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CELLULAR AND MOLECULAR
i/o Proteins Containing a Pertussis Toxin-Insensitive Mutation
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan
Received October 7, 2005; accepted January 23, 2006.
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Abstract |
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i/o proteins expressed in C6µ cells were used to examine the hypothesis that there are agonist-specific conformational states of the µ-opioid receptor with coupling preferences to different Gi/o subtypes, as measured by the degree of stimulation of [35S]guanosine 5'-O-(3-thio)triphosphate (GTP
S) binding. Binding of [35S]GTPS to endogenous Gi/o proteins stimulated by the full µ-opioid agonist [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) was completely blocked by overnight treatment with 100 ng/ml PTX. Treatment for 4 h with lower concentrations led to a PTX-dependent reduction in the maximal effect of DAMGO but no alteration in the potency of DAMGO or morphine nor in the relative maximal effect (relative efficacy) of the partial agonists morphine and buprenorphine compared with the full agonist DAMGO. Using PTX-insensitive G mutants in which the PTX-sensitive cysteine was replaced with isoleucine, the potency for a series of µ-opioid agonists was highest in cells expressing Gi3 and Go and lowest with Gi1 and Gi2, with no significant change in the order of potency, namely, etorphine >> endomorphin-1 = DAMGO = endomorphin-2 = fentanyl = morphine >> meperidine. The order of agonist relative efficacy, etorphine = DAMGO = endomorphin-1 = endomorphin-2 = fentanyl 
morphine meperidine > buprenorphine nalbuphine, was also the same across all of the PTX-insensitive Gi/o subtypes. Highest relative efficacy to stimulate [35S]GTPS binding was seen with Gi3. Consequently, reported observations of agonist-directed trafficking at µ-opioid receptors most likely involve non-PTX-sensitive G protein mechanisms.
i/o family when occupied by agonist (Laugwitz et al., 1993
; Chakrabarti et al., 1995). These early studies used the highly efficacious peptidergic agonist DAMGO. However, there is growing evidence at a variety of GPCRs for the formation or stabilization of agonist-specific conformations that have the potential to activate different downstream signaling pathways (Kenakin, 1995, 2003). For example, at the µ-opioid receptor, there is evidence for differential binding of peptide and nonpeptide agonists (Fukuda et al., 1995; Wang et al., 1995; Xue et al., 1995; Chaipatikul et al., 2003), and with the use of human µ-opioid receptor-G fusion protein constructs (hMOR-G) expressed in human embryonic kidney cells and Escherichia coli, differences have been observed between µ-opioid agonists in the ability to stimulate [35S]GTPS binding (Stanasila et al., 2000, Massotte et al., 2002). In addition, the ability of µ-opioid agonists to cause receptor internalization does not correlate with their ability to activate other G protein-mediated events (Whistler and von Zastrow, 1998; Borgland et al., 2003).
Agonist-dependent activation of G subunits could have downstream consequences, resulting in agonist-specific trafficking of intracellular signals (Kenakin, 1995, 2003). This could occur because Gi2 is thought to couple more efficiently to adenylyl cyclase than Go (McKenzie and Milligan, 1990; Moon et al., 2001), whereas Go may couple more effectively to inhibition of Ca2+ currents (Hescheler et al., 1987). Such differences may have behavioral outcomes. Thus, antisense knockdown of Gi2 but not of the other Gi/o proteins significantly attenuates µ-opioid-induced supraspinal antinociception in the mouse (Raffa et al., 1994), and Garzón and colleagues have demonstrated that knockdown of several G proteins differentially affects supraspinal antinociception induced by a variety of µ-agonists in the mouse (Sánchez-Blázquez et al., 2001).
