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Endogenous Regulators of G Protein Signaling Differentially Modulate Full and Partial µ-Opioid Agonists at Adenylyl Cyclase as Predicted by a Collision Coupling Model

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan (M.J.C., J.R.T.); and Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan (J.J.L.)

Received November 15, 2007; accepted February 19, 2008

	Abstract

Top Abstract Materials and Methods Results Discussion Appendix References

 
Regulator 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 Gβ 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 µ-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 µ-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 with 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.

The effect of G protein cycle kinetics on agonist potency and maximal response at adenylyl cyclase has been formulated for G_s by Stickle and Barber (1992) and Whaley et al. (1994) based on the collision coupling model (Tolkovsky and Levitzki, 1978), which included parameters for the G protein cycle of GDP-GTP exchange and GTP hydrolysis. According to this model, both the fractional activation of adenylyl cyclase and the agonist potency at steady state are dependent on the rate constant for G protein activation, the rate constant for inactivation, and the number of receptors. The activation rate constant is dependent on the collision frequency between receptor and G protein (a function of diffusivity and the numbers of molecules), receptor occupancy, and agonist intrinsic efficacy (Whaley et al., 1994; Krumins et al., 1997). The inactivation rate constant is dependent on the rate of GTP hydrolysis, which is modulated by RGS proteins, thus providing a simple model for predicting the effects of RGS proteins on agonist responses.

In this study, we tested the hypothesis that endogenously expressed RGS proteins affect maximal agonist response and potency in a manner consistent with the collision coupling model and that this explains the differential sensitivity of full and partial agonists to RGS action. Moreover, because different G_i/o subtypes may have different rate constants for activation and inactivation by agonist-occupied receptors and RGS proteins may differentially affect the G subtypes because of their different GAP activities (Posner et al., 1999; Posner et al., 1999; Cavalli et al., 2000; Welsby et al., 2002; Hooks et al., 2003), we also test the hypothesis that the endogenous RGS proteins may be more effective at G_o than the G_i subtypes.

By expressing G_i/o subunits with a mutation at the pertussis toxin (PTX)-sensitive cysteine (C351I) in C6 glioma cells expressing the µ-opioid receptor, we are able to study the action of the exogenously expressed G subtype on inhibition of adenylyl cyclase after treatment with PTX to uncouple the endogenous G subunits. There is evidence for the expression of certain RGS proteins in C6 cells (Snow et al., 2002), but a full description is not available. Therefore, to examine the effects of the GAP activity of the total complement of endogenous RGS proteins present in C6 cells, we expressed the PTX-insensitive G subunits with or without an additional mutation at the RGS binding site (G184S). This prevents binding between G and the RGS box of all RGS proteins without affecting the endogenous GTPase activity of the G. This eliminates RGS-stimulated GTPase-activity (Clark et al., 2003; Huang et al., 2006), resulting in an accumulation of active G-GTP and Gβ signaling molecules, which is observed as an increase in agonist potency and/or an increase in the maximal agonist effect (Clark et al., 2003).

Our results demonstrate that, as predicted by the collision coupling model, endogenous RGS proteins have a greater effect on the maximal effect of partial µ-opioid agonists than full agonists at inhibition of adenylyl cyclase but similarly reduce agonist potency for both full and partial agonists.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion Appendix References

 
Materials. [³H]Diprenorphine and [³⁵S]GTPS were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). Tissue culture medium, fetal bovine serum, trypsin, LipofectAMINE Plus reagent, LipofectAMINE 2000, G-418 (Geneticin), and Zeocin were from Invitrogen (Carlsbad, CA). Trizma, GDP, forskolin, 3-isobutyl-1-methylxantihine (IBMX), DAMGO, and other biochemicals were from Sigma Chemical Co (St. Louis, MO). Morphine, buprenorphine, nalbuphine, and naloxone were obtained through the Opioid Basic Research Center at the University of Michigan (Ann Arbor, MI).

RGS-Insensitive Mutation and FLAG-Epitope Tagging. Human G containing a cysteine-to-isoleucine mutation at the PTX-sensitive Cys (G_oA^C351I, G_i2^C352I, G_i3^C351I) in the pcDNA3.1+ vector were purchased from the Missouri University of Science and Technology cDNA Resource Center (http://www.cdna.org). For insertion into the Zeocin-resistant vector, pcDNAzeo^-, the restriction enzymes Nhe I and Xba I (Promega, Madison, WI) were used to remove G_oA^C351I or G_i3^C351I from the original vector and Pme I (New England Biolabs, Ipswich, MA) was used for G_i2^C352I, followed by ligation with calf intestinal alkaline phosphatase-treated pcDNAzeo^- vector. A Quik-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to replace the glycine at the RGS binding site (G_oA, G_i2: G184S; G_i3: G183S) to a serine using the following primers: 5'-GGGTCAAAACCACTTCCATCGTAGAAAC-3' for G_oA^C351I, 5'-GTAAAGACCACGTCGATCGTGGAG-3' for G_i2^C352I, and 5'-CGAGAGTGAAGACCACATCCATTGTAGAAAC-3' for G_i3^C351I (underlining indicates mutation sites).

