Modulation of μ-Opioid Receptor Signaling by RGS19 in SH-SY5Y Cells

Abstract

Regulator of G-protein signaling protein 19 (RGS19), also known as Ga-interacting protein (GAIP), acts as a GTPase accelerating protein for Gaz as well as Gai/o subunits. Interactions with GAIP-interacting protein N-terminus and GAIP-interacting protein C-terminus (GIPC) link RGS19 to a variety of intracellular proteins. Here we show that RGS19 is abundantly expressed in human neuroblastoma SH-SY5Y cells that also express mand d-opioid receptors (MORs and DORs, respectively) and nociceptin receptors (NOPRs). Lentiviral delivery of short hairpin RNA specifically targeted to RGS19 reduced RGS19 protein levels by 69%, with a similar reduction in GIPC. In RGS19-depleted cells, there was an increase in the ability of 5 -enkephalin or SNC80) agonists increased RGS19 and GIPC protein levels in a time-and concentration-dependent manner. The MOR-induced increase in RGS19 protein was prevented by pretreatment with pertussis toxin or the opioid antagonist naloxone. Protein kinase C (PKC) activation alone increased the level of RGS19 and inhibitors of PKC 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo [2,3-a] pyrrolo [3,4-c]carbazole-12-propanenitrile and mitogen-activated protein kinase kinase 1 2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one, but not protein kinase A (H89), completely blocked DAMGO-induced RGS19 protein accumulation. The findings show that RGS19 and GIPC are jointly regulated, that RGS19 is a GTPase accelerating protein for MOR with selectivity over DOR and NOPR, and that chronic MOR or DOR agonist treatment increases RGS19 levels by a PKC and the MAPK pathway-dependent mechanism.

Heat-shock protein 90 (Hsp90) promotes opioid-induced anti-nociception by an ERK mitogen-activated protein kinase (MAPK) mechanism in mouse brain

“…We first sought to explore the role of Hsp90 in regulating MOR signaling in four in vitro models. These include Chinese hamster ovary (CHO), human embryonic kidney (HEK), and human bone osteosarcoma (U2OS) cell lines stably expressing the human MOR (see "Experimental procedures" for cell line details) and the human neuroblastoma cell line SH-SY5Y, which endogenously expresses the MOR and ␦-opioid receptor (DOR) (23). All cells were treated with 1 M 17-AAG for 24 h and then stimulated with the MOR full agonist DAMGO ([D-Ala 2 , N-MePhe 4 , Gly 2 -ol]-enkephalin) for 10 min; signaling protein expression and signal transduction were analyzed by Western blotting.…”

Regulator of G-Protein Signaling (RGS) Protein Modulation of Opioid Receptor Signaling as a Potential Target for Pain Management

“…Antisense knockdown of central RGSZ17 levels in male mice was seen to increase morphine and DAMGO antinociception, but also increased the rate of tolerance development (Garzón et al, 2005). Knockdown of RGS19 in SH-SY5Y cells enhances MOR agonistinduced MAPK stimulation and adenylyl cyclase inhibition (Wang and Traynor, 2013). Consequently, knockdown of RGS19 and RGS20, enhances the antinociceptive effects of morphine and DAMGO (Garzón et al, 2004).…”

“…Purified RGS19, like RGS4 acts as a GAP for DOR signaling in NG108-15 cells (Hepler et al, 1997). In contrast DOR signaling in SH-SY5Y cells to adenylate cyclase or MAPK was not sensitive to knockdown of RGS19 (Wang and Traynor, 2013) and knockdown of RGS19 failed to modulate antinociceptive responses to DOR agonists, DPDPE and deltorphin (Garzón et al, 2004). This could suggest the experiments in NG108-15 cells that RGS19 is acting as a non-selective GAP.…”

Genetic Analysis of Rare Human Variants of Regulators of G Protein Signaling Proteins and Their Role in Human Physiology and Disease

“…RGS19 expression levels are reportedly upregulated in several disease states, including multiple sclerosis (Igci et al, 2016) and ovarian cancers (Tso et al, 2011). RGS19 is also highly expressed in human neuroblastoma SH-SY5Y cells (Wang and Traynor, 2013). However, in other instances, RGS19 has been reported to inhibit Ras activation by upregulating Nm23, a tumor metastasis suppressor .…”