Discovery of two novel branched peptidomimetics containing endomorphin-2 and RF9 pharmacophores: Synthesis and neuropharmacological evaluation

It is well known that opioid analgesics produce side effects including tolerance and constipation. Since neuropeptide FF (NPFF) receptor antagonists reversed opioid-induced hyperalgesia and analgesic tolerance, the present work was performed to synthetize two branched peptidomimetics, EKR and RKE, containing the opioid peptide endomorphin-2 (EM-2) and the NPFF receptor antagonist RF9. Our data obtained from the in vitro cyclic adenosine monophosphate experiment demonstrated that EKR functioned as a mixed mu-, delta-opioid receptors agonist and NPFF₁ receptor antagonist/NPFF₂ receptor partial agonist, whereas RKE acted as a multi-functional peptidomimetic with the mu-opioid agonism and the NPFF₁ antagonism/NPFF₂ partial agonism. Furthermore, EKR and RKE completely blocked the NPFF₂ receptor-mediated neurite outgrowth of Neuro 2A cells. In vivo antinociception studies found that supraspinal administration of EKR and RKE dose-dependently produced potent antinociception via the mu-opioid receptor in the tail-flick test. In carrageenan inflammatory pain model, spinal administration of EKR and RKE induced dose-related analgesia, which was significantly reduced by the opioid antagonist naloxone and the NPFF antagonist RF9. Notably, compared with morphine, intracerebroventricular repeated administration of EKR and RKE maintained prolonged antinociceptive effectiveness. In addition, at the antinociceptive doses, these two branched peptidomimetics did not significantly inhibit gastrointestinal transit. Taken together, the present work suggest that EKR and RKE behave as multi-functional ligands with the opioid agonism and the NPFF₁ antagonism/NPFF₂ partial agonism, and produce prolonged antinociception with limited side effects. Moreover, our results imply that EKR and RKE might be interesting pharmacological tools for further investigating the biological function of the NPFF and opioid systems.     

Opposite Effects of Neuropeptide FF on Central Antinociception Induced by Endomorphin-1 and Endomorphin-2 in Mice

Neuropeptide FF (NPFF) is known to be an endogenous opioid-modulating peptide. Nevertheless, very few researches focused on the interaction between NPFF and endogenous opioid peptides. In the present study, we have investigated the effects of NPFF system on the supraspinal antinociceptive effects induced by the endogenous µ-opioid receptor agonists, endomorphin-1 (EM-1) and endomorphin-2 (EM-2). In the mouse tail-flick assay, intracerebroventricular injection of EM-1 induced antinociception via µ-opioid receptor while the antinociception of intracerebroventricular injected EM-2 was mediated by both µ- and κ-opioid receptors. In addition, central administration of NPFF significantly reduced EM-1-induced central antinociception, but enhanced EM-2-induced central antinociception. The results using the selective NPFF₁ and NPFF₂ receptor agonists indicated that the EM-1-modulating action of NPFF was mainly mediated by NPFF₂ receptor, while NPFF potentiated EM-2-induecd antinociception via both NPFF₁ and NPFF₂ receptors. To further investigate the roles of µ- and κ-opioid systems in the opposite effects of NPFF on central antinociception of endomprphins, the µ- and κ-opioid receptors selective agonists DAMGO and {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593, respectively, were used. Our results showed that NPFF could reduce the central antinociception of DAMGO via NPFF₂ receptor and enhance the central antinociception of {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 via both NPFF₁ and NPFF₂ receptors. Taken together, our data demonstrate that NPFF exerts opposite effects on central antinociception of endomorphins and provide the first evidence that NPFF potentiate antinociception of EM-2, which might result from the interaction between NPFF and κ-opioid systems.     

Neuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells

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Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF₂) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF₂ receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distribution in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.     

Anti-analgesia of a selective NPFF2 agonist depends on opioid activity

NPFF agonists designed to be selective NPFF(2) receptor probes were synthesized. D.Asn-Pro-(N-Me)Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH(2) (dNPA) displays a very high affinity (0.027nM) for NPFF(2) receptors transfected in CHO cells, and a very high selectivity with a discrimination ratio greater than 100 versus NPFF(1) receptors. dNPA acts as a potent and selective agonist in [(35)S]GTPgammaS binding experiments and inhibits intracellular cAMP production with the same efficacy as NPA-NPFF. In SH-SY5Y cells expressing NPFF(2) receptors dNPA, in the presence of carbachol, stimulates Ca(2+) release from the intracellular stores. In vivo, after intracerebroventricular injection dNPA increases body temperature in mice and reverses the morphine-induced analgesia. Also, dNPA displays anti-opioid activity after systemic administration. So far, dNPA exhibits the highest affinity and selectivity for NPFF(2) receptors and reveals that its behavioral anti-opioid activity depends on the degree of opioid-induced analgesia.     