A FLAG tag was added to the N terminus of PTX-insensitive G in the Zeocin-resistant vector using the QuikChange site-directed mutagenesis kit with the following primers: for G_oA^C351I, 5'-CCCTTGATCGGTACCACCATGGACTACAAAGACGATGACGACAAG-ATGGGATGTACTCTG-3'; for G_i2^C352I, 5'-CCCTTGATCGGTACCACCATGGACTACAAAGACGATGACGACAAG-ATGGGCT GCACCGTG-3'; and for G_i3^C351I 5'-CCCTTGATCGGTACCACCATGGACTACAAAGACGATGACGACAAG-ATGGGCTGCACGTTG-3'.

Cell Culture and Expression of PTX-Insensitive G. Human µ-opioid receptor in the pcDNA3.1 vector (http://www.cdna.org) was stably transfected into HEK293 cells using LipofectAMINE 2000 and selected using 0.8 mg/ml G-418. C6 cells stably expressing the µ-opioid receptor (C6µ; Emmerson et al., 1996) or HEK293 cells stably expressing the µ-opioid receptor (HEK293µ) were stably transfected with PTX-insensitive G using LipofectAMINE Plus and selected using 0.4 mg/ml Zeocin for C6µ or 0.2 mg/ml Zeocin for HEK293µ. Selected clones were maintained in DMEM containing 10% fetal bovine serum, 0.4 mg/ml (HEK293µ) or 0.25 mg/ml (C6µ) G-418, and 0.2 mg/ml (HEK293µ) or 0.4 mg/ml (C6µ) Zeocin.

Membrane Preparation. Cells were treated overnight with 100 ng/ml PTX before harvesting and preparation of plasma membranes as described previously (Clark et al., 2003).

Western Blots. Membrane proteins (5 µg of HEK293µ-FLAGG^CI; 60 µg of C6µG^CI/CIGS) were separated by 12% SDS-polyacrylamide gel electrophoresis (Protogel; National Diagnostics, Inc., Atlanta, GA) and transferred to a nitrocellulose membrane (45 µm; Pierce, Rockford, IL). Membranes with C6µ G^CI/CIGS were blocked overnight with 5% dry milk and probed with 1:200 anti-G_o (Santa Cruz Biotechnology, Santa Cruz, CA) or 1:6000 anti-G_i2 (Calbiochem, San Diego, CA), followed by 1:15,000 goat anti-rabbit IgG-HRP in 5% dry milk (Santa Cruz Biotechnology). Membranes with HEK293µ-FLAGG^CI were blocked over-night with 5% dry milk and probed with 1:2000 anti-M2 FLAG and 1:1000 anti-tubulin (Sigma Chemical Co., St. Louis, MO) in 5% dry milk followed with 1:12,000 goat anti-mouse IgG-HRP in 5% dry milk (Santa Cruz Biotechnology). Bands were visualized using Super Signal West Pico enhanced chemiluminescence (Pierce) and quantified using a KODAK Image Station (Carestream Health, Rochester, NY). We were unable to find a satisfactory antibody to a region of G_i3 without the PTX- or RGS-insensitive mutations.

Receptor Binding Assay. Membranes (5-20 µg) were incubated with 2 to 3 nM [³H]diprenorphine with or without 50 µM naloxone (to determine nonspecific binding) in 50 mM Tris-HCl, pH 7.4, as described previously (Clark et al., 2003), to determine receptor number.

[³⁵S]GTPS Binding Assay. Membranes (5-20 µg) were incubated for 60 min with 0.1 nM [³⁵S]GTPS, 30 µM GDP, 100 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol, 20 mM Tris-HCl, pH 7.4, and 0.01 to 10 µM DAMGO or distilled water and filtered as described previously (Clark et al., 2003) to determine bound [³⁵S]GTPS.

cAMP Accumulation Assay. Cells were plated to 80% confluence 2 days before the assay and treated with 100 ng/ml PTX. To start the assay, medium was replaced with DMEM containing 5 µM forskolin, 1 mM IBMX, and 0.001 to 10 µM DAMGO, morphine, nalbuphine, buprenorphine, or distilled water. After 10 min at 37°C, the reaction was stopped by replacing the medium with ice-cold 3% perchloric acid. After at least 30 min at 4°C, 0.4 ml was removed from each sample, neutralized with 0.08 ml of 2.5 M KHCO₃, vortexed, and centrifuged at 15,000g for 1 min. A 20-µl aliquot was taken from the supernatant of each sample, and accumulated cAMP was quantified using a [³H]cAMP assay kit according to manufacturer's instructions (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Inhibition was determined as the percentage decrease of forskolin-stimulated cAMP accumulation in the absence of opioid agonist.

View larger version (7K): [in this window] [in a new window]   Fig. 1. Predicted effect of RGS proteins on the relationship between the overall activation rate constant (kact) and agonist inhibition of adenylyl cyclase. The maximal effect (%) at adenylyl cyclase was plotted for a range of values of kact (eqs. 1 and 2) with the rate constant for inactivation by hydrolysis (khydrol) as 0.1 s-1 in the absence of RGS proteins and 5 s-1 in the presence of RGS proteins.

