A novel regulatory role of RGS4 in δ‐opioid receptor mediated neuronal outgrowth and differentiation

Abstract

Regulators of G proteins signaling (RGS) despite their initial role as GTPase activating proteins, serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription to tumorgenesis. Evidence suggests the cytoplasmic/nuclear distribution and translocation of RGS proteins is mediated by interactions with other proteins which mediate cross talk between G proteins and other cellular processes. RGS4 a member of the B/R4 family of RGS proteins has been found to be a universal negative regulator of μ, δ and κ‐opioid receptor signaling that confers selectivity for G protein coupling to these receptors by directly interacting with them (Georgoussi et al., 2006, 2012; Leontiadis et al. 2009; Papakonstantinou et al., 2015). It was also shown that δ‐opioid receptor (δ‐OR) forms a multicomponent signaling complex, consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription STAT5B, that leads to neurite outgrowth and neuronal differentiation via a phosphoryalted‐STAT5B‐Gαi/o pathway (Georganta et al., 2010, 2013). Knowing that RGS4 is a multitask protein we questioned whether RGS4 could be implicated in neuronal differentiation and neurite outgrowth upon δ‐OR activation through a similar signaling pathway. We demonstrate that RGS4 directly interacts with STAT5B in vitro and in living cells independently of δ‐OR presence. 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. Measurements of DSLET‐dependent neurite outgrowth of neuroblastoma cells is also blocked by RGS4 expression, whereas primary cortical cultures of RGS4 knock out mice (RGS4−/−) exhibit enhanced neuronal sprouting after δ‐OR activation. Finally, neuronal progenitor cultures from RGS4−/− mice exhibit enhanced proliferation with concomitant increases in the mRNA levels of the anti‐apoptotic genes Bcl2 and Bcl‐xl STAT5B target genes. These observations suggest that RGS4 plays a significant regulatory role in opioid dependent neuronal differentiation and neurite outgrowth via a “non‐canonical” signaling pathway involving STAT5B‐directed responses.Support or Funding InformationThis work was supported by the Excellence II‐3722, (“NO‐ALGOS”) of the Hellenic General Secretariat of Research and Technology and was carried out in facilities of the OPENSCREEN‐GR National (Greece) Research Infrastructure. Z.G participates in the EU COST Action CM1207 (GLISTEN).