Solubilization and functional reconstitution of human neuropeptide FF2 receptors

Neuropeptide FF (NPFF, FLFQPQRFamide) receptors modulate endogenous opioid functions. Here, we report the solubilization of the human NPFF₂ receptor expressed in Chinese hamster ovary (CHO) cells by the zwitterionic detergent Chaps. Chaps solubilization resulted in the abolishment of specific agonist binding activity, which was restored by a polyethylene glycol (PEG) precipitation method. Reincorporation after the precipitation step into liposomes made of endogenous lipids issued from CHO membranes or exogenous lipids significantly enhanced the specific agonist binding activity and G-protein coupling. This method of solubilization and lipid reconstitution could be useful for studies of NPFF receptors.     

Modulation by Neuropeptide FF of the interaction of Mu-opioid (MOP) receptor with G-proteins

The Neuropeptide FF (NPFF) system is known to modulate the effects of opioids in vivo and in vitro. In the present study, we have investigated the effect of NPFF agonists on the coupling of the Mu-opioid (MOP) receptor to G-proteins in a model of SH-SY5Y cells transfected with NPFF₂ receptor, in which the neuronal anti-opioid activity of NPFF was previously reproduced. Activation of G-proteins was monitored by [³⁵S]GTPγS binding assay and analysis of G-protein subunits associated with MOP receptors was performed by Western blotting after immunoprecipitation of the receptor. The results demonstrate that concentrations of NPFF agonists that produce a cellular anti-opioid effect, did not affect the ability of the opioid agonist DAMGO to activate G-proteins. However, at saturating concentration of agonist or when expression of receptor was high, opioid and NPFF agonists did not stimulate [³⁵S]GTPγS binding in an additive manner, indicating that both receptors share a common fraction of a G-protein pool. In addition, stimulation of NPFF receptors in living cells modified the G-protein environment of MOP receptor by favoring its interaction with α_s, α_i2 and β subunits. This change in G-protein coupling to MOP receptor might participate in the mechanism by which NPFF agonists reduce the inhibitory activity of opioids.     

Prokineticin receptors interact unselectively with several G protein subtypes but bind selectively to β-arrestin 2

Prokineticin 1 (pk1) and prokineticin 2 (pk2) interact with two structurally related G-protein coupled receptors, prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2). Cellular signalling studies show that the activated receptors can evoke Ca²⁺-mobilization, pertussis toxin-sensitive ERK phosphorylation, and intracellular cAMP accumulation, which suggests the partecipation of several G protein subtypes, such as G_q/11, G_i/o and G_s. However, direct interactions with these transduction proteins have not been studied yet. Here we measured by bioluminescence resonance energy transfer (BRET) the association of PKR1 and PKR2 with different heterotrimeric Gα proteins in response to pk1 and pk2 activation. Using host-cell lines carrying gene deletions of Gα_q/11 or Gα_s, and pertussis toxin treatment to abolish the receptor interactions with Gα_i/o, we determined that both receptors could couple with comparable efficiency to G_q/11 and G_i/o, but far less efficiently to G_s or other pertussis toxin-insensitive G proteins. We also used BRET methodology to assess the association of prokineticin receptors with β-arrestin isoforms. Fluorescent versions of the isoforms were transfected both in HEK293 cells and in double KO β-arrestin 1/2 mouse fibroblasts, to study receptor interaction with the reconstituted individual β-arrestins without background expression of the endogenous genes. Both receptors formed stable BRET-emitting complexes with β-arrestin 2 but not with β-arrestin 1, indicating strong selectivity for the former. In all the studied transducer interactions and in both receptors, pk2 was more potent than pk1 in promoting receptor binding to transduction proteins.     

Identification and Functional Characterization of the Phosphorylation Sites of the Neuropeptide FF2 Receptor

The neuropeptide FF₂ (NPFF₂) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF₂ receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF₂ receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, ⁴¹²TNST⁴¹⁵ at the end of the C terminus of the receptor, and additional sites involved in desensitization (³⁷²TS³⁷³) and internalization (Ser³⁹⁵). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF₂ receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns.     