View larger version (8K): [in this window] [in a new window]   Fig. 2. Predicted effect of RGS proteins on the relationship between the overall activation rate constant (kact) and agonist potency at adenylyl cyclase. Agonist potency was plotted for a range of rate constants of G protein activation (eqs. 2 and 3) with the agonist Kd set at 250 nM and the rate constant for inactivation by hydrolysis (khydrol) as 0.1 s-1 in the absence of RGS proteins and 5 s-1 in the presence of RGS proteins.

	Results

Top Abstract Materials and Methods Results Discussion Appendix References

 
Collision Coupling Model Predictions of Maximal Agonist Response and Potency with and without RGS Proteins. The prediction of the collision coupling model for the maximum agonist response at an effector, f, is

(1)

where k_a is the rate constant for activation, R_TOT is the total number of receptors, f_active is the fraction of bound receptors in the active form (a measure of agonist intrinsic efficacy), and k_hydrol is the rate constant for hydrolysis of G-GTP (Kinzer-Ursem et al., 2006; see Appendix for details). For simplicity of notation, we define an overall activation rate constant k_act as:

(2)

Thus, the activation level of the effector (maximal response) is determined by the overall activation rate constant k_act and the hydrolysis rate constant k_hydrol. To predict the effect of RGS proteins on the maximum agonist response, we used the estimated values for rate constants of hydrolysis (k_hydrol) of 0.1 s^-1 in the absence of RGS proteins and 5 s^-1 in the presence of RGS proteins (Shea et al., 2000). The value of the rate constant for activation, k_a, can be estimated from Monte Carlo simulations of receptor and G protein diffusion to be on the order of 10^-5 to 10^-3 (number per cell)^-1s^-1 (Mahama and Linderman, 1994; Shea et al., 1997) and the units of R_TOT are (number per cell). Values for maximal response were calculated for a range of values for the overall activation rate constant (k_act) (Fig. 1). When k_hydrol is small relative to k_act, then agonist response approaches 100% and when k_hydrol is larger relative to k_act, then agonist effect approaches zero. Thus, the model predicts that RGS proteins will reduce agonist response most effectively when k_hydrol is similar to k_act. Partial agonists have previously been shown to have slower activation rates than full agonists due to the activation of fewer receptors (Traynor et al., 2002). Therefore, the model predicts that RGS proteins will have less effect on the maximal effect of full agonists than partial agonists.

The prediction of the collision coupling model for potency is

(3)

(see Appendix for details). The effect of RGS proteins on agonist potency is predicted using the estimated values for rates of inactivation (k_hydrol) of 0.1 s^-1 in the absence of RGS proteins and 5 s^-1 in the presence of RGS proteins. Values for potency (EC₅₀) were calculated for a K_d of 250 nM with a range of values for the overall activation rate constant (k_act) (Fig. 2). This model predicts that RGS proteins will reduce potency (increase EC₅₀) across a wide range of k_act values, but when k_act is small relative to k_hydrol, which would occur for example with a very-low-efficacy agonist, then EC₅₀ approaches the K_d and RGS proteins will have diminished effect on agonist potency. Therefore, the potency of full agonists could be expected to be decreased by RGS proteins similarly to or more than partial agonists, depending on the rate of activation.

To test this model, we have studied RGS modulation of µ-opioid inhibition of adenylyl cyclase. Because we do not know which RGS proteins are involved in opioid inhibition of adenylyl cyclase and there are no inhibitors of RGS proteins available, we have made use of RGS-insensitive G_i/o proteins to prevent all RGS protein activity. To avoid a potential confound due to effects of receptor and G protein levels, we have compared sets of clones expressing similar levels of receptor and similar levels of PTX-insensitive G with or without RGS sensitivity.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Expression levels of PTX-insensitive G in C6µ cells. Membranes were prepared from untransfected C6µ cells or C6µ cells stably expressing PTX-insensitive Gi2CI or GoCI with or without the RGS-insensitive mutation (GS) and subjected to Western blot analysis as described under Materials and Methods. Shown is the mean intensity of each band(s) relative to the intensity of the band(s) for the untransfected cells performed on the same blot (n = 3). Representative blots for Gi2 and Go are shown. Note that C6 cells endogenously express mainly Gi2 (Charpentier et al., 1993).

View this table: [in this window] [in a new window]   TABLE 1 Receptor number and [35S]GTPS binding in cells expressing RGS-sensitive and -insensitive G proteins RGS-sensitive (CI) and RGS-insensitive (CIGS) clones were matched for G expression levels and receptor number. [3H]diprenorphine and [35S]GTPS binding in C6µ cells stably expressing PTX-insensitive G with or without the RGS-insensitive mutation were measured as described under Materials and Methods and given as the mean ± S.E.M. from at least three separate experiments.

