacting specifically with Tc10) interacts with the SSTR5 (somatostatin receptor 5) and is responsible for its intracellular localization. Here, we show that PIST is expressed in pancreatic beta-cells and interacts with SSTR5 in these cells. PIST expression in MIN6 insulinoma cells is reduced by somatostatin (SST). After stimulation with SST, SSTR5 undergoes internalization together with PIST. MIN6 cells over-expressing PIST display enhanced glucose-stimulated insulin secretion and a decreased sensitivity to SST-induced inhibition of insulin secretion. These data suggest that PIST plays an important role in insulin secretion by regulating SSTR5 availability at the plasma membrane.     

Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers

The existence of receptor dimers has been proposed for several G protein-coupled receptors. However, the question of whether G protein-coupled receptor dimers are necessary for activating or modulating normal receptor function is unclear. We address this question with somatostatin receptors (SSTRs) of which there are five distinct subtypes. By using transfected mutant and wild type receptors, as well as endogenous receptors, we provide pharmacological, biochemical, and physical evidence, based on fluorescence resonance energy transfer analysis, that activation by ligand induces SSTR dimerization, both homo- and heterodimerization with other members of the SSTR family, and that dimerization alters the functional properties of the receptor such as ligand binding affinity and agonist-induced receptor internalization and up-regulation. Double label confocal fluorescence microscopy showed that when SSTR1 and SSTR5 subtypes were coexpressed in Chinese hamster ovary-K1 cells and treated with agonist they underwent internalization and were colocalized in cytoplasmic vesicles. SSTR5 formed heterodimers with SSTR1 but not with SSTR4 suggesting that heterodimerization is a specific process that is restricted to some but not all receptor subtype combinations. Direct protein interaction between different members of the SSTR subfamily defines a new level of molecular cross-talk between subtypes of the SSTR and possibly related receptor families.     

Resistance of the human beta 1-adrenergic receptor to agonist-mediated down-regulation. Role of the C terminus in determining beta-subtype degradation

Prolonged agonist stimulation results in down-regulation of most G protein-coupled receptors. When we exposed baby hamster kidney cells stably expressing the human beta1-adrenergic receptor (beta 1AR) to agonist over a 24-h period, we instead observed an increase of approximately 30% in both beta 1AR binding activity and immune-detected receptors. In contrast, beta 2AR expressed in these cells exhibited a decrease of > or =50%. We determined that the basal turnover rates of the two subtypes were similar (t(1/2) approximately 7 h) and that agonist stimulation increased beta 2AR but not beta 1AR turnover. Blocking receptor trafficking to lysosomes with bafilomycin A1 had no effect on basal turnover of either subtype but blocked agonist-stimulated beta 2AR turnover. As beta 1AR mRNA levels increased in agonist-stimulated cells, beta 1AR up-regulation appeared to result from increased synthesis with no change in degradation. To explore the basis for the subtype differences, we expressed chimeras in which the C termini had been exchanged. Each chimera responded to persistent agonist stimulation based on the source of its C-tail; beta 1AR with a beta 2AR C-tail underwent down-regulation, and beta 2AR with a beta 1AR C-tail underwent up-regulation. The C-tails had a corresponding effect on agonist-stimulated receptor phosphorylation and internalization with the order being beta 2AR > beta 1AR with beta 2AR C-tail > beta 2AR with a beta 1AR C-tail > beta 1AR. As internalization may be a prerequisite for down-regulation, we addressed this possibility by co-expressing each subtype with arrestin-2. Although beta 1AR internalization was increased to that of beta 2AR, down-regulation still did not occur. Instead, beta 1AR accumulated inside the cells. We conclude that in unstimulated cells, both subtypes appear to be turned over by the same mechanism. Upon agonist stimulation, both subtypes are internalized, and beta 2AR but not beta 1AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail.     

Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to beta-arrestin-dependent internalization

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a beta-arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize beta-arrestin. Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring beta-arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not beta-arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail. We thus investigated a role for CKII sites in determining GPCR internalization via beta-arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and beta-arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail of a beta-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of beta-arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of beta-arrestin-dependence was observed. Visualization of beta-arrestin/GFP redistribution confirmed a loss or gain of beta-arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent beta-arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for beta-arrestin-dependent internalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by beta-arrestin, thus providing further support for the involvement of CKII. This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the beta-arrestin-dependent pathway.     

