A novel regulatory role of RGS4 in STAT5B activation, neurite outgrowth and neuronal differentiation.

The Regulator of G protein Signalling 4 (RGS4) is a multitask protein that interacts with and negatively modulates opioid receptor signalling. Previously, we showed that the δ-opioid receptor (δ-OR) forms a multiprotein signalling complex consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription 5B (STAT5B) that leads to neuronal differentiation and neurite outgrowth upon δ-ΟR activation. Here, we investigated whether RGS4 could participate in signalling pathways to regulate neurotropic events. We demonstrate that RGS4 interacts directly with STAT5B independently of δ-ΟR presence both in vitro and in living cells. This interaction involves the N-terminal portion of RGS4 and the DNA-binding SH3 domain of STAT5B. Expression of RGS4 in HEK293 cells expressing δ-OR and/or erythropoietin receptor results in inhibition of [D-Ser2, Leu5, Thr6]-enkephalin (DSLET)-and erythropoietin-dependent STAT5B phosphorylation and subsequent transcriptional activation. DSLET-dependent neurite outgrowth of neuroblastoma cells is also blocked by RGS4 expression, whereas primary cortical cultures of RGS4 knockout mice (RGS4-/-) exhibit enhanced neuronal sprouting after δ-OR activation. Additional studies in adult brain extracts from RGS4-/- mice revealed increased levels of p-STAT5B. Finally, neuronal progenitor cultures from RGS4-/- mice exhibit enhanced proliferation with concomitant increases in the mRNA levels of the anti-apoptotic STAT5B target genes bcl2 and bcl-xl. These observations suggest that RGS4 is implicated in opioid dependent neuronal differentiation and neurite outgrowth via a "non-canonical" signaling pathway regulating STAT5B-directed responses.

RGS2 and RGS4 proteins: New modulators of the κ-opioid receptor signaling.

Previous studies have shown that RGS4 associates with the C-termini of µ- and δ-opioid receptors in living cells and plays a key role in Gi/Go protein coupling selectivity and signalling of these receptors [12,20]. To deduce whether similar effects also occur for the κ-opioid receptor (κ-ΟR) and define the ability of members of the Regulators of G protein Signaling (RGS) of the B/R4 subfamily to interact with κ-ΟR subdomains we generated glutathione S-transferase fusion peptides encompassing the carboxyl-termini of κ-OR (κ-CT). Results from pull down experiments indicated that RGS2 and RGS4 directly interact within different domains of the κ-CT. Co-precipitation studies in living cells indicated that RGS2 and RGS4 associate with κ-ΟR constitutively and upon receptor activation and confer selectivity for coupling with a specific subset of G proteins. Expression of both members, RGS2 and/or RGS4, in 293F cells attenuated κ-agonist mediated-adenylyl cyclase inhibition and extracellular signal regulated kinase (ERK1,2) phosphorylation with a different amplitude in their modulatory effect in κ-ΟR signaling. Our findings demonstrate that RGS2 and RGS4 are new interacting partners that play key roles in G protein coupling to negatively regulate κ-ΟR signaling.

Selective interactions of spinophilin with the C-terminal domains of the δ- and µ-opioid receptors and G proteins differentially modulate opioid receptor signaling.

Previous studies have shown that the intracellular domains of opioid receptors serve as platforms for the formation of a multi-component signaling complex consisting of various interacting partners (Leontiadis et al., 2009, Cell Signal. 21, 1218-1228; Georganta et al., 2010, Neuropharmacology, 59(3), 139-148). In the present study we demonstrate that spinophilin a dendritic-spine enriched scaffold protein associates with δ- and µ-opioid receptors (δ-ΟR, µ-OR) constitutively in HEK293 an interaction that is altered upon agonist administration and enhanced upon forskolin treatment for both µ-OR and δ-ΟR. Spinophilin association with the opioid receptors is mediated via the third intracellular loop and a conserved region of the C-terminal tails. The portion of spinophilin responsible for interaction with the δ-OR and µ-OR is narrowed to a region encompassing amino acids 151-444. Spinophilin, RGS4, Gα and Gßγ subunits of G proteins form a multi-protein complex using specific regions of spinophilin and a conserved amino acid stretch of the C-terminal tails of both δ-µ-ORs. Expression of spinophilin in HEK293 cells potentiated DPDPE-mediated adenylyl-cyclase inhibition of δ-OR leaving unaffected the levels of cAMP accumulation mediated by the µ-OR. Moreover, measurements of extracellular signal regulated kinase (ERK1,2) phosphorylation indicated that the presence of spinophilin attenuated agonist-driven ERK1,2 phosphorylation mediated upon activation of the δ-OR but not the µ-OR. Collectively, these findings suggest that spinophilin associates with both δ- and µ-ΟR and G protein subunits in HEK293 cells participating in a multimeric signaling complex that displays a differential regulatory role in opioid receptor signaling.