Introduction

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Many hormones and neurotransmitters exert their effects through GPCRs that activate heterotrimeric G-proteins and, thereby, change the activity of effector systems (Gilman, 1987; Kobilka, 1992). Binding of an agonist to a GPCR induces distinct conformational changes in the receptor protein that enable GPCR to promote GDP release from G (Wess, 1997; Gether and Kobilka, 1998). The agonist-occupied GPCR forms a high-affinity ternary complex with guanine nucleotide-free G (Kent et al., 1980; Kobilka, 1992; Seifert et al., 1998a,b). Upon binding of GTP or the hydrolysis-resistant GTP analog GTPS to G, the high-affinity ternary complex is disrupted, and both G and G can regulate the activity of effector systems (Kent et al., 1980; Gilman, 1987; Kobilka, 1992; Seifert et al., 1998b). G-protein deactivation is accomplished by the GTPase activity of G (Gilman, 1987).

Several models have been developed to conceptualize the as yet incompletely understood mechanisms of GPCR activation (for a historical perspective, see Kenakin, 1996a; Colquhoun, 1998). The ternary complex model of GPCR activation states that there is a correlation between the efficacy of agonists at stabilizing the ternary complex and at promoting multiple G-protein activation/deactivation cycles (Kent et al., 1980). The ternary complex model has been elaborated into the extended ternary complex model to explain the finding that GPCRs can activate G-proteins even in the absence of agonist and that certain receptor ligands, namely, inverse agonists, suppress G-protein activation by agonist-free GPCRs (Costa and Herz, 1989; Lefkowitz et al., 1993; Gether and Kobilka, 1998). The agonist-independent GPCR activity is referred to as "constitutive activity". The extended ternary complex model assumes that agonists stabilize GPCR in the active (R*) state, while inverse agonists stabilize the inactive (R) state. It has been proposed that for constitutively active mutant (CAM) GPCRs, the equilibrium between R and R* is shifted toward R* as a result of diminished structural constraints that control spontaneous R to R* transition. Experimentally, this results in increased agonist affinity and potency and increased efficacy of partial agonists at CAM-GPCRs compared with wild-type GPCRs (Samama et al., 1993). In addition, the relative inhibitory effects of inverse agonists at CAM-GPCRs are larger than at wild-type GPCRs (Samama et al., 1994; Gether and Kobilka, 1998).

Of interest, an increasing number of experimental results cannot readily be explained by the extended ternary complex model. As an example, certain ₂AR ligands behave like "protean drugs", i.e., they act as agonist in one setting, but as inverse agonist in another (Chidiac et al., 1994). Additionally, the efficacy and potency of ₂AR agonists is strongly dependent on the specific purine nucleotide present for G-protein activation and the specific G-protein to which the ₂AR couples (Seifert et al., 1999a; Wenzel-Seifert and Seifert, 2000). Moreover, the agonist-free and agonist-occupied ₂AR differ from each other with respect to their ability to activate different cellular effectors (Zhou et al., 1999). These and other experimental findings (Keith et al., 1996; Blake et al., 1997; Krumins and Barber, 1997; Zuscik et al., 1998; Thomas et al., 2000) could be reconciled by a multistate model of GPCR activation in which ligands stabilize unique and ligand-specific GPCR conformations, which enable GPCR to modulate its cognate G-protein(s) in a ligand-specific manner (Kenakin, 1996a,b; Tucek, 1997; Gether and Kobilka, 1998).

The aim of this study was to examine the hypothesis that the functional properties of partial agonists are due to their ability to stabilize distinct conformational states in GPCRs. Therefore, we characterized functional differences between full agonists and partial agonists using fusion proteins between the wild-type ₂AR and G_s (₂ARG_s) and ₂AR_CAM (Samama et al., 1993; Gether et al., 1997) and G_s (₂AR_CAMG_s) expressed in Sf9 insect cells. We and others have shown that fusion proteins are a very sensitive experimental system for studying receptor/G-protein interactions (Seifert et al., 1999b; Milligan, 2000). Because of the defined 1:1 stoichiometry of GPCR to G, fusion proteins eliminate bias in the analysis of ligand potencies and efficacies caused by varying ratios of receptor to G-protein (Hoyer and Boddeke, 1993; Kenakin, 1997). In addition, fusion proteins allow precise determination of agonist and inverse agonist efficacy in an expression level-independent manner by measurement of steady-state GTP hydrolysis and ternary complex formation (Seifert et al., 1999b; Milligan, 2000). The results of our present study suggest that agonists have different efficacies with respect to their ability to promote different steps of the G-protein activation/deactivation cycle and provide evidence for the existence of multiple ligand-specific conformational GPCR states. Finally, our results suggest that one possible mechanism for partial agonism is strong stabilization of the ternary complex, thereby reducing G-protein turnover.

	Experimental Procedures

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Materials. The DNA encoding ₂AR_CAM was kindly donated by Dr. R. J. Lefkowitz (Duke University, Durham, NC) (Samama et al., 1993). Sources of other materials have been described elsewhere (Seifert et al., 1998a,b).

Construction of ₂AR_CAMG_s Fusion Protein DNAs. The fusion proteins used in our present study contained the long splice variant of G_s. The construction of ₂ARG_s DNA was described recently (Seifert et al., 1998a,b). For construction of ₂AR_CAMG_s DNA, the KpnI/EcoRV-fragment of the ₂AR was excised from pGEM-3Z-₂ARG_s and replaced by the corresponding KpnI/EcoRV-fragment of ₂AR_CAM DNA. The KpnI/EcoRV fragment of ₂AR_CAM DNA differs from the corresponding ₂AR DNA fragment by mutations that encode for four discrete amino acid substitutions in the third intracellular loop of the receptor (Samama et al., 1993). The ₂AR_CAMG_s DNA was cloned into the baculovirus expression vector pVL 1392.

Cell Culture and Membrane Preparation. Recombinant baculoviruses were generated in Sf9 cells using the BaculoGOLD transfection kit (Pharmingen, San Diego, CA). After initial transfection, recombinant baculoviruses were isolated by plaque purification. High-titer virus stocks were generated by three sequential amplifications of plaque-purified virus colonies. Culture of Sf9 cells and membrane preparation were performed as described recently (Seifert et al., 1998a,b).

[³H]DHA Binding. Membranes were thawed and sedimented by a 15-min centrifugation at 4°C and 15,000g to remove residual endogenous guanine nucleotides as far as possible and resuspended in binding buffer (12.5 mM MgCl₂, 1 mM EDTA, and 75 mM Tris/HCl, pH 7.4). Expression levels of fusion proteins were determined by incubating Sf9 membranes (15-25 µg of protein per tube) in the presence of [³H]DHA at a concentration of 10 nM. Nonspecific binding was determined in the presence of [³H]DHA (10 nM) plus 10 µM (±)-alprenolol. The total volume of the binding reaction was 500 µl. Incubations were performed for 90 min at 25°C and shaking at 250 rpm. Competition binding experiments were carried out with Sf9 membranes expressing ₂ARG_s or ₂AR_CAMG_s (15-40 µg of protein per tube), 1 nM [³H]DHA and agonists at various concentrations with or without GTPS (10 µM). Ligand-competition studies were performed in triplicates. Bound [³H]DHA was separated from free [³H]DHA by filtration through GF/C filters using a 48-well harvester (model M-48R; Brandel, Gaithersburg, MD), followed by three washes with 2 ml of binding buffer (4°C). Filter-bound radioactivity was determined by liquid scintillation counting using Cytoscint cocktail from ICN (Irvine, CA). The experimental conditions chosen ensured that not more than 10% of the total amount of [³H]DHA added to binding tubes was bound to filters.

Steady-State GTPase Activity. Membranes were thawed and sedimented by a 15-min centrifugation at 4°C and 15,000g to remove residual endogenous guanine nucleotides as far as possible and resuspended in 10 mM Tris/HCl, pH 7.4. Assay tubes contained Sf9 membranes expressing ₂ARG_s or ₂AR_CAMG_s (10 µg of protein per tube), 1.0 mM MgCl₂, 0.1 mM EDTA, 0.1 mM ATP, 1 mM adenylyl imidodiphosphate, 100 nM GTP, 5 mM creatine phosphate, 40 µg of creatine kinase, 0.2% (w/v) bovine serum albumin in 50 mM Tris/HCl, pH 7.4, and ₂AR ligands at various concentrations. Reaction mixtures (80 µl) were incubated for 3 min at 25°C before the addition of 20 µl of [-³²P]GTP (0.2-0.5 µCi/tube). Reactions were conducted for 20 min at 25°C. Reactions were terminated by the addition of 900 µl of a slurry consisting of 5% (w/v) activated charcoal and 50 mM NaH₂PO₄, pH 2.0. Charcoal absorbs nucleotides but not P_i. Charcoal-quenched reaction mixtures were centrifuged for 15 min at room temperature at 15,000g. Seven hundred microliters of the supernatant fluid of reaction mixtures was carefully removed to avoid any aspiration of charcoal, and ³²P_i was determined by liquid scintillation counting. Enzyme activities were corrected for spontaneous degradation of [-³²P]GTP. Spontaneous [-³²P]GTP degradation was determined in tubes containing all of the above-described components plus a very high concentration of unlabeled GTP (1 mM) that, by competition with [-³²P]GTP, prevents [-³²P]GTP hydrolysis by enzymatic activities present in Sf9 membranes. Spontaneous [-³²P]GTP degradation was <1% of the total amount of radioactivity added. The experimental conditions chosen ensured that not more than 10% of the total amount of [-³²P]GTP added was converted to ³²P_i.