Somatostatin inhibits cell migration and reduces cell counts of human keratinocytes and delays epidermal wound healing in an ex vivo wound model

The peptide hormone somatostatin (SST) and its five G protein-coupled receptors (SSTR1-5) were described to be present in the skin, but their cutaneous function(s) and skin-specific signalling mechanisms are widely unknown. By using receptor specific agonists we show here that the SSTRs expressed in keratinocytes are functionally coupled to the inhibition of adenylate cyclase. In addition, treatment with SSTR4 and SSTR5/1 specific agonists significantly influences the MAP kinase signalling pathway. As epidermal hormone receptors in general are known to regulate re-epithelialization following skin injury, we investigated the effect of SST on cell counts and migration of human keratinocytes. Our results demonstrate a significant inhibition of cell migration and reduction of cell counts by SST. We do not observe an effect on apoptosis and necrosis. Analysis of signalling pathways showed that somatostatin inhibits cell migration independent of its effect on cAMP. Migrating keratinocytes treated with SST show altered cytoskeleton dynamics with delayed lamellipodia formation. Furthermore, the activity of the small GTPase Rac1 is diminished, providing evidence for the control of the actin cytoskeleton by somatostatin receptors in keratinocytes. While activation of all receptors leads to redundant effects on cell migration, only treatment with a SSTR5/1 specific agonist resulted in decreased cell counts. In accordance with reduced cell counts and impaired migration we observe delayed re-epithelialization in an ex vivo wound healing model. Consequently, our experiments suggest SST as a negative regulator of epidermal wound healing.     

Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis

Background

Somatostatin (SST) via five G_i coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3.

Results

Although in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits G_i-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27^Kip1 suggests a cytostatic effect.

Conclusion

Our study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology.     

Cell growth inhibition and functioning of human somatostatin receptor type 2 are modulated by receptor heterodimerization

Somatostatin (SST) analogs have been successfully used in the medical treatment of acromegaly, caused by GH hypersecreting pituitary adenomas. Patients on SST analogs rarely develop tachyphylaxis despite years of continuous administration. It has been recently proposed that a functional association between SST receptor (SSTR) subtypes 2 and 5 exists to account for this behavior; however, a physical interaction has yet to be identified. Using both coimmunoprecipitation and photobleaching fluorescence resonance energy transfer microscopy techniques, we determined that SSTR2 and SSTR5 heterodimerize. Surprisingly, selective activation of SSTR2 and not SSTR5, or their costimulation, modulates the association. The SSTR2-selective agonist L-779,976 is more efficacious at inhibiting adenylate cyclase, activating ERK1/2, and inducing the cyclin-dependent kinase inhibitor p27(Kip1) in cells expressing both SSTR2 and SSTR5 compared with SSTR2 alone. Furthermore, cell growth inhibition by L-779,976 treatment was markedly extended in coexpressing cells. Trafficking of SSTR2 is also affected upon heterodimerization, an attribute corresponding to modifications in beta-arrestin association kinetics. Activation of SSTR2 results in the recruitment and stable association of beta-arrestin, followed by receptor internalization and intracellular receptor pooling. In contrast, heterodimerization increases the recycling rate of internalized SSTR2 by destabilizing its interaction with beta-arrestin. Given that SST analogs show preferential binding to SSTR2, these data provide a mechanism for their effectiveness in controlling pituitary tumors and the absence of tolerance seen in patients undergoing long-term administration.     

Internalization-defective mutants of somatostatin receptor subtype 2 exert normal signaling functions in hematopoietic cells

The regulatory peptide somatostatin (SST) acts via a family of G-protein-coupled receptors comprising five subtypes (SSTR1-5). G-protein-coupled receptors activate multiple signaling mechanisms, which variably depend on internalization and intracellular routing of activated receptors. We have recently demonstrated that hematopoietic precursors express SSTR2 and that SST is a chemoattractant for these cells. Herein, we characterize critical regions in SSTR2 involved in endocytosis and describe how ligand-induced internalization impacts on two major signaling functions of SSTR2 in hematopoietic cells, the activation of the Erk pathway and the induction of promigratory responses.     

Heterooligomerization of human dopamine receptor 2 and somatostatin receptor 2 Co-immunoprecipitation and fluorescence resonance energy transfer analysis

Somatostatin and dopamine receptors are well expressed and co-localized in several brain regions, suggesting the possibility of functional interactions. In the present study we used a combination of pharmacological, biochemical and photobleaching fluorescence resonance energy transfer (pbFRET) to determine the functional interactions between human somatostatin receptor 2 (hSSTR2) and human dopamine receptor 2 (hD2R) in both co-transfected CHO-K1 or HEK-293 cells as well as in cultured neuronal cells which express both the receptors endogenously. In monotransfected CHO-K1 or HEK-293 cells, D2R exists as a preformed dimer which is insensitive to agonist or antagonist treatment. In control CHO-K1 cells stably co-transfected with hD2R and hSSTR2, relatively low FRET efficiency and weak expression in co-immunoprecipitate from HEK-293 cells suggest the absence of preformed heterooligomers. However, upon treatment with selective ligands, hD2R and hSSTR2 exhibit heterodimerization. Agonist-induced heterodimerization was accompanied by increased affinity for dopamine and augmented hD2R signalling as well as prolonged hSSTR2 internalization. In contrast, cultured striatal neurons display constitutive heterodimerization between D2R and SSTR2, which were agonist-independent. However, heterodimerization in neurons was completely abolished in the presence of the D2R antagonist eticlopride. These findings suggest that hD2R and hSSTR2 operate as functional heterodimers modulated by ligands in situ, which may prove to be a useful model in designing new therapeutic drugs.     

