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

Abstract

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.

Keywords: Deoxycholate (PubChem CID: 91896239); Differentiation; Erythropoietin; MgCl2(PubChem CID:5360315); Neurite outgrowth; Opioid receptor; PMSF (PubChem CID: 4784); RGS4; Retinoic acid (PubChem CID: 444795); STAT5B; Sodium orthovanadate (PubChem CID: 6167); Triton X-100 (PubChem CID: 5590); [(3)H]-diprenorphine (PubChem CID: 71719127); [D-Ser(2),Leu5, Thr6]-enkephalin (PubChem CID: 107847).