In the present study using indirect immunofluorescence immunohistochemistry, co-immunoprecipitation and western blot analysis we determined the colocalization of dopamine receptors 1–5 and dopamine ...and cAMP-regulated phosphoprotein (DARPP-32) in rat brain cortex and striatum. All five DR subtypes and DARPP-32 were expressed in rat brain cortex and striatum. DARPP-32 positive neurons displayed comparative colocalization with DR1-5. In cingulate cortex, the colocalization of DR subtypes was greatly different from frontal or temporal cortex. D1R is one of the most predominant subtypes which colocalized with DARPP-32 in cortex as well as striatum and followed by D2R, D3R, D4R and D5R. Amongst all DR subtypes D5R was coexpressed the least with DARPP-32 positive neurons. Consistent with immunohistochemical data, western blot analysis also reveals comparable distribution of DR subtypes and DARPP-32 in cortex and striatum. Colocalization studies were also supported by using co-immunoprecipitate assay displaying DARPP-32 expression in DR immunoprecipitate from tissue lysate prepared from cortex and striatum. Taken together our data support receptor specific association of DARPP-32 with DR subtypes that might shed new information in drugs of abuse and pathophysiology of neurodegenerative diseases as well as neuropsychiatric disorders such as schizophrenia.
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.
Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of ...mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.
► In this study we demonstrate the activation of SSTRs inhibits epidermal growth factor mediated EGFR phosphorylation and mitogen-activated protein kinases. ► The presence of SSTRs blocks the epidermal growth factor mediated PI3K/AKT cell survival pathway. ► SSTRs in receptor-specific manner impede the epidermal growth factor stimulated heterodimerization between EGFR and ErbB2.
In the present study we describe heterodimerization, trafficking, coupling to adenylyl cyclase and signaling in HEK-293 cells cotransfected with human-somatostatin receptor 5 (hSSTR5) and ...β1-adrenergic receptor (β1AR). hSSTR5/β1AR exists as heterodimers in basal conditions which was further enhanced upon synergistic activation of both receptors. Activation of either β1AR or hSSTR5 displayed dissociation of heterodimerization. In cotransfectants, β1AR effect on cAMP was predominant; however, blocking β1AR with antagonist resulted in 60% inhibition of forskolin-stimulated cAMP in the presence of hSSTR5 agonists. cAMP/PKA pathway in cotransfected cells was regulated in receptor-specific manner, in contrast, the status of pERK1/2 and pPI3K/AKT was predominantly regulated by hSSTR5. The expression levels of phosphorylated NFAT remained unchanged indicating blockade of calcineurin-mediated dephosphorylation and nuclear translocation of NFAT, the process predominantly regulated by pJNK in SSTR5 dependent manner. Taken together, the functional consequences of results described here might have relevance in the cardiovascular system where SSTR and AR subtypes play important roles.
In the present study, we describe heterodimerization between human-Somatostatin Receptor 5 (hSSTR5) and β2-Adrenergic Receptor (β2AR) and its impact on the receptor trafficking, coupling to adenylyl ...cyclase and signaling including mitogen activated protein kinases and calcineurin-NFAT pathways.
We used co-immunoprecipitation, photobleaching- fluorescence resonance energy transfer and Fluorescence assisted cell sorting analysis to characterize heterodimerization between SSTR5 and β2AR.
Our results indicate that hSSTR5/β2AR exist as preformed heterodimers in the basal condition which is enhanced upon co-activation of both receptors. In contrast, the activation of individual receptors leads to the dissociation of heterodimers. Receptor coupling to adenylyl cyclase displayed predominant effect of β2AR, however, somatostatin mediated inhibition of cAMP was enhanced upon blocking β2AR. Our results indicate hSSTR5 mediated significant activation of ERK1/2 and inhibition of phospho-p38. The phospho-NFAT level was enhanced in cotransfected cells indicating the blockade of calcineurin mediated dephosphorylation of NFAT upon receptor heterodimerization.
These data for the first time unveil a novel insight for the role of hSSTR5/β2AR in the modulation of signaling pathways which has not been addressed earlier.
The novel inhibitory role of somatostatin receptor subtype 5 on EGF-mediated heterodimerization of ErbBs and modulation of cell survival promoting signaling pathways is discussed.
Epidermal growth ...factor through the stimulation of epidermal growth factor receptor (EGFR) plays a critical role in the activation of MAPKs and phosphatidylinositol-3-protein kinase/AKT cell survival pathways attributed in many pathological conditions. At the cellular level, such functions involve EGFR overactivation and phosphorylation. In the present study, we describe that human embryonic kidney-293 cells transfected with somatostatin (SST) receptor 5 (SSTR5) exhibit inhibition of EGFR phosphorylation and modulate MAPK and phosphatidylinositol-3-protein kinase/AKT cell survival signaling. Furthermore, suppression of EGFR by using small interference RNA and an antagonist (AG1478) potentiates the SST effect via activation of SSTR5 on signaling molecules. In wild-type human embryonic kidney-293 cells, EGFR/ErbB2 exists as constitutive heterodimers. The presence of SSTR5 leads to the dissociation of the heteromeric complex of EGFR/ErbB2 and display preferential heterodimerization between SSTR5 and EGFR in an agonist-dependent manner. These findings highlight a new undiscovered mechanism and potential role of SSTR5 to attenuate the EGFR-mediated signaling pathways involved in tumorigenesis. Our data indicate that the activation and/or overexpression of SST receptors along with the inhibition of EGFR will serve as an important therapeutic approach in the treatment of ErbB-positive tumors.