Miscellaneous. Protein was determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Immunoblotting studies were performed as described (Seifert et al., 1998a,b). Ligand competition curves and concentration-response curves were analyzed by nonlinear regression, using the Prism program (GraphPad, San Diego, CA).

	Results

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Expression of ₂ARG_s and ₂AR_CAMG_s in Sf9 Membranes. Similar to nonfused ₂AR and ₂AR_CAM (Gether et al., 1997), the maximum expression level of ₂AR_CAMG_s in Sf9 cells was ~2.5-fold lower than the expression level of ₂ARG_s (₂ARG_s, 7.2 ± 2.1 pmol/mg; ₂AR_CAMG_s, 3.2 ± 1.2 pmol/mg). However, the different expression levels of the fusion proteins were not of relevance for our studies since the efficacies of ligands at stabilizing the ternary complex and at promoting GTP hydrolysis are expression level-independent (Seifert et al., 1999; Milligan, 2000). Immunoblotting studies with the M1 antibody recognizing the N-terminal FLAG epitope of ₂AR and ₂AR_CAM (Gether et al., 1995, 1997) and anti-G_s Ig confirmed that ₂ARG_s and ₂AR_CAMG_s expressed in Sf9 membranes were structurally intact (data not shown).

Agonist and Inverse Agonist Regulation of Steady-State GTPase Activity in Sf9 Membranes Expressing ₂ARG_s and ₂AR_CAMG_s. An important hallmark of constitutive GPCR activation is the increased relative inhibitory effect of inverse agonist at CAM-GPCR compared with wild-type GPCR (Lefkowitz et al., 1993; Gether and Kobilka, 1998). In addition, the agonist-affinity of CAM-GPCRs is increased relative to wild type-GPCRs, while the inverse agonist-affinity is decreased. The full agonist ()-ISO was more potent at activating GTP hydrolysis at ₂AR_CAMG_s (EC₅₀ = 2.4 ± 1.1 nM) than at ₂ARG_s, (EC₅₀ = 13 ± 3 nM), while the inverse agonist ICI was less potent at ₂AR_CAMG_s (IC₅₀ = 8.5 ± 2.5 nM) than at ₂ARG_s (IC₅₀ = 2.8 ± 1.0 nM) (Fig. 1). For ₂ARG_s, 17% of the ligand-regulated GTPase activity (the difference between maximum ()-ISO-stimulated and minimum ICI-inhibited GTP hydrolysis) was attributable to the inverse agonist, while for ₂AR_CAMG_s, 48% of the ligand-regulated GTPase activity was attributable to ICI.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effects of ()-ISO and ICI on GTPase activity in Sf9 membranes expressing 2ARGs and 2ARCAMGs. GTPase activity in membranes expressing 2ARGs (6.5 pmol/mg) (A) or 2ARCAMGs (2.5 pmol/mg) (B) was determined as described under Experimental Procedures. Reaction mixtures contained ()-ISO or ICI at the concentrations indicated on the abscissa. The dashed lines are extrapolations of basal GTPase activities to illustrate the relative contributions of ()-ISO and ICI at the ligand-regulated enzyme activity. To reliably quantitate the inhibitory effect of ICI on GTPase in membranes expressing 2ARGs, this construct was expressed at a higher level than 2ARCAMGs. Therefore, the absolute GTPase activities in A are higher than in B. To facilitate comparison of the two constructs, the scales of the ordinates in A and B are different. Data shown are the means ± S.D. of three independent experiments performed in duplicate.