The studies discussed above did not examine Gi3, which is reported to couple most efficiently to the DAMGO-activated µ-opioid receptor (Laugwitz et al., 1993; Chakrabarti et al., 1995), and generally, only a limited number of agonists have been studied that do not cover a wide range of agonist potency or efficacy. Here, we test the hypothesis that different agonists induce activation states of the µ-opioid receptor that differ in their ability to activate Gi/o protein subtypes and that this can result in changes in the rank order of potency or efficacy as measured at the level of G protein activation. To study individual G subtypes, PTX-insensitive mutants of the G subtypes were expressed in C6 glioma cells stably expressing the µ-opioid receptor (C6µ), and agonist stimulation of [35S]GTPS binding was measured in membranes prepared from PTX-treated cells to uncouple endogenous G proteins, which mainly comprise Gi2 (Charpentier et al., 1993). PTX-insensitive subunits have been used successfully to study receptor activation of different Gi/o proteins and have been shown to be able to couple to downstream effectors (Hunt et al., 1994, Senogles, 1994; Wise et al., 1997; Yamaguchi et al., 1997). The potency and efficacy of several µ-opioid agonists of differing structure, including an alkaloid (morphine), small synthetic or semisynthetic compounds (etorphine, fentanyl, meperidine, and nalbuphine), and peptides (DAMGO, endomorphin-1, and endomorphin-2) to activate the different Gi/o subtypes were determined. The results show that µ-opioid agonists were most potent at activating Gi3 and Go compared with Gi1 and Gi2 and had the highest efficacy, relative to the full agonist DAMGO, at Gi3, with similar relative efficacy across the other G subtypes. However, there was no change in the rank order of potency or relative efficacy for the series of ligands across the different Gi/o subunits, indicating a lack of agonist-specific activation of Gi/o proteins.
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Materials and Methods |
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S was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Morphine, etorphine, fentanyl, meperidine, buprenorphine, and nalbuphine were obtained through the Narcotic Drug and Opioid Peptide Basic Research Center at the University of Michigan (Ann Arbor, MI). DAMGO, endomorphin-1, endomorphin-2, GDP, Trizma base, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Human PTX-insensitive (PTXi) G protein cDNAs were purchased from UMA cDNA Resource Center (www.cDNA.org). The GF/B 4 x 6 glass fiber filtermats and filtermat bags were purchased from PerkinElmer Life and Analytical Sciences. EcoLume scintillation cocktail was obtained from ICN (Aurora, OH).
Cell Culture and Membrane Preparation. cDNA for human C351I GaoA, Gi1, Gi3, or C352I Gi2 was inserted into pcDNA3.1zeo- vectors (Invitrogen). The identity of each of the Gi/o cDNAs was confirmed by sequencing at the University of Michigan sequencing core. Transient transfection into C6 glioma cells stably expressing the rat µ-opioid receptor (C6µ) was performed using Lipofectamine Plus and 4 µg of appropriate cDNA (Clark et al., 2003). In one set of experiments, different quantities of human C351I Gi3 cDNA (4, 6, or 8 µg) and vector to a total of 8 µg of total DNA were transfected. Cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum under 5% CO2. Transiently transfected cells were collected for membrane preparation 48 h after transfection. Cells were treated for 4 h or overnight with PTX before membranes were prepared as described previously (Clark et al., 2003).
[35S]GTPS Binding. Cell membranes (10-20 µg) were incubated for 60 min in a shaking water bath at 25°C with 30 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 100 mM NaCl, 0.1 mM dithiothreitol (freshly prepared), 30 µM GDP, 0.1 nM [35S]GTPS, and varying concentrations of ligand or double-distilled H2O as described by Traynor and Nahorski (1995). Samples were filtered through a glass-fiber filtermat mounted in a Brandel cell harvester and rinsed three times with ice-cold 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, and 100 mM NaCl. Filtermats were dried, and 0.3 ml of EcoLume scintillation cocktail was added to each sample area to soak the filter (total 7.2 ml). Each filtermat was placed in a polyethylene bag, which was heat-sealed, and radioactivity retained in each sample area was counted in a Wallac 1450 MicroBeta liquid scintillation and luminescence counter.
Data Analysis. Concentration-response data were fitted to sigmoidal curves using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA) to determine EC50 and maximal stimulation. Data from at least three separate experiments, each carried out in duplicate, are presented as mean ± S.E.M. Comparisons between drugs or across G subunits were made by one-way analysis of variance followed by Tukey's multiple comparison test.
