Co-Expression of Regulator of G Protein Signaling 4 (RGS4) and the MU Opioid Receptor in Regions of Rat Brain: Evidence That RGS4 Attenuates MU Opioid Receptor Signaling

摘要

Regulators of G protein Signaling (RGS) proteins influence G protein- coupled receptor signal transduction by enhancing the intrinsic GTPase activity of G proteins. The RGS-enhanced GTPase activity of G proteins may be responsible for the desensitization of certain G protein-coupled receptors, including the mu opioid receptor. The goal of this research was to evaluate the ability of recombinant RGS4 to affect mu opioid receptor-mediated cellular signaling and to identify regions of the rat brain in which both RGS4 and the mu opioid receptor are co-expressed. We evaluated the ability of recombinant RGS4 to affect D-Ala2, N-Me-Phe4, glyol enkephalin (DAMGO)-mediated inhibition of adenylyl cyclase activity in membranes of SH-SY5Y cells, a cell line that express endogenous mu receptors. Recombinant RGS4 caused a concentration- dependent attenuation of DAMGOmediated inhibition of adenylyl cyclase activity. RGS4 diminished the efficacy, but not the potency, of DAMGO in inhibiting adenylyl cyclase activity. In contrast, RGS4 had no effect on the ability of GTPgS, a nonhydrolyzable analogue of GTP, to inhibit adenylyl cyclase activity. RGS4 also had no effect on DAMGO stimulated 35SgtpgS binding in SH-SY5Y membranes. Additionally, RGS4 was tested for its ability to affect 3Hdamgo binding to the mu receptor. RGS4 failed to affect either the KD of the Bmax of 3Hdamgo in saturation binding experiments. Antibodies generated against rat RGS4 and the rat mu opioid receptor were used in immunohistochemical staining to identify specific regions of rat brain where the two proteins are co- expressed. Both RGS4 and mu opioid receptor proteins were present in many of the same regions of the brain. Further, we demonstrated that RGS4 is primarily localized to the nucleus, but that administration of fentanyl, a potent mu opioid agonist, induces translocation out of the nucleus, to the cytoplasm in the hippocampal CA(number sign) pyramidal neurons.