H-Tyr(Me)-OH

H-Tyr(Me)-OH

Role for engagement of beta-arrestin2 by the transactivated EGFR in agonist-specific regulation of delta receptor activation of ERK1/2.[Pubmed:26211551]

Br J Pharmacol. 2015 Oct;172(20):4847-63.

BACKGROUND AND PURPOSE: beta-Arrestins function as signal transducers linking GPCRs to ERK1/2 signalling either by scaffolding members of ERK1/2s cascades or by transactivating receptor tyrosine kinases through Src-mediated release of transactivating factor. Recruitment of beta-arrestins to the activated GPCRs is required for ERK1/2 activation. Our previous studies showed that delta receptors activate ERK1/2 through a beta-arrestin-dependent mechanism without inducing beta-arrestin binding to the delta receptors. However, the precise mechanisms involved remain to be established. EXPERIMENTAL APPROACH: ERK1/2 activation by delta receptor ligands was assessed using HEK293 cells in vitro and male Sprague Dawley rats in vivo. Immunoprecipitation, immunoblotting, siRNA transfection, intracerebroventricular injection and immunohistochemistry were used to elucidate the underlying mechanism. KEY RESULTS: We identified a new signalling pathway in which recruitment of beta-arrestin2 to the EGFR rather than delta receptor was required for its role in delta receptor-mediated ERK1/2 activation in response to H-Tyr-Tic-Phe-Phe-OH (TIPP) or morphine stimulation. Stimulation of the delta receptor with ligands leads to the phosphorylation of PKCdelta, which acts upstream of EGFR transactivation and is needed for the release of the EGFR-activating factor, whereas beta-arrestin2 was found to act downstream of the EGFR transactivation. Moreover, we demonstrated that coupling of the PKCdelta/EGFR/beta-arrestin2 transactivation pathway to delta receptor-mediated ERK1/2 activation was ligand-specific and the Ser(363) of delta receptors was crucial for ligand-specific implementation of this ERK1/2 activation pathway. CONCLUSIONS AND IMPLICATIONS: The delta receptor-mediated activation of ERK1/2 is via ligand-specific transactivation of EGFR. This study adds new insights into the mechanism by which delta receptors activate ERK1/2.

Effects of Tyroserleutide on phosphatidylinositol 3'-kinase/AKT pathway in human hepatocellular carcinoma cell.[Pubmed:24147456]

J Drug Target. 2014 Feb;22(2):146-55.

Tyroserleutide (YSL) is an active, low-molecular-weight polypeptide with in vitro and in vivo anticancer effects on human hepatocellular carcinoma BEL-7402 cells. In this study, we studied the effects of YSL on PI3K/AKT in the BEL-7402 cells to explore its anti-tumor mechanism. Results showed that YSL could up-regulate the mRNA and protein expression of tumor suppressor PTEN and increase their activities, meanwhile inhibited the mRNA and protein expression of oncogene AKT and decreased the kinase activities of AKT and PDK1. The resuming balance effect of YSL between PTEN and AKT could prevent the transmission of tumor cell proliferation signals in the PI3K/AKT pathway. Inhibition of AKT would change the status of downstream effectors in the PI3K/AKT pathway: (1) inhibition of AKT up-regulated expression of cell cycle regulatory factors of downstream - P21 and P27 which repressed cell cycle and inhibited proliferation of tumor cells. (2) Inhibition of AKT decreased the phosphorylation level of MDM2, and then increased the protein level of P53 which would accelerate death proceeding of tumor cells. (3) Inactivation of AKT removed its inhibition effect on phosphorylation of Bad, which might decrease protein level of apoptosis inhibitor Bcl-2 and Bcl-XL, damaging mitochondria of tumor cells and inducing apoptosis.

Two delta opioid receptor subtypes are functional in single ventral tegmental area neurons, and can interact with the mu opioid receptor.[Pubmed:28645621]

Neuropharmacology. 2017 Sep 1;123:420-432.

The mu and delta opioid receptors (MOR and DOR) are highly homologous members of the opioid family of GPCRs. There is evidence that MOR and DOR interact, however the extent to which these interactions occur in vivo and affect synaptic function is unknown. There are two stable DOR subtypes: DPDPE sensitive (DOR1) and deltorphin II sensitive (DOR2); both agonists are blocked by DOR selective antagonists. Robust motivational effects are produced by local actions of both MOR and DOR ligands in the ventral tegmental area (VTA). Here we demonstrate that a majority of both dopaminergic and non-dopaminergic VTA neurons express combinations of functional DOR1, DOR2, and/or MOR, and that within a single VTA neuron, DOR1, DOR2, and MOR agonists can differentially couple to downstream signaling pathways. As reported for the MOR agonist DAMGO, DPDPE and deltorphin II produced either a predominant K(+) dependent hyperpolarization or a Cav2.1 mediated depolarization in different neurons. In some neurons DPDPE and deltorphin II produced opposite responses. Excitation, inhibition, or no effect by DAMGO did not predict the response to DPDPE or deltorphin II, arguing against a MOR-DOR interaction generating DOR subtypes. However, in a subset of VTA neurons the DOR antagonist TIPP-Psi augmented DAMGO responses; we also observed DPDPE or deltorphin II responses augmented by the MOR selective antagonist CTAP. These findings directly support the existence of two independent, stable forms of the DOR, and show that MOR and DOR can interact in some neurons to alter downstream signaling.

N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile.[Pubmed:23932788]

Bioorg Med Chem Lett. 2013 Sep 15;23(18):5082-5.

Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related delta opioid antagonists showed delta partial agonist or mixed delta partial agonist/mu partial agonist activity. Guanidinylation of the mixed mu agonist/delta antagonists H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) and H-Dmt-TicPsi[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Psi]) converted them to mixed mu agonist/delta agonists. A docking study revealed distinct positioning of DIPP-NH2 and Guan-DIPP-NH2 in the delta receptor binding site. Lys(3)-analogues of DIPP-NH2 and DIPP-NH2[Psi] (guanidinylated or non-guanidinylated) turned out to be mixed mu/kappa agonists with delta antagonist-, delta partial agonist- or delta full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of cocaine addiction.

A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells.[Pubmed:23856011]

J Pharmacol Toxicol Methods. 2013 Nov-Dec;68(3):340-5.

INTRODUCTION: In contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for the generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability. METHODS: We fused tdT to the KOPR C-terminus. The KOPR-tdT cDNA construct was transfected into a Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking. RESULTS: KOPR-tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR-tdT enables visualization of the time course of agonist-induced internalization of KOPR in real time for 60 min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4min and plateaued at about 30 min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H. DISCUSSION: tdT is an alternative to, or even a better tool than, GFPs for fusion to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to the reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity that the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance.