Differential modulation of mu-opioid receptor signaling to adenylyl cyclase by regulators of G protein signaling proteins 4 or 8 and 7 in permeabilised C6 cells is Galpha subtype dependent

Abstract

Regulators of G protein signaling (RGS) proteins act as GTPase-accelerating protein to negatively modulate G protein signaling and are defined by a conserved RGS domain with considerable amino acid diversity. To determine the effects of specific, purified RGS proteins on mu-opioid signaling, C6 cells stably expressing a mu-opioid receptor were rendered permeable to proteins by treatment with digitonin. Mu-opioid inhibition of forskolin-stimulated adenylyl cyclase by [D-Ala(2),N-Me-Phe(4),Gly-ol]-enkephalin (DAMGO), a mu-specific opioid peptide, remained fully intact in permeabilized cells. Purified RGS domain of RGS4 added to permeabilized cells resulted in a twofold loss in DAMGO potency but had no effect in cells expressing RGS-insensitive G proteins. The inhibitory effect of DAMGO was reduced to the same extent by purified RGS4 and RGS8. In contrast, the RGS domain of RGS7 had no effect and inhibited the action of RGS8 as a result of weak physical association with Galphai2 and minimal GTPase-accelerating protein activity in C6 cell membranes. These data suggest that differences in conserved RGS domains of specific RGS proteins contribute to differential regulation of opioid signaling to adenylyl cyclase and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein-coupled receptor signaling.

Fig. 1

Inhibition of forskolin-stimulated AC activity by the mu opioid receptor agonist DAMGO in digitonin-permeabilized cells. C6μ cells incubated with (closed circles) or without (open circles) 20 µM digitonin were incubated with increasing concentrations of DAMGO. Adenylyl cyclase activity was measured as pmol cAMP per mg protein*min in the presence of 1 µM IBMX. EC₅₀ values were calculated by non-linear regression analysis of three independent experiments. Data presented are the mean ± SEM (n=3).

Fig. 2

ΔN-RGS4 (RGS4-box) alters mu-opioid inhibition of forskolin-stimulated AC activity. A) Purified ΔN-RGS4 (lacking N-terminal 18 amino-acids) decreased the inhibitory effect of DAMGO (100 nM) in a concentration-dependent manner. Cells were stimulated with 10 µM forskolin (Fsk) ± DAMGO and/or purified RGS protein. cAMP was measured as described. Data are expressed as the percentage of the Fsk response and are the mean ± SEM (n=5–8). B) Increasing concentrations of forskolin (Fsk) were used to stimulate AC activity in the presence (filled circles) or absence (open circles) of a maximal concentration of ΔN-RGS4 (1 µM). Data are expressed as the pmol of cAMP per mg protein*min and are the mean ± SEM from at least four independent experiments (n=4).

Fig. 3

Activity of ΔN-RGS4 in cells expressing RGS-insensitive Gαi2. (A) [³⁵S]GTPγS binding was determined in membranes from cells stably expressing either RGS-sensitive (RGS-s) or RGS-insensitive (RGS-i) Gαi2. Overnight treatment with 100 ng/ml pertussis toxin (PTX) was used to block endogenous Gi/o protein activity. Data are derived from four assays, each carried out in duplicate, and are expressed as a percentage of basal binding. Inset: levels of RGS-i and RGS-s Gαi2 expression were verified by SDS-PAGE (20 µg of membranes or 20 ng of Gα_i2 standard (Gαi2)). Proteins were transferred to a nitrocellulose membrane and probed with anti-Gα_i2 antibody. Shown is a representative blot from three separate blots. (B) Increasing concentrations of DAMGO were also used to inhibit AC activity stimulated by 10 µM forskolin (Fsk) in RGS-s and RGS-i Gαi2-expressing cells that were permeabilized with digitonin. Again cells were treated overnight with PTX (100 ng/ml) to block coupling to endogenous Gα. Data are the percentage of the Fsk response and are the mean ± SEM from at least three independent experiments. (C) Near maximal concentrations of DAMGO were used to inhibit Fsk-stimulated AC activity in digitonin-permeabilized RGS-s and RGS-i Gαi2-expressing cells (1 µM and 0.1 µM, respectively). Addition of ΔN-RGS4 (1 µM) reduced the inhibitory effect of DAMGO in cells expressing RGS-sensitive Gαi2 but not RGS-insensitive Gαi2. Data are expressed as the percentage of the Fsk response and are the mean ± SEM from at least four independent experiments; *p < 0.05 compared to DAMGO-treated RGS-s Gαi2 expressing cells as indicated (determined by one-way ANOVA with Tukey’s post-hoc test).

Fig. 4

Purified RGS4 and RGS8 negatively regulate opioid coupling to AC. (A) DAMGO inhibition of forskolin-stimulated AC was determined in the presence of maximal concentrations (1 µM) of RGS4 (circles) or RGS8 (squares). Each RGS protein decreased DAMGO potency and efficacy to a similar extent. Data are expressed as the percentage of the Fsk response and are the mean ± SEM from at least four independent experiments. EC₅₀ values were calculated by non-linear regression (GraphPad Prism). (B) Inhibition of forskolin-stimulated AC by DAMGO (100 nM) was measured in the presence of increasing concentrations of RGS4 (circles), RGS8 (squares), or RGS7-box (triangles). Data are expressed as the percentage of the Fsk response and are the mean ± SEM from four to six independent experiments. (C) Single turnover GTP hydrolysis of Gαo was measured with (filled circles) and without RGS7-box (open circles) to confirm GAP activity of the RGS7-box preparation. Measurements were taken in the presence of 200 nM Gαo ± 125 nM RGS7 with a 3-fold molar excess of γ[³²P]GTP. [³²P]P_i released at each time point was fit to an exponential function: [³²P] Pi counts(t) = counts_(t=0) + counts_{(t=30 min)}*(1-e^−kt), to calculate the rate constant (k). Fitting constraints included setting counts_(t=0) for each curve to the average of the counts_(t=0) for the experiment, and setting counts_{(t=30 min)} to the same value for all curves in an experiment. Data are the mean ± SEM of four independent experiments performed in triplicate.

Fig. 5

Direct Regulation of Gαi and Gαo by RGS Proteins. GAP activity of RGS8 and RGS7-box was measured by DAMGO-stimulated ³²Pi release in C6μ cells stably expressing similar levels of mu-opioid receptor and Gαo (A) or Gαi2 (B). RGS proteins were added at a concentration of 1 µM with 10 µM DAMGO prior to the addition of [γ32P]GTP. DAMGO-stimulated Pi released is shown as the mean ± SEM from at least 3 assays measured in duplicate. (C) RGS7-box binds to and GAPs Gαo but has reduced affinity for Gαi2 compared to RGS4. The binding of fluorescently labeled Gαo (Alexa Fluor 532®) to LumAvidin-conjugated RGS4 was measured by flow cytometry. Activity and effective concentration of Gαo was determined post-labeling using [³⁵S]GTPγS. The RGS-Gαo interaction was measured on a Luminex 100IS flow cytometer as the median fluorescence intensity. Increasing concentrations of unlabeled Gαi2 were added to compete for RGS protein binding. Data are the mean ± SEM of three separate experiments each performed in duplicate.

Fig. 6

Antagonism of RGS8 activity by RGS7-box. Inhibition of forskolin-stimulated AC by DAMGO (100 nM) was measured in the presence of either RGS7-box (5 uM), RGS8 (1 uM) or both (RGS7 + RGS8). Data are expressed as the percentage of the Fsk response and are the mean ± SEM (n=3–4).