Brain somatostatin receptors are up-regulated in somatostatin-deficient mice

The peptide somatostatin (SST) is widely synthesized in the brain and periphery and acts through a family of five receptors (SSTR1-5) to exert numerous effects. A gene product related to SST, cortistatin (CST), also interacts with SSTR1-5. Here we have investigated the regulation of SSTR1-5 and of CST in SST knockout (SSTKO) mice. The five SSTRs were quantitated individually by subtype-selective binding analysis, by immunocytochemistry, and by mRNA measurement and showed, in the brain of SSTKO mice, up-regulation of subtypes 1, 2, 4, and 5, and down-regulation of SSTR3. Peripheral tissues displayed both subtype- and tissue-specific changes in SSTR1-5 mRNA levels of expression. Lack of SST did not up-regulate normal CST expression in brain nor did it induce its expression in the periphery. SST-like immunoreactivity, however, was induced in the proximal midgut in SSTKO animals, suggesting intestinal expression of a novel SST-like gene.     

Somatostatin receptors 1 and 5 heterodimerize with epidermal growth factor receptor: Agonist-dependent modulation of the downstream MAPK signalling pathway in breast cancer cells

The role of somatostatin (SST) and epidermal growth factor (EGF) in breast cancer is undisputed; however, the molecular mechanisms underlying their antiproliferative or proliferative effects are not well understood. We initially confirmed that breast tumour tissues express all five somatostatin receptors (SSTR1-5) and four epidermal growth factor receptors (ErbB1-4). Subsequently, to gain insight into the function of SSTRs and ErbBs in oestrogen receptor (ER)-positive (MCF-7) or ERα-negative (MDA-MB-231) breast cancer cells, we defined SSTR1, SSTR5 and ErbB1 mRNA and protein expression in these two tumour cell lines. Consistent with previous studies showing SSTR1/SSTR5 heterodimerization and having seen cell-specific and ligand-selective alterations in receptor expression, we next elucidated whether SSTR1 and SSTR5 functionally interact with ErbB1 using pbFRET analysis. We subsequently determined the effects of SST and EGF either alone, or in combination, on selected downstream signalling molecules such as erk1/2, p38 and JNK. Here, we showed that both SST and EGF influenced erk1/2 phosphorylation and that SST modulated the effects of EGF in a cell-specific manner. We also demonstrated agonist-, time and cell-dependent regulation of p38 phosphorylation. We further investigated modulation of Grb2, SOS, Shc, SH-PTP1 and SH-PTP2. ErbB1 adaptor proteins known to play a role in MAPK activation, Shc, Grb2 and SOS, changed in an agonist- and cell-specific manner whereas, SH-PTP1 and SH-PTP2, adaptor proteins reported to interact with SSTRs, translocated from the cytosol to membrane in a cell-specific manner following SST and/or EGF treatment. Although several previous studies have shown crosstalk between RTKs and GPCRs, there are no reports describing SSTR (GPCR) modulation of ErbBs (RTK) in breast cancer. To the best of our knowledge, this is the first report describing crosstalk/interactions between SSTRs and ErbBs.     

Selective stimulation of somatostatin receptor subtypes: differential effects on Ras/MAP kinase pathway and cell proliferation in human neuroblastoma cells

In previous studies we have showed that somatostatin (SST) inhibits cell division, mitogen-activated protein (MAP) kinase and Ras activity in the human neuroblastoma cell line SY5Y. In the present study, we have assessed the role of a series of SST analogs, three of which were selective for SSTR1, SSTR2 or SSTR5, in these cellular events. All the analogs inhibited forskolin-induced cAMP accumulation. Selective stimulation of SSTR1 or SSTR2 but not of SSTR5 inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation. The three analogs inhibited PDGF-stimulated MAP kinase activity, at least at an early time. In contrast, none of the analogs used individually was able to inhibit PDGF-stimulated Ras activity. A combined stimulation of SSTR2 and SSTR5 was necessary to obtain a significant inhibitory effect, suggesting the possibility of receptor heterodimerization. These results indicate that SST inhibition of Ras and MAP kinase activities takes place via different pathways and that SST inhibition of PDGF-induced cell proliferation occurs via a Ras-independent pathway.