View larger version (10K): [in this window] [in a new window]   Fig. 4. DAMGO inhibition of forskolin-stimulated adenylyl cyclase. C6µ cells stably expressing PTX-insensitive G were plated onto 24-well plates and treated with 100 ng/ml PTX for 2 days before assay. The media was replaced with serum-free DMEM containing 5 µM forskolin, 1 mM IBMX, and 0 to 10 µM DAMGO to start the assay, which was allowed to proceed for 10 min at 37°C before termination by replacing the assay media with 3% perchloric acid. Accumulation of cAMP was measured as described under Materials and Methods in C6µ cells expressing PTX-insensitive (GCI, closed symbols) or PTX- and RGS-insensitive (GCIGS, open symbols). a, Gi2 (clone A) at higher levels; b, Gi2 (clone B) at lower levels; c, Go; d, Gi3. The accumulated cAMP level in the presence of DAMGO was normalized as a fraction of the cAMP level in the absence of DAMGO for that assay (mean ± S.E.M; n = 3 in duplicate). Arrows and accompanying values indicate the degree of shift in the DAMGO concentration effect curves measured as the EC50 values. Forskolin-stimulated cAMP for the RGS-sensitive and -insensitive clones, respectively, were (picomoles of cAMP per microgram of protein): 2.2 ± 0.3 and 2.6 ± 0.4 for Gi2CI/CIGS/A; 1.0 ± 0.1 and 3.8 ± 0.1 for Gi2CI/CIGS/B; 1.7 ± 0.2 and 3.2 ± 0.5 for GoCI/CIGS; or 1.0 ± 0.2 and 1.1 ± 0.1 for Gi3CI/CIGS). Alternative Go clones matched for their forskolin stimulated level of cAMP (GoCI 0.9 ± 0.08 pmol cAMP/µg of protein; GoCIGS 1.2 ± 0.1 pmol cAMP/µg of protein) gave similar maximal inhibition by DAMGO (GoCI, 40.2%; GoCIGS, 37 ± 4%), but DAMGO was 8.4 times more potent in the GoCIGS-expressing clones (EC50 values: GoCI, 9.2 ± 4.0 nM; GoCIGS, 1.1 ± 0.5 nM). Note that the clones matched for cAMP were not matched for receptor number and G protein expression.

µ-Opioid Agonist Inhibition of Forskolin-Stimulated cAMP Accumulation. Maximal inhibition of 5 µM forskolin-stimulated cAMP accumulation by the full agonist, DAMGO, was significantly higher with the RGS-insensitive mutation in the cells expressing either matched pair of G_i2^CI and G_i2^CIGS (Fig. 4, Table 2). The potency of DAMGO to inhibit forskolin-stimulated cAMP accumulation was also increased (Fig. 4). The potency and efficacy of DAMGO to stimulate [³⁵S]GTPS binding in the matched G_i2^CI and G_i2^CIGS clones were the same (Table 1), which confirms an effect of endogenous RGS proteins in decreasing the concentration of the active G-GTP. In contrast, in cells expressing G_o^CI or G_o^CIGS, the maximal inhibition of forskolin-stimulated cAMP accumulation by DAMGO was not affected by the RGS-insensitive mutation (Fig. 4c). The potency of DAMGO, however, was increased by the RGS-insensitive mutation in G_o^CI-expressing cells (Fig. 4c). Likewise, the RGS-insensitive mutation increased DAMGO potency in cells expressing G_i3^CI without affecting maximal inhibition of forskolin-stimulated cAMP accumulation (Fig. 4d). The increase in DAMGO potency with or without an increase in maximal response with the RGS-insensitive mutation agrees with the predictions based on the collision coupling model under conditions of a fast k_act. Although the cells were matched within G subtypes, there was a difference in the level of forskolin-stimulated cAMP between the matched RGS-sensitive and -insensitive clones, which was especially noticeable for clones expressing lower levels of the mutant G_i2 proteins (Fig. 4b) and clones expressing the mutant G_o (Fig. 4c). This may relate to an increased adenylyl cyclase sensitization in the presence of the more efficient G signaling (Clark and Traynor, 2006). Consequently, we repeated the experiment in cells expressing mutant G_i2 cells in the presence of fetal bovine serum in the assay medium to prevent expression of sensitization (Clark and Traynor, 2006). Under these conditions, the level of forskolin-stimulated cAMP accumulation in the G_i2^CI/B and G_i2^CIGS/B clones was similar (1.38 ± 0.11 and 0.98 ± 0.15 pmol/mg of protein, respectively), but the maximal degree of inhibition by DAMGO (34 ± 8 and 85 ± 2%, respectively) and potencies (14 ± 9 and 1.4 ± 0.3 nM, respectively) were unchanged compared with conditions in which the level of forskolin-stimulated cAMP was widely different (Table 2). In confirmation of this, G_o^CI and G_o^CIGS clones that gave a similar level of forskolin-stimulated cAMP gave similar DAMGO-mediated degree of inhibition but retained the shift in potency (see legend to Fig. 4c). In addition, the maximal DAMGO inhibition was not dependent on the forskolin concentration, for example, with the G_i2^CI/B clone, maximal DAMGO inhibition was similar (31 ± 9%) using 30 µM forskolin as with 5 µM forskolin (37%; Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Inhibition of forskolin-stimulated cAMP accumulation in cells expressing RGS-sensitive or RGS-insensitive G proteins µ-Opioid agonist inhibition of forskolin-stimulated cAMP accumulation was measured for 10 or 30 min in C6µ cells stably expressing PTX-insensitive G with (CIGS) or without (CI) the RGS-insensitive mutation as described under Materials and Methods and given as mean ± S.E.M. from at least three separate experiments.