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Results |
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S binding in these cells was 33 ± 5 fmol/mg protein, and the full µ-opioid agonist DAMGO stimulated [35S]GTPS binding to 175 ± 11 fmol/mg protein. In C6µ cells expressing wild-type G proteins, the rank order of efficacy, as measured by the maximal ability to stimulate [35S]GTPS binding, was DAMGO = etorphine = endomorphin-1 = endomorphin-2 = fentanyl morphine meperidine > buprenorphine nalbuphine (Table 1). Potency for the series of compounds followed the order etorphine >> endomorphin-1 = DAMGO = endomorphin-2 = fentanyl = morphine >> meperidine (Table 1). Potency data for buprenorphine and nalbuphine could not be reliably obtained due to the small degree of stimulation. DAMGO stimulation of [35S]GTPS binding in C6µ cells expressing wild-type G proteins (485 ± 76%) was completely abolished (0.8 ± 2.7%) by overnight (18 h) treatment with 100 ng/ml PTX, confirming that the µ-opioid receptor was acting only through PTX-sensitive Gi/o proteins.
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Effect of G Protein Concentration on µ-Opioid Agonist Relative Efficacy and Potency. In studies with different PTX-insensitive G subtypes, it is not possible to directly compare the absolute level of G expressed. Consequently, comparisons of agonist potency and efficacy in cells expressing different G subtypes can only be made if these parameters are independent of the level of G expression. To test this concept, the number of functional endogenous G proteins was reduced in wild-type C6µ cells by treatment with varying concentrations of PTX for 4 h before preparation of membranes. The maximal degree of stimulation of [35S]GTPS binding by the highly efficacious µ-opioid agonist DAMGO was reduced by PTX treatment (Fig. 1A) in direct proportion to the amount of PTX used, resulting in a 3-fold loss of maximal stimulation after exposure to 100 ng/ml PTX for 4 h. The maximal degree of [35S]GTPS binding stimulated by the µ-opioid partial agonists morphine and buprenorphine was similarly decreased by PTX treatment so that the maximal stimulation by the two compounds remained constant relative to DAMGO (Fig. 1B). The potencies of DAMGO and morphine, as measured by their EC50 values to stimulate [35S]GTPS binding, were not significantly altered by reducing the number of functional G proteins with PTX (Fig. 1C). Therefore, µ-opioid agonist relative efficacy, as measured by maximal [35S]GTPS stimulation relative to DAMGO, and absolute as well as relative potencies can be compared in cells expressing different expression levels of G subunits. In all subsequent studies that use cells expressing PTX-insensitive G, cells were treated with 100 ng/ml PTX for 18 h to prevent activation of endogenous G proteins.
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proteins in C6µ cells would interfere with the exogenously expressed PTX-insensitive G subunits, we compared stable clones expressing PTX-insensitive GoA (C351I) or Gi3 (C351I) with and without PTX treatment. The maximal level of DAMGO-stimulated [35S]GTPS incorporation over basal was greater in the non-PTX-treated cells (GoA = 503 ± 29%; Gi3 = 500 ± 53%) than the PTX-treated cells (GoA = 205 ± 20%; Gi3 = 63 ± 5%), suggesting an additive effect with endogenous G subunits. However, the potency obtained with non-PTX-treated cells expressing GoA (205 ± 20 nM) and Gi3 (49.4 ± 5.7 nM) was reduced compared with PTX-treated cells (GoA = 56 ± 7 nM; Gi3 = 7.5 ± 2.4 nM). Thus, if endogenous G proteins do interfere with the exogenously expressed PTX-insensitive G subunits, this interference is inhibited by the PTX treatment.
Comparison of Agonist Potency and Relative Efficacy of µ-Opioid Agonists at Different G Protein Subtypes. C6µ cells were transiently expressed with PTX-insensitive GoA, Gi1, Gi2, or Gi3 subunits with a C351(2)I mutation and treated overnight with 100 ng/ml PTX, such that there was no response to opioid agonists in nontransfected cells. The maximal stimulation of [35S]GTPS binding by DAMGO was at least 6 times greater with the C351(2)I mutation for each of the Gi/o subtypes than with the C351(2)G mutation that we have previously used (Clark et al., 2003; data not shown), in agreement with studies at the 2A-adrenoceptors (Bahia et al., 1998). The C351(2)I mutation was used in subsequent studies.
