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

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 dopioid 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 RGS19depleted cells, there was an increase in the ability of MOR (morphine) but not of DOR [(4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC80)] or NOPR (nociceptin) agonists to inhibit forskolinstimulated adenylyl cyclase and increase mitogen-activated protein kinase (MAPK) activity. Overnight treatment with either MOR [D-Ala, N-Me-Phe, Gly-ol-enkephalin (DAMGO) or morphine] or DOR (D-Pen-enkephalin or SNC80) agonists increased RGS19 and GIPC protein levels in a timeand 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)-4Hchromen-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 MORwith 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.