To further examine the model experimentally, relative agonist response was determined for partial agonists providing lower k_act values as a percentage of the maximal DAMGO inhibition in the same assay. The relative efficacy of morphine, which behaved as a full agonist in these experiments, was not significantly affected by the RGS-insensitive mutation in cells expressing G_o^CI, G_i2^CI, or G_i3^CI (Fig. 5). On the other hand, the relative response of the partial agonists, buprenorphine and nalbuphine, was increased by 1.3- and 1.9-fold, respectively, in the RGS-insensitive mutation in the G_i2^CI-expressing cells, 1.3- and 2.6-fold in the RGS-insensitive G_o^CI-expressing cells, and 2.3- and 6.7-fold in the G_i3^CI-expressing cells (Fig. 5). The potencies of morphine and buprenorphine were also increased by the RGS-insensitive mutation in each of the G subtypes (Table 2). These increased potencies and maximal responses with the RGS-insensitive mutation agree with the predictions of the collision coupling model under conditions of slower k_act.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Relative µ-opioid agonist inhibition of forskolin-stimulated adenylyl cyclase. C6µ cells stably expressing PTX-insensitive G were plated onto 24-well plates and treated with 100 ng/ml PTX for 2 days before assay. cAMP accumulation was determined as described in legend to Fig. 4 in the presence of 0 to 10 µM DAMGO, morphine, buprenorphine, or nalbuphine. Maximal percentage of inhibition of forskolin-stimulated cAMP accumulation by each agonist was determined from concentration-response curves and normalized as a percentage of the maximal level of DAMGO inhibition in cells expressing PTX-insensitive GCI (filled bars) or PTX- and RGS-insensitive GCIGS (open bars). a, Gi2 (clone A) at higher levels; b, Gi2 (clone B) at lower levels; c, Go; d, Gi3. Shown are the combined data from three assays carried out in duplicate. *, p < 0.05; ***, p < 0.001 compared with RGS-sensitive clone.

Calculations of Overall Activation Rate Constants. The RGS-insensitive G subunits do not bind RGS proteins, so the rate constant of inactivation (k_hydrol) for these G subunits will be the same as if there were no RGS proteins present. Using this assumption, we used the collision coupling model to calculate the overall activation rate constants (k_act) for the RGS-insensitive clones using the experimental EC₅₀ values for DAMGO inhibition of adenylyl cyclase (Table 2), a rate constant of inactivation of 0.1 s^-1 (k_hydrol), and K_i values measured in the presence of NaCl and GDP (from Emmerson et al., 1996) for the K_d values. These values are presented in Table 3. As predicted by the model, the calculated k_act for DAMGO was slower with decreased receptor number in the clones expressing the same G subtype. When matched for receptor number, the k_act for DAMGO was almost 2-fold faster with G_o^CIGS than with G_i2^CIGS. In agreement with the collision coupling concept of lower agonist intrinsic efficacy reducing the number of active receptors, and therefore productive "collisions," the overall activation rate constants (k_act) for the partial agonist, morphine, were at least 7 times slower than for DAMGO at each of the RGS-insensitive clones. However, k_act values could not be calculated for nalbuphine or buprenorphine because the EC₅₀ values were higher than the K_i value measured in the presence of NaCl and GDP (Emmerson et al., 1996).

View this table: [in this window] [in a new window]   TABLE 3 Rates of activation calculated for cells expressing RGS-insensitive G proteins Kact was calculated from the EC50 for inhibition of forskolin-stimulated cAMP accumulation (equation 11). Ki values measured in the presence of NaCl and GDP were used for Kd (DAMGO Ki = 279 nM; morphine Ki = 132 nM; from Emmerson et al., 1996) and khydrol = 0.1 s–1 for absence of RGS proteins (from Shea et al., 2000).

