Delta opioid receptor (DOR) agonists (e.g. DADLE) exert opposing effects on T-cells: alone, these agonists can activate specific pathways involved in signal transduction; however, when exposure to these agonists precedes T-cell receptor (TCR)+/-CD28- dependent activation of T-cells, the same signal transduction pathways can be partially inhibited. These investigations wilt determine the mechanism(s) underlying the opposing effects of DORs on enriched primary splenic T-cells, by focusing on two Tcell functions: (i) interleukin-2 (IL-2) mRNA production and (ii) the expression of CXCR4, a chemokine receptor which is a coreceptor for the entry of human immunodeficiency vires-1 (HIV-I) into CD4 v T-cells. Since DADLE appears to inhibit TCR driven IL-2 mRNA production by downregulating AP-1 and NF-kappaB DNA binding activities (preliminary data), in SA#1 the effects of anti-CD3 +/- DADLE on IL-2 mRNA stability and transcription will be elucidated by measuring the following: IL-2 mRNA half-life; the rote of IL-2 gene transcription by run-on assay; transcription factor binding activity by electrophoretic mobility shift analyses (EMSA); and changes in the amounts of AP-1 and NF-kappaB actually present on the IL-2 chromatin intracellularly, using the chromatin immunoprecipitation (CHIP) assay. In SA#2, we will determine the effects of DADLE on key signaling intermediates that induce AP-1 and NF-kappaB-mediated IL-2 transcription when the TCR is triggered. Since one of these signaling intermediates, phosphoinositide 3-kinase (PI3K), is required for the effects of DORs alone on c-Jun phosphorylation and CXCR4 downregulation, we propose that DADLE induces the translocation and temporary sequestration of PI3K from the cytosolic pool. Therefore, following pretreatment with DADLE, PI3K would be less available for translocation into the immune synapse that contains the molecular machinery which propagates TCR-dependent signals, activating AP-1 and NF-kappaB through Vav and protein kinase C-theta ( PKC-theta). Using co-immunoprecipitation and immunoblotting, SA#2 studies will define the effects of DADLE +/- anti-CD3 on the translocation, phosphorylation and activity of PI3K, Vav and PKC-theta. In SA#3, the effects of PI3K on DOR-induced downregulation of CXCR4 will be determined in primary human CD4 + T-calls. The following studies will be conducted: flow cytometry to compare the downregulation of DORs and CXCR4; immunoblotting to determine DOR agonist-induced CXCR4 phosphorylation; co-immunoprecipitation to assess the association of CXCR4 with beta-arrestins, which link G-protein-coupled receptors to proteins mediating intenaali7ation; pharmacological inhibition to assess the role of other serine/threonine kinases in the DOR-dependent phosphorylation and downregulation of CXCR4. In summary, by focusing on the attenuation of TCR signaling and the downregulation of CXCR4, these studies will provide a mechanistic basis for understanding the opposing effects of DORS.
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