Signal Transducer and Activator of Transcription 3 Activation by the δ-Opioid Receptor via Gα14 Involves Multiple Intermediates

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Abstract
The hematopoietic-specific Gα14 links a variety of G protein-coupled receptors to phospholipase Cβ (PLCβ) stimulation. Recent studies reveal that several Gα subunits are capable of activating signal transducer and activator of transcription (STAT) proteins. In the present study, we investigated the mechanism by which Gα14 mediates receptor-induced stimulation of STAT3. In human embryonic kidney 293 cells, coexpression of Gα14 with δ-opioid receptor supported [d-Pen2, d-Pen5]enkephalin (DPDPE)-induced STAT3 phosphorylations at both Tyr705 and Ser727 in a pertussis toxin-insensitive manner. The constitutively active Gα14QL mutant also induced STAT3 phosphorylations at these sites and promoted STAT3-dependent luciferase activity. Requirements for PLCβ, protein kinase C (PKC), and calmodulin-dependent kinase II (CaMKII) in Gα14QL-induced STAT3 activation were demonstrated by their respective inhibitors as well as by coexpression of their dominant-negative mutants. Inhibition of c-Src and Janus kinase 2 and 3 activities abolished STAT3 activation induced by Gα14QL, but no physical association between Gα14QL and c-Src could be detected by coimmunoprecipitation. Various intermediates along the extracellular signal-regulated kinase signaling cascade were apparently required for Gα14QL-induced STAT3 activation; they included Ras/Rac1, Raf-1, and mitogen-activated protein kinase kinase-1/2. In contrast, functional blockade of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol-3 kinase had no effect on Gα14QL-induced responses. PLCβ, PKC, and CaMKII were shown to be involved in Gα14QL-mediated c-Src phosphorylation. Similar results were obtained with human erythro-leukemia cells upon DPDPE treatment. These results demonstrate for the first time that Gα14 activation can lead to STAT3 stimulation via a complex signaling network involving multiple intermediates.