Received for publication November 16, 2007.
Revised February 18, 2008.
Accepted for publication February 19, 2008.

Endogenous RGS Proteins Differentially Modulate Full and Partial Mu-Opioid Agonists at Adenylyl Cyclase as Predicted by a Collision Coupling Model

Abstract

Regulators of G protein signaling (RGS) proteins accelerate the endogenous GTPase activity of G _i/o proteins to increase the rate of deactivation of active G-GTP and G signaling molecules. Previous studies have suggested that RGS proteins are more effective on less efficiently coupled systems such as with partial agonist responses. To determine the role of endogenous RGS proteins in functional responses to mu-opioid agonists of different intrinsic efficacy, G_i/o subunits with a mutation at the pertussis toxin (PTX) sensitive cysteine (C351I) and with or without a mutation at the RGS binding site (G184S) were stably expressed in C6 glioma cells expressing a mu-opioid receptor. Cells were treated overnight with PTX to inactivate endogenous G proteins. Maximal inhibition of forskolin-stimulated adenylyl cyclase by the low efficacy partial agonists buprenorphine and nalbuphine was increased in cells expressing RGS-insensitive G_o^CIGS, G_i2^CIGS, or G_i3^CIGS compared with their G^CI counterparts, but the RGS-insensitive mutation had little or no effect on the maximal inhibition by the higher efficacy agonists, DAMGO and morphine. The potency of all the agonists to inhibit forskolin-stimulated adenylyl cyclase was increased in cells expressing RGS-insensitive G_o^CIGS,G_i2^CIGS, or G_i3^CIGS, regardless of efficacy. These data are comparable to predictions based on a collision coupling model. In this model the rate of G protein inactivation, which is modulated by RGS proteins, and the rate of G protein activation, which is affected by agonist intrinsic efficacy, determine the maximal agonist response and potency at adenylyl cyclase under steady state conditions.

Key words: Gi family, cAMP, RGS proteins, Thermodynamic and kinetic processes and modeling, Mutagenesis/Chimeric approaches, Opioids