The potency of DAMGO and other µ-opioid agonists to stimulate [35S]GTPS binding to the C351(2)I G subtypes decreased in the order Gi3 GoA > Gi1 > Gi2 (Table 2). When compared as potencies relative to DAMGO, there were no differences confirming that absolute values can be used. The overall agonist potency in membranes expressing Gi3 was 4.6 ± 0.4-fold higher than in membranes from cells expressing Gi2 across all seven agonists examined (Table 2). In contrast, treatment of wild-type cells with 100 ng/ml PTX for 4 h, which reduced maximal DAMGO stimulation of [35S]GTPS binding and, presumably, functional G subunits by at least 3-fold (Remmers et al., 2000) (Fig. 1) only reduced the potency of DAMGO and morphine potency by a nonsignificant 1.4-fold (Fig. 1C).
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The degree of maximal stimulation of [35S]GTPS binding by DAMGO and the other µ-opioid agonists varied with the different G subtypes in the decreasing order GoA > Gi2 > Gi1 > Gi3 (Fig. 2), possibly as a consequence of differential expression. Consequently, the maximal stimulation for the different agonists was compared with that obtained with DAMGO. The rank order of maximal stimulation of [35S]GTPS binding across the C351(2)I mutant G subtypes was constant in the order: DAMGO = etorphine = endomorphin-1 = endomorphin-2 = fentanyl morphine meperidine > buprenorphine nalbuphine (Fig. 3). The same rank order was obtained in membranes from cells stably expressing the less efficient C351(2)G G mutants (data not shown). Although the rank order of µ-agonist-mediated [35S]GTPS-stimulated binding was unchanged across the various Gi/o subunits, the maximal [35S]GTPS stimulation relative to DAMGO was generally greatest with Gi3. However, this difference was only significant for endomorphin-1, endomorphin-2, morphine, and buprenorphine (Fig. 3).
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To test whether the low degree of [35S]GTPS incorporation into Gi3 was responsible for the higher relative agonist efficacy and increased potency of agonists at Gi3, stimulation of [35S]GTPS binding was examined in C6µ cells transfected with increasing amounts of Gi3 cDNA. The results showed that the maximal effect of DAMGO increased with increasing cDNA, but neither the potency of DAMGO nor the relative efficacy of morphine and buprenorphine was altered (Table 3).
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Discussion |
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subunits and C6 cells stably expressing a µ-opioid receptor to test the hypothesis that structurally dissimilar µ-opioid agonists stabilize distinct activation states of the receptor, which differentially activate Gi/o subtypes. Following overnight treatment with a high concentration of PTX, the ability of µ-opioid agonists to stimulate [35S]GTPS binding was completely lost but could be rescued by transfection with PTX-insensitive mutants of Gi/o subunits, although agonist potency to stimulate [35S]GTPS binding varied in the order Gi3 Go > Gi1 Gi2. Across a wide range of µ-opioid agonists, there was no change in the rank order of potency or relative maximal ability to stimulate [35S]GTPS binding to these different PTX-insensitive G subunits.