Characterization of two novel tritiated radioligands for labelling Neuropeptide FF (NPFF1 and NPFF2) receptors

The binding characteristics of [³H]-NPVF and [³H]-EYF, the two first tritiated probes for the respective labelling of NPFF₁ and NPFF₂ receptors, are presented. In membranes from CHO cells transfected with the human NPFF₁ receptor, [³H]-NPVF labelled one class of binding sites with a high affinity (Bmax = 4 pmol/mg protein, Kd = 2.65 nM). In membranes from CHO cells transfected with the human NPFF₂ receptor, [³H]-EYF labelled one class of binding sites with a high affinity (Bmax = 16 pmol/mg protein, Kd = 0.54 nM). Both radioligands exhibited time-dependent binding, low (10–20%) non-specific binding and poor cross-reactivity towards the related receptor subtype. The potency of different NPFF ligands to displace [³H]-NPVF and [³H]-EYF binding profiles was in good agreement with the profile previously measured by using ¹²⁵I-probes (NPFF₁ receptor: NPVF ≥ 1DMe = SPA-NPFF > NPFF = SQA-NPFF = QFW-NPSF > NPSF > RF9; NPFF₂ receptor: SPA-NPFF > > SQA-NPFF = QFW-NPSF = 1DMe = NPFF  NPSF = NPVF > RF9). Therefore, [³H]-NPVF and [³H]-EYF are new valuable tools for performing binding on NPFF receptors.     

A high-affinity,radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine

A new radioiodinated photoaffinity compound, [¹²⁵I]YE(Bpa)WSLAAPQRFNH₂, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF₂ receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (K_D = 0.24 nM) with a single class of binding sites. It was also able to affinity label NPFF₂ receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [¹²⁵I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF₂ receptor with apparent molecular weights of 140 and 95 kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF₂ receptor is observed. [¹²⁵I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF₂ receptors in vitro.     

The heparan sulfate proteoglycan agrin modulates neurite outgrowth mediated by FGF‐2

Although the role of agrin in the formation of the neuromuscular junction is well established, other functions for agrin have remained elusive. The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the heparin‐binding growth factor basic fibroblast growth factor (FGF‐2), which we have shown previously to bind to agrin with high affinity and that has been shown to mediate neurite outgrowth from a number of neuronal cell types. Using both an established neuronal cell line, PC12 cells, and primary chick retina neuronal cultures, we find that agrin potentiates the ability of FGF‐2 to stimulate neurite outgrowth. In PC12 cells and retinal neurons agrin increases the efficacy of FGF‐2 stimulation of neurite outgrowth mediated by the FGF receptor, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF‐2. We also examined possible mechanisms by which agrin may modulate neurite outgrowth, analyzing ERK phosphorylation and c‐fos phosphorylation. These studies indicate that agrin augments a transient early phosphorylation of ERK in the presence of FGF‐2, and augments and sustains FGF‐2 mediated increases in c‐fos phosphorylation. These data are consistent with established mechanisms where heparan sulfate proteoglycans such as agrin may increase the affinity between FGF‐2 and the FGF receptor. In summary, our studies suggest that neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF‐2. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 261–277, 2003     

Regulation of homocysteine-induced MMP-9 by ERK1/2 pathway

Homocysteine (Hcy) induces matrix metalloproteinase (MMP)-9 in microvascular endothelial cells (MVECs). We hypothesized that the ERK1/2 signaling pathway is involved in Hcy-mediated MMP-9 expression. In cultured MVECs, Hcy induced activation of ERK, which was blocked by PD-98059 and U0126 (MEK inhibitors). Pretreatment with BAPTA-AM, staurosporine (PKC inhibitor), or Gö6976 (specific inhibitor for Ca²⁺-dependent PKC) abrogated ERK phosphorylation, suggesting the role of Ca²⁺ and Ca²⁺-dependent PKC in Hcy-induced ERK activation. ERK phosphorylation was suppressed by pertussis toxin (PTX), suggesting the involvement of G protein-coupled receptors (GPCRs) in initiating signal transduction by Hcy and leading to ERK activation. Pretreatment of MVECs with genistein, BAPTA-AM, or thapsigargin abrogated Hcy-induced ERK activation, suggesting the involvement of the PTK pathway in Hcy-induced ERK activation, which was mediated by intracellular Ca²⁺ pool depletion. ERK activation was attenuated by preincubation with N-acetylcysteine (NAC) and SOD, suggesting the role of oxidation in Hcy-induced ERK activation. Pretreatment with an ERK1/2 blocker (PD-98059), staurosporine, folate, or NAC modulated Hcy-induced MMP-9 activation as measured using zymography. Our results provide evidence that Hcy triggers the PTX-sensitive ERK1/2 signaling pathway, which is involved in the regulation of MMP-9 in MVECs. calcium signaling; protein kinase C; Src; G protein-coupled receptor; nonreceptor tyrosine kinase; protein G_i; protein G_q; protein tyrosine kinase 2; microvascular endothelial cell; cardiovascular remodeling     