Pain relief is the principal action of opioids. Somatostatin (SST), a growth hormone inhibitory peptide is also known to alleviate pain even in cases when opioids fail. Recent studies have shown that ...mice are prone to sustained pain and devoid of analgesic effect in the absence of somatostatin receptor 4 (SSTR4). In the present study, using brain slices, cultured neurons and HEK-293 cells, we showed that SSTR4 and delta-Opioid receptor (deltaOR) exist in a heteromeric complex and function in synergistic manner. SSTR4 and deltaOR co-expressed in cortical/striatal brain regions and spinal cord. Using cultured neuronal cells, we describe the heterogeneous complex formation of SSTR4 and deltaOR at neuronal cell body and processes. Cotransfected cells display inhibition of cAMP/PKA and co-activation of SSTR4 and deltaOR oppose receptor trafficking induced by individual receptor activation. Furthermore, downstream signaling pathways either associated with withdrawal or pain relief are modulated synergistically with a predominant role of SSTR4. Inhibition of cAMP/PKA and activation of ERK1/2 are the possible cellular adaptations to prevent withdrawal induced by chronic morphine use. Our results reveal direct intra-membrane interaction between SSTR4 and deltaOR and provide insights for the molecular mechanism for the anti-nociceptive property of SST in combination with opioids as a potential therapeutic approach to avoid undesirable withdrawal symptoms.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are mediators of inflammation D.L. Dewitt, W.L. Smith, Primary structure of ...prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence, Proc. Natl. Acad. Sci. USA 85 (1988) 1412–1416,
1. It exists mainly in two isoforms COX-1 and COX-2 A. Raz, A. Wyche, N. Siegel, P. Needleman, Regulation of fibroblast cyclooxygenase synthesis by interleukin-1, J. Biol. Chem. 263 (1988) 3022–3028,
2. The conventional non-steroidal anti-inflammatory drugs (NSAIDs) have adverse gastrointestinal side-effects, because they inhibit both isoforms T.D. Warner, F. Guiliano, I. Vojnovic, A. Bukasa, J.A. Mitchell, J.P. Vane, Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis, Proc. Natl. Acad. Sci. USA 96 (1999) 7563–7568,
3; L.J. Marnett, A.S. Kalgutkar, Cyclooxygenase 2 inhibitors: discovery, selectivity and the future, Trends Pharmacol. Sci. 20 (1999) 465–469,
4; J.R. Vane, NSAIDs, Cox-2 inhibitors, and the gut, Lancet 346 (1995) 1105–1106,
5. Therefore drugs which selectively inhibit COX-2, known as coxibs were developed. Recent reports on the harmful cardiovascular and renovascular side-effects of the anti-inflammatory drugs have led to the quest for a novel class of COX-2 selective inhibitors. Keeping this in mind, we have used the X-ray crystal structures of the complexes of the COX-1 and COX-2 with the known inhibitors for a rational, structure based approach to design a small peptide, which is potent inhibitor for COX-2. The peptides have been checked experimentally by in-vitro kinetic studies using surface plasmon resonance (SPR) and other biochemical methods. We have identified a tripeptide inhibitor which is a potential lead for a new class of COX-2 inhibitor. The dissociation constant (
K
D) determined for COX-2 with peptide WCS is 1.90
×
10
−10
M, the kinetic constant (
K
i) determined by spectrophotometry is 4.85
×
10
−9
M and the IC
50 value is 1.5
×
10
−8
M by ELISA test.
Phospholipase A2 (PLA2; EC 3.1.1.4) is a key enzyme involved in the production of proinflammatory mediators known as eicosanoids. The binding of the substrate to PLA2 occurs through a well-formed ...hydrophobic channel. To determine the viability of PLA2 as a target molecule for the structure-based drug design against inflammation, arthritis, and rheumatism, the crystal structure of the complex of PLA2 with a known anti-inflammatory compound oxyphenbutazone (OPB), which has been determined at 1.6 Å resolution. The structure has been refined to an R factor of 0.209. The structure contains 1 molecule each of PLA2 and OPB with 2 sulfate ions and 111 water molecules. The binding studies using surface plasmon resonance show that OPB binds to PLA2 with a dissociation constant of 6.4 × 10-8 M. The structure determination has revealed the presence of an OPB molecule at the binding site of PLA2. It fits well in the binding region, thus displaying a high level of complementarity. The structure also indicates that OPB works as a competitive inhibitor. A large number of hydrophobic interactions between the enzyme and the OPB molecule have been observed. The hydrophobic interactions involving residues Tyr52 and Lys69 with OPB are particularly noteworthy. Other residues of the hydrophobic channel such as Leu3, Phe5, Met8, Ile9, and Ala18 are also interacting extensively with the inhibitor. The crystal structure clearly reveals that the binding of OPB to PLA2 is specific in nature and possibly suggests that the basis of its anti-inflammatory effects may be due to its binding to PLA2 as well.
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