Because the PTX-insensitive [C351(2)I] mutation is in a region of G involved in receptor coupling, it was necessary to show that functional coupling to the µ-opioid receptor is retained and that there is no interference by endogenous G proteins. The ability of DAMGO to stimulate [35S]GTPS binding was much greater in cells expressing both wild-type and PTX-insensitive G protein before treatment with PTX, suggesting that both endogenous and exogenous G subunits were activated. This result agrees with findings that the C351I mutant of Gi1 couples extremely well to the 2A-adrenergic receptor (Bahia et al., 1998). Moreover, as in other studies (Wise et al., 1997; Clark et al., 2003) additional G
subunits were not required, suggesting that the G subunits expressed are able to use endogenous G. In contrast, agonist potency was higher in PTX-treated cells compared with non-PTX-treated cells, suggesting that competition between endogenous and exogenous G subunits occurred, but this was removed by PTX-mediated ADP-ribosylation of the endogenous G. Finally, it is possible that the relatively high-receptor expression level used in the study (Clark et al., 2003) may mask differences in relative agonist efficacies and potencies of the different ligands. Receptor number does affect relative agonist efficacy (MacEwan et al., 1995; Selley et al., 2000), and differences in rank order of efficacy and potency have been demonstrated with different receptor numbers (Brink et al., 2000; Cordeaux et al., 2000). However, this should not be a concern because many of the compounds tested, namely morphine, meperidine, buprenorphine, and nalbuphine, are confirmed to be partial agonists, indicating that a response ceiling had not been reached.
In C6µ cells expressing endogenous G subunits, the ability of the efficacious agonist DAMGO to stimulate [35S]GTPS was completely lost by overnight treatment with 100 ng/ml PTX, confirming that the response was due to coupling to PTX-sensitive Gi/o proteins. This effect was rescued by transfection with each of the PTX-insensitive [C352(2)I] G subunits, confirming the expression of these subunits. However, the degree of DAMGO-mediated stimulation varied across the subtypes, possibly due to differential expression. Therefore, to be able to study agonists across G subtypes, we compared relative agonist efficacy as maximal stimulation of [35S]GTPS binding relative to DAMGO as well as agonist potency. To do this, we first confirmed that different levels of G expression had no effect on these parameters. This confirmation was achieved in two ways. First, reduction in the number of functional Gi/o proteins by partial treatment with PTX lessened the maximal DAMGO stimulation of [35S]GTPS binding. Importantly, this treatment did not alter the potency of DAMGO or morphine or the efficacy of morphine and buprenorphine relative to DAMGO. The result agrees with data using 2-adrenergic receptors expressed in NIH-3T3 fibroblasts in which no change in the relative efficacy of clonidine in relation to epinephrine was observed with progressive PTX treatment (Yang and Lanier, 1999). Second, increasing the amount of Gi3 cDNA transfected resulted in an increased ability of DAMGO to stimulate [35S]GTPS binding, with no change in its potency and no change in the relative efficacy of morphine and buprenorphine. Together, these experiments show that comparisons of both absolute and relative potency and relative efficacy across µ-opioid agonists can be made between cells expressing PTX-insensitive G subtypes at different levels.
The highest potency and relative efficacy of the agonists were seen with Gi3 GoA = Gi1 Gi2, suggesting that µ-opioid receptors are most efficiently coupled to Gi3 and GoA and least efficiently to Gi1 and Gi2. This observation confirms work showing that DAMGO-stimulated incorporation of [-32P]azidoanilide into G subunits was more potent for Gi3 than for Gi1 in SH-SY5Y cells (Laugwitz et al., 1993) and with potency in the order Gi3 > Go > Gi2 in Chinese hamster ovary cells expressing a µ-opioid receptor (Chakrabarti et al., 1995). However, the same rank order of agonist relative efficacy and potency was maintained across cells expressing GoA,Gi1,Gi2,orGi3. This contrasts with agonist-specific differences observed in systems expressing receptor-G fusion proteins. For example, in human embryonic kidney cells expressing a hMOR-Go fusion protein, morphine showed similar efficiency to stimulate [35S]GTPS incorporation with both hMOR-Gi1 and hMOR-Gi2, yet endomorphin-1 and -2 promoted [35S]GTPS binding to hMOR-Gi1 but very poorly, if at all, to hMOR-Gi2 (Massotte et al., 2002). The endomorphins, together with morphine and the synthetic peptide agonist DAMGO, were equipotent in stimulating [35S]GTPS binding in E. coli expressing a hMOR-Go fusion protein, but morphine and endomorphin-1 were more potent than DAMGO or endomorphin-2 in cells expressing hMOR-Gi2 (Stanasila et al., 2000). The differences between our findings and those of the fusion systems may be accounted for by the very different systems used. However, the differences may not be so definite because, even in the present system, morphine is equipotent at Gi1 and Gi2, whereas endomorphin-1 is more potent than endomorphin-2. Consequently, subtle differences between ligands may be apparent, but these are not sufficient to alter agonist rank order.