[³⁵S]GTPS Binding and cAMP Accumulation Using FLAG-Tagged G_o^CI, G_i2^CI, and G_i3^CI. To determine whether the faster rates of activation calculated for the G_o^CIGS cells were due to higher levels of expression, G subunits were FLAG-tagged at the N terminus so that expression levels of different G subtypes could be directly compared by Western blot using the same anti-FLAG antibody. FLAG-G_i2^CI, FLAG-G_o^CI, or FLAG-G_i3^CI were stably expressed in HEK293 cells stably expressing the µ-opioid receptor, and clones expressing similar levels of receptor and FLAG-G were selected (Fig. 6, a and b). None of the FLAG-G_i3^CI clones expressed higher levels. Maximal stimulation of [³⁵S]GTPS binding by DAMGO after overnight treatment with PTX was 3-fold higher in the FLAG-G_o^CI-expressing clone than in the FLAG-G_i2^CI or FLAG-G_i3^CI clones (Fig. 6c). This suggests that the G_o^CIGS cells used to calculate the rates of activation (Table 3) actually had lower levels of PTX-insensitive G than the G_i2^CIGS clones because they had similar levels of stimulation of [³⁵S]GTPS binding by DAMGO.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Comparison of FLAG-Gi2CI-, FLAG-GoCI-, or FLAG-Gi3CI-expressing HEK293µ cells. PTX-insensitive GCI was FLAG-tagged and stably expressed in HEK293µ cells. Cells matched for receptor number (femtomoles per milligram of protein: FLAG-Gi2CI, 860 ± 80; FLAG-GoCI, 850 ± 50; FLAG-Gi3CI, 950 ± 100) were treated with 100 ng/ml PTX overnight. a and b, Western blot analysis with anti-M2 FLAG antibody and anti-tubulin as described under Materials and Methods. a, representative blot; b, combined data from at least three blots normalized to tubulin loading control. c, maximal stimulation of 0.1 nM [35S]GTPS binding by DAMGO in presence of 30 µM GDP for 60 min at 25°C as described under Materials and Methods. The EC50 of DAMGO to stimulate [35S]GTPS binding was lower at FLAG-GoCI (68 ± 5 nM) and FLAG-Gi3CI (72 ± 24 nM) than at FLAG-Gi2CI (169 ± 14 nM). Basal levels of [35S]GTPS binding were similar for FLAG-GoCI (13 ± 2 fmol/mg), FLAG-Gi2CI (11 ± 2 fmol/mg), or FLAG-Gi3CI (9 ± 2 fmol/mg). d, maximal inhibition of forskolin-stimulated cAMP accumulation by 10 µM DAMGO measured as described in legend to Fig. 4. The EC50 of DAMGO to inhibit cAMP accumulation was 42 ± 22 nM at FLAG-GoCI, 23 ± 5 nM at FLAG-Gi2CI, and 95 ± 35 nM at FLAG-Gi3CI. The levels of forskolin-stimulated cAMP accumulation in the absence of µ-opioid agonist (picomoles per milligram of protein) were similar for FLAG-GoCI (1.0 ± 0.1), FLAG-Gi2CI (2.2 ± 0.7), or FLAG-Gi3CI (1.4 ± 0.3). **, p < 0.01 compared with FLAG-GoCI. A second set of clones expressing similar levels of FLAG-tagged GoCI and Gi2CI showed similar results with DAMGO stimulation of [35S]GTPS (EC50 and maximal stimulation: 69 ± 9 nM and 215 ± 27% for GoCI; 157 ± 21 nM and 104 ± 10% for Gi2CI) and inhibition of cAMP accumulation (EC50 and maximal inhibition: 29 ± 11 nM and 35 ± 3% for GoCI; 68 ± 31 nM and 42 ± 5% for Gi2CI).

Effect of Different G Protein Levels. The simple collision coupling model used here assumes that the G proteins are closely associated with adenylyl cyclase and that agonist potency is independent of G protein and adenylyl cyclase concentrations as shown in turkey erythrocyte membranes (Tolkovsky et al., 1982). In this study, we used clones matched for receptor and G protein levels to avoid the possible confound of transient G protein and adenylyl cyclase interactions. However, to determine whether G and adenylyl cyclase are indeed precoupled, agonist potency at inhibition of forskolin-stimulated cAMP accumulation was compared in clones expressing different levels of FLAG-G_o^CI but similar levels of µ-opioid receptor (Table 4). DAMGO potency to inhibit cAMP accumulation was decreased at lower levels of FLAG-G_o^CI expression, whereas potency to stimulate [³⁵S]GTPS binding was not affected, suggesting that G and adenylyl cyclase are not precoupled in these cells.

View this table: [in this window] [in a new window]   TABLE 4 Effect of FLAG-GoCI expression level on DAMGO stimulation of [35S]GTPS binding and inhibition of forskolin-stimulated cAMP accumulation Expression levels of FLAG-GoCI stably expressed in HEK293µ cells were determined by Western blot analysis as described under Materials and Methods. DAMGO stimulation of [35S]GTPS binding and inhibition of forskolin-stimulated cAMP accumulation were measured as described under Materials and Methods.

Comparison of Calculated and Experimentally Determined EC₅₀ Values. Using the calculated k_act values for the RGS-insensitive clones and the k_hydrol of 5 s^-1 for the presence of RGS proteins, the expected decrease in potency for the RGS sensitive clones is predicted to range from 29- to 38-fold for DAMGO. This is higher than the experimental range of a 4- to 15-fold decrease in DAMGO potency across all of the RGS-sensitive clones compared with their matched RGS-insensitive clone (Table 2). The predicted potency shift for morphine based on the k_act values is 5- to 16-fold, which is closer to the measured range of a 2- to 6-fold decrease in morphine potency. In contrast, using previously published data comparing G_o^CGGS and G_o^CG (Clark et al., 2003) with the less efficiently coupled CG PTX-insensitive mutation (Bahia et al., 1998; Clark et al., 2006), the measured 8- to 35-fold shift in DAMGO potency was similar to the 16-fold shift predicted from the calculated k_act values of 2.1 and 2.3 s^-1. The measured morphine potency shift of 8-fold was also similar to the 3- to 6-fold shift predicted from the calculated k_act values of 0.51 and 0.20 s^-1. This would suggest that rate constant of hydrolysis in the presence of RGS proteins of 5 s^-1, which was used for the predictions in this study, seems appropriate in these cells.

	Discussion

Top Abstract Materials and Methods Results Discussion Appendix References

 
This study, using RGS-insensitive mutants of G proteins, demonstrates that endogenous RGS proteins reduce agonist potency for the inhibition of adenylyl cyclase similarly for full and partial agonists. The maximal agonist response, however, was reduced more by RGS proteins under conditions of slower rates of G protein activation, such as with partial agonists and was not affected by RGS proteins under conditions of faster rates of G protein activation, as with full agonists. These results agree with the predictions of a collision coupling model.