Minireview: Differential Targeting and Retention of G Protein-Coupled Receptors in Polarized Epithelial Cells

Abstract

Localization of receptors in discrete cellular microdomains undoubtedly contributes to their interaction with particular effectors and receptor targets. For G protein-coupled receptors, virtually nothing is known about the mechanisms and structural features responsible for their targeting to and retention in varying surface domains. We have shown that the G_j/G_o-coupled α_2A-adrenergic receptor (α_2AAR) is directly targeted to the lateral subdomain of MDCK II cells. Mutational analysis has revealed that regions in or near the bilayer are likely critical for α_2AAR targeting, whereas endofacial domains contribute to α_2AAR retention on the lateral surface. Although the α_2BAR also is enriched on the lateral subdomain at steady-state, its polarization occurs after initial random delivery to both apical and basolateral surfaces followed by a selective accumulation on the lateral subdomain. The α_2CAR also is expressed on the lateral subdomain and achieves its localization via direct delivery to the basolateral surface; however, the α_2CAR also exists in an as yet not fully characterized intracellular compartment. Interestingly, another G_j/G_o-coupled receptor, the A₁ adenosine receptor, is enriched on the apical surface of MDCK II calls and achieves this localization by direct apical delivery. These findings indicate that receptor delivery to polarized surfaces is not determined by receptor coupling to a specific subpopulation of G proteins.     

Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells

Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X₇ receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined. We characterized the role of P2X₇ receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X₇ receptors. Functional inhibition of P2X₇ receptors by P2X₇ receptor selective antagonists, 5′-triphosphate, periodate-oxidized 2′,3′-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X₇ receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were decreased in either RA- or oATP-differentiated or P2X₇ receptor knockdown N2a cells. Simply cultured N2a cells in low Ca²⁺ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X₇ receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X₇ receptors are involved in neuronal differentiation. We provide additional evidence shown that the ATP release-activated P2X₇ receptor is important in maintaining cell survival of N2a neuroblastoma cells.     

Up‐regulation and activation of the P2Y2 nucleotide receptor mediate neurite extension in IL‐1β‐treated mouse primary cortical neurons

The pro‐inflammatory cytokine interleukin‐1β (IL‐1β), whose levels are elevated in the brain in Alzheimer's and other neurodegenerative diseases, has been shown to have both detrimental and beneficial effects on disease progression. In this article, we demonstrate that incubation of mouse primary cortical neurons (mPCNs) with IL‐1β increases the expression of the P2Y₂ nucleotide receptor (P2Y₂R) and that activation of the up‐regulated receptor with UTP, a relatively selective agonist of the P2Y₂R, increases neurite outgrowth. Consistent with the accepted role of cofilin in the regulation of neurite extension, results indicate that incubation of IL‐1β‐treated mPCNs with UTP increases the phosphorylation of cofilin, a response absent in PCNs isolated from P2Y₂R^?/? mice. Other findings indicate that function‐blocking anti‐α_vβ_3/5 integrin antibodies prevent UTP‐induced cofilin activation in IL‐1β‐treated mPCNs, suggesting that established P2Y₂R/α_vβ_3/5 interactions that promote G₁₂‐dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL‐1β‐treated mPCNs is also decreased by inhibitors of Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), suggesting a role for P2Y₂R‐mediated and G_q‐dependent calcium mobilization in neurite outgrowth. Taken together, these studies indicate that up‐regulation of P2Y₂Rs in mPCNs under pro‐inflammatory conditions can promote cofilin‐dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases.     

G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E₂), couple to the G proteins G_s and G_i/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E₂ and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of G_i/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of G_i/o and retains receptors in the plasma membrane.