Overall, studies suggest that Gi3 is the Gi/o protein most efficiently coupled to the µ-opioid receptor. The preference of activated µ-opioid receptor for Gi3 potentially provides for agonist-directed selectivity of second messenger signaling. There is evidence that Gi3 is a better coupling partner for G in inwardly rectifying potassium channel activation than other Gi/o subunits in Xenopus oocytes (Ivanina et al., 2004), and Go is involved in inhibition of voltage-gated Ca 2+ channels, at least in neuronal cells (Hescheler et al., 1987). In contrast, Gi1, Gi2, and Gi3 are all able to mediate inhibition of adenylyl cyclase (Gerhardt and Neubig, 1991). However, maximal DAMGO-stimulated [35S]GTPS binding was much lower in Gi3-expressing cells than in Go-expressing cells, and the potency difference for agonist stimulation of [35S]GTPS incorporation into the different G subunits is not large. Therefore, the relative concentration of G proteins may be more influential in governing downstream signaling. Indeed, Go is expressed at very high levels in brain and is considered to be the most important protein for transducing opioid signals (Jiang et al., 2001).
The present findings do not provide evidence for agonist-specific conformations of the µ-receptor that activate specific Gi/o proteins and so potentially lead to agonist-directed trafficking of intracellular signaling. Instead, differential activation of second-messenger pathways could occur by a strength of a signaling mechanism (Kenakin, 1995) dependent on the degree of agonist efficacy. A full agonist, such as DAMGO, might activate all Gi/o proteins expressed in a cell and the downstream pathways to which they are coupled, but a partial agonist may only be able to stimulate enough of the most preferred or abundant G protein and thus activate signaling through a lesser number of downstream pathways. In contrast, there are reports of agonist-directed trafficking of intracellular signal pathways via the µ-opioid receptor. In particular, there appears to be no relationship between the ability of µ-opioids to mediate receptor internalization and down-regulation compared with other events, including inhibition of Ca2+ currents (Borgland et al., 2003), inhibition of adenylyl cyclase (Keith et al., 1996; Whistler and von Zastrow, 1998), and activation of inwardly rectifying K+ channels (Celver et al., 2004). However, differences between µ-opioid ligands in mediating internalization and down-regulation may be due to agonist-dependent differences in the degree of phosphorylation of the µ-opioid receptor, leading to differences in the ability to recruit -arrestin 1 and -arrestin 2 for internalization (Bohn et al., 2004). Such actions may be independent of G protein. For example, down-regulation has a PTX-insensitive component (Yabaluri and Medzihradsky, 1997) and the ability of DAMGO but not of morphine to stimulate phospholipase D2 by a mechanism involving small G proteins may contribute to internalization (Koch et al., 2003).
In summary, the findings confirm that the agonist-activated µ-opioid receptor couples to Gi3 more efficiently than to other Gi/o subtypes. However, there was no change in the rank order of relative agonist efficacy or potency among the different Gi/o subtypes to indicate that different agonists generate specific activation states of the receptor. These results imply that reported examples of agonist-specific signaling may involve the ability of µ-opioid receptors to activate downstream signaling through a non-PTX-sensitive G protein mechanism rather than by differential activation of the structurally similar Gi/o proteins.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GPCR, G-protein-coupled receptor; PTX, pertussis toxin; DAMGO, [D-Ala2,MePhe4,Gly5-ol]enkephalin; C6µ, rat C6 glioma cells stably expressing the µ-opioid receptor; [35S]GTPS, guanosine-5'-O-(3-[35S]thio)triphosphate; PTXi, PTX-insensitive; C351(2), 351Cys or 352Cys in Gi/o proteins.
Address correspondence to: Dr. John R. Traynor, Department of Pharmacology, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail: jtraynor{at}umich.edu
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