Agonist potency was increased similarly by the RGS-insensitive mutation for G_i2- and G_o-expressing cells. Previous studies have shown that the rate of GTP hydrolysis is faster with G_o than with G_i2 with RGS4 (Posner et al., 1999; Cavalli et al., 2000; Hooks et al., 2003), or with RGS6, -7, -9, or -11 (Hooks et al., 2003). There are several explanations for our finding. There could be RGS proteins present that do not act very efficiently as GAPS at G_o, or colocalization of the receptors and G proteins with the RGS proteins may be different. Although RGS proteins that might be considered as prime candidates to interact with µ-opioid signaling in vivo [i.e., RGS4 and RGS9 (Traynor and Neubig, 2005)] are not expressed in C6 cells (Snow et al., 2002), a number of RGS proteins have been identified (RGS 2, 8, 10, 12, and 14; Snow et al., 2002), and several of these are known to effectively act as GAPS for G_i/o proteins coupled to µ-opioid receptors (reviewed in Xie and Palmer, 2005). Finally, the PTX-insensitive G^CI mutants have faster rates of activation than wild-type G (Bahia et al., 1998), which may hide differences in the sensitivity of G proteins to endogenous RGS proteins. Indeed, with the less efficiently coupled G^CG PTX-insensitive mutation, morphine is a partial agonist at inhibition of adenylyl cyclase (Clark et al., 2003), whereas in this study with the G^CI PTX-insensitive mutation, morphine is a full agonist.

The increase in potency with the RGS-insensitive PTX-insensitive G_o^CG was in accordance with the calculations based on the collision coupling model, whereas the potencies in the PTX-insensitive G^CI-expressing cells used in this study were not increased to the extent predicted. This may be due to a physical diffusion-controlled limitation on EC₅₀ and k_act. This would limit the differences with and without RGS proteins in a highly efficient G^CI system with an already low EC₅₀ to less than the theoretical predictions (see Fig. 2, inset). Alternatively, if RGS proteins do not modulate some of the G^CI pool, the effective k_hydrol in the RGS sensitive cells would actually reflect a combination of k_hydrol with and without RGS proteins, so the shift in potency with the G^CIGS mutant would be less than predicted.

The maximal effect of the full agonist DAMGO was increased in cells expressing RGS-insensitive G_i2 but not G_o or G_i3 proteins. This suggests that the k_act for G_i2 is lower so that G_i2 is more susceptible to the effects of RGS proteins according to the collision coupling model. Using FLAG-tagged G subunits with similar expression levels, we measured a higher efficiency of coupling for G_o in the [³⁵S]GTPS assay, confirming that the µ-opioid receptor couples more efficiently to G_o and G_i3 than G_i2 (Clark et al., 2006). The rate limiting step in the [³⁵S]GTPS binding assay is the exchange of GDP for [³⁵S]GTPS, so this higher equilibrium level of DAMGO-stimulated [³⁵S]GTPS binding at G_o could be explained by a faster rate of GDP/[³⁵S]GTPS exchange compared with the slow off rate of [³⁵S]GTPS. Indeed, the rate constant of activation calculated for G_o was faster. Other studies have shown that G_o has a faster nucleotide exchange rate than G_i1 (Remmers et al., 1999; Kimple et al., 2001) or G_i2 (Parker et al., 1991; Zhang et al., 2002). This is also reflected in the higher basal steady state GTPase, which has been observed for G_o compared with G_i1, G_i2, or G_i3 (Hooks et al., 2003). The endogenous basal rate of GTP hydrolysis, as measured by single turnover assay, has been shown to be similar for G_i2 and G_o (Posner et al., 1999), supporting the suggestion that the faster steady-state GTPase rates for G_o are due to faster nucleotide exchange rates.

Although we see a more efficient coupling of the µ-opioid receptor with FLAG G_o than FLAG G_i2 or G_i3, the degree of inhibition of forskolin-stimulated cAMP accumulation by DAMGO after overnight treatment with PTX was similar across the clones. The difference in the kinetics of the G subtypes may not be sufficient to significantly affect the maximal response and potency of the full agonist. Alternatively, G_o could have a lower efficacy than G_i2 for inhibiting the adenylyl cyclases present in these cells, despite a faster rate of G protein activation. HEK293 cells are reported to contain ACI, ACIII, and ACVI; G_o has been shown to inhibit ACI but not ACVI, whereas G_i is able to inhibit ACI and ACVI (Taussig et al., 1994). There could also be RGS proteins in HEK293 cells that do have a significantly differential effect on G_o and G_i2 (such as RGS6). RGS proteins, receptors, and different adenylyl cyclase subtypes may also colocalize or interact differently with the different G subtypes, allowing for differential effects within the same cell.

