Opioid-induced Down-Regulation of RGS4 ROLE OF UBIQUITINATION AND IMPLICATIONS FOR RECEPTOR CROSS-TALK

Qin Wang; John R. Traynor

doi:10.1074/jbc.M110.160911

Opioid-induced Down-Regulation of RGS4

ROLE OF UBIQUITINATION AND IMPLICATIONS FOR RECEPTOR CROSS-TALK*

Qin Wang^‡ and
John R. Traynor^‡^§,¹

From the ^‡Department of Pharmacology and
^§Substance Abuse Research Center, University of Michigan, Ann Arbor, Michigan 48109

1 To whom correspondence should be addressed: Dept. of Pharmacology, 1301 MSRB III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-5632. Fax: 734-763-4450; E-mail: jtraynor{at}umich.edu.

Abstract

Regulator of G protein signaling protein 4 (RGS4) acts as a GTPase accelerating protein to modulate μ- and δ- opioid receptor (MOR and DOR, respectively) signaling. In turn, exposure to MOR agonists leads to changes in RGS4 at the mRNA and/or protein level. Here we have used human neuroblastoma SH-SY5Y cells that endogenously express MOR, DOR, and RGS4 to study opioid-mediated down-regulation of RGS4. Overnight treatment of SH-SY5Y cells with the MOR agonist DAMGO or the DOR agonist DPDPE decreased RGS4 protein by ∼60% accompanied by a profound loss of opioid receptors but with no change in RGS4 mRNA. The decrease in RGS4 protein was prevented by the pretreatment with pertussis toxin or the opioid antagonist naloxone. The agonist-induced down-regulation of RGS4 proteins was completely blocked by treatment with the proteasome inhibitors MG132 or lactacystin or high concentrations of leupeptin, indicating involvement of ubiquitin-proteasome and lysosomal degradation. Polyubiquitinated RGS4 protein was observed in the presence of MG132 or the specific proteasome inhibitor lactacystin and promoted by opioid agonist. The loss of opioid receptors was not prevented by MG132, demonstrating a different degradation pathway. RGS4 is a GTPase accelerating protein for both Gα_i/o and Gα_q proteins. After overnight treatment with DAMGO to reduce RGS4 protein, signaling at the Gα_i/o-coupled DOR and the Gα_q-coupled M₃ muscarinic receptor (M₃R) was increased but not signaling of the α₂ adrenergic receptor or bradykinin BK₂ receptor, suggesting the development of cross-talk between the DOR and M₃R involving RGS4.