The simple collision coupling model we have used was originally formulated for G_s-mediated stimulation of adenylyl cyclase (Stickle and Barber, 1992; Whaley et al., 1994). The model assumes that the G proteins are closely associated with adenylyl cyclase and that agonist potency is independent of G protein and adenylyl cyclase concentrations (Tolkovsky et al., 1982). On the other hand, there is the possibility that the interaction between the G protein and adenylyl cyclase is transient, so that the kinetics of that encounter need to be considered. A "shuttle mechanism" has been incorporated into the collision coupling model by adding a rate constant for the activation of adenylyl cyclase by G protein and parameters for the number of G proteins and adenylyl cyclase molecules (Krumins et al., 1997). According to this shuttle mechanism, agonist potency at adenylyl cyclase will be increased by increasing the number of G proteins if the rate of activation of adenylyl cyclase by G protein is fast compared with the rate of inactivation (Krumins et al., 1997). In this study, we used clones matched for receptor and G protein levels to avoid the possible confound of transient G protein and adenylyl cyclase interactions, although data with different FLAG-G expression levels does suggest that the system is not precoupled. Similar to the present findings, GIRK channel activation by m2 receptor activation of G_i2 has also been shown to be consistent with the collision coupling model, whereas m2 activation of GIRK mediated by G_o was not (Zhang et al., 2002). The authors propose that expression conditions were favorable to formation of a precoupled receptor-G_o-GIRK channel complex. The possibility of precoupled or colocalized receptor and G protein instead of collision coupling was also indicated by the lack of relationship between agonist potency for inhibition of adenylyl cyclase and the number of 5-HT_1A receptors in NIH-3T₃ cells (Varrault et al., 1992). In addition, there is evidence for a continuous interaction between β-adrenergic receptor and G protein for the stimulation of adenylyl cyclase in turkey erythrocyte membranes (Ugur and Onaran, 1997). This led to the suggestion that an equilibrium model of G protein and receptor states, such as the extended ternary complex model (Samama et al., 1993), needs to be considered along with the G protein kinetics of the collision coupling model. This has been done by combining the kinetic parameters of G protein activation and inactivation to the cubic ternary complex model to formulate the cubic ternary complex activation model (Shea et al., 2000). Unlike the collision coupling model, this more comprehensive model will provide predictions for agonist maximal response and potency in the absence or presence of RGS proteins, whether or not the receptor and G protein are precoupled.

View larger version (12K): [in this window] [in a new window]   Fig. 7. Collision coupling model.

	Appendix

Top Abstract Materials and Methods Results Discussion Appendix References

 
Here we derive the equations for maximum agonist response and potency for a simple collision coupling model at steady state, as diagrammed in Figure 7. Receptors and ligands are assumed to bind with equilibrium dissociation constant K_d. Receptor/ligand complexes exist in active (LR_a) or inactive (LR_i) forms, referring to their ability to activate G proteins, and these complexes are assumed to distribute according to an equilibrium constant K_eq (Kinzer-Ursem et al., 2006). It is assumed that there is no precoupling of (unbound) receptors and G proteins and no constitutive activity. The total numbers of receptors (R_TOT) and G proteins (G_TOT) on the cell surface that can participate in signaling are assumed constant (i.e., no internalization, up-regulation, or desensitization). G proteins are activated after diffusion and collision with a ligand-bound receptor with rate constant k_a. Thus, the value of k_a depends on the diffusivity of receptors and G proteins in the membrane and also on the number and distribution of G proteins available for activation (which determines the necessary distance that must be traversed via diffusion) (Mahama and Linderman, 1994; Shea et al., 1997). G proteins are inactivated after GTP hydrolysis with rate constant k_hydrol, the value of which depends on the presence or absence of RGS. At steady state, the rates of G protein activation and deactivation are equal. Thus, the starting equations for this model are simply

(4)

(5)

(6)

(7)

After algebraic manipulation, one can find that the amount of G protein activation is

(8)

where the fraction of bound receptors in the active form, a measure of the agonist intrinsic efficacy, is

(9)

Thus, the fractional activation of G proteins at high ligand concentrations (maximal response), assumed equal in this model to the fractional activation of adenylyl cyclase, is

(10)

Agonist potency predicted by this model is given by

(11)

When all bound receptors are active (K_eq >>1 or f_active = 1), eqs. 10 and 11 are identical to the results of Whaley et al. (1994).

	Acknowledgements

 
We thank Christopher Brinkerhoff for helpful discussions and Richard Neubig for critical reading of the manuscript.

	Footnotes

 
This work was supported by National Institutes of Health grant DA04087

ABBREVIATIONS: RGS, regulator of G protein signaling; GAP, GTPase accelerating protein; PTX, pertussis toxin; [³⁵S]GTPS, 5'-O-(3-[³⁵S]thiotriphosphate); IBMX, 3-isobutyl-1-methylxanthine; C6µ, C6 cells stably expressing the µ-opioid receptor; HEK, human embryonic kidney; HEK293µ, HEK293 cells stably expressing the µ-opioid receptor; DAMGO, [D-Ala²,N-Me-Phe⁴, Gly⁵-ol]enkephalin; GIRK channels, G proteingated inwardly rectifying K⁺ channels; CI, RGS-sensitive clone; CIGS, RGS-insensitive clone.

Address correspondence to: John R. Traynor, 1301 MSRB III/Pharmacology Dept., 1150 W. Medical Center Dr., Ann Arbor, MI 48109-5632. E-mail: jtraynor{at}umich.edu

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