Ligand Binding Properties of ₂ARG_s and ₂AR_CAMG_s. Sf9 membranes expressing ₂ARG_s and ₂AR_CAMG_s bound the ₂AR antagonist [³H]DHA in a monophasic and saturable manner and with similar K_d values (₂ARG_s, 0.36 ± 0.03 nM; ₂AR_CAMG_s, 0.24 ± 0.05 nM). We also studied the agonist and inverse agonist binding properties of ₂ARG_s and ₂AR_CAMG_s. The ligand competition curves are shown in Figs. 2-4, and Table 1 summarizes the nonlinear regression analysis of the binding data. In membranes expressing ₂ARG_s, strong agonists [()-ISO, (+)-ISO, SAL, and DOB] efficiently stabilized the ternary complex as is shown by the GTPS-sensitive high-affinity agonist binding. With these ligands, ~40% of the ₂ARs displayed high agonist affinity. For the partial agonist EPH, distinct high-affinity binding sites at ₂ARG_s could not be discriminated, but GTPS could still shift the EPH competition curve ~5-fold to the right. The binding of another partial agonist, DCI, to ₂ARG_s, was virtually GTPS insensitive. GTPS did not shift the ICI-competition curve in membranes expressing ₂ARG_s.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Competition by ()-ISO, SAL, and DOB of [3H]DHA binding in Sf9 membranes expressing 2ARGs or 2ARCAMGs: effect of GTPS. [3H]DHA binding in Sf9 membranes was performed as described under Experimental Procedures. Reaction mixtures contained Sf9 membranes (15-40 µg of protein per tube) expressing 2ARGs (3.3-7.5 pmol/mg) (A-C) or 2ARCAMGs (2.5-4.9 pmol/mg) (D-F), 1 nM [3H]DHA, and ligands at the concentrations indicated on the abscissa. Reaction mixtures additionally contained distilled water (control) or GTPS (10 µM). Data shown are the means ± S.D. of four to seven independent experiments performed in triplicate.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Competition by EPH, DCI, and ICI of [3H]DHA binding in Sf9 membranes expressing 2ARGs or 2ARCAMGs: effect of GTPS. [3H]DHA binding in Sf9 membranes was performed as described under Experimental Procedures. Reaction mixtures contained Sf9 membranes (15-40 µg of protein per tube) expressing 2ARGs (3.3-7.5 pmol/mg) (A-C) or 2ARCAMGs (2.5-4.9 pmol/mg) (D-F), 1 nM [3H]DHA, and ligands at the concentrations indicated on the abscissa. Reaction mixtures additionally contained distilled water (control) or GTPS (10 µM). Data shown are the means ± S.D. of four to seven independent experiments performed in triplicate.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Competition by ()-ISO and (+)-ISO of [3H]DHA binding in Sf9 membranes expressing 2ARGs or 2ARCAMGs: effect of GTPS. [3H]DHA binding in Sf9 membranes was performed under Experimental Procedures. Reaction mixtures contained Sf9 membranes (15-40 µg of protein per tube) expressing 2ARGs (3.3-7.5 pmol/mg) (A and B) or 2ARCAMGs (2.5-4.9 pmol/mg) (C and D), 1 nM [3H]DHA, and ligands at the concentrations indicated on the abscissa. Reaction mixtures additionally contained distilled water (control) or GTPS (10 µM). Data shown are the means ± S.D. of four to seven independent experiments performed in triplicate.

View this table: [in this window] [in a new window]   TABLE 1 Ligand binding properties of 2ARGs and 2ARCAMGs expressed in Sf9 membranes: effect of GTPS [3H]DHA binding was determined as described under Experimental Procedures in Sf9 membranes expressing 2ARGs (3.3-7.5 pmol/mg) or 2ARCAMGs (2.5-4.9 pmol/mg). The ligand competition binding curves shown in Figs. 2, 5, and 7 were analyzed by nonlinear regression for best fit to single-site or two-site binding. Kh and Kl designate the dissociation constants for high- and low-affinity agonist binding, respectively. %Rh indicates the percentage of receptors displaying high agonist affinity. The corresponding values obtained in the presence of GTPS (10 µM) are designated KhGTPS, KlGTPS, and %RhGTPS, respectively. Dissociation constants for ICI are listed below Kl and KlGTPS, respectively. Data shown represent the means ± S.D. of four to seven independent experiments performed in triplicate.

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Efficacies of Partial Agonists on Steady-State GTPase Activity in Sf9 Membranes Expressing ₂ARG_s and ₂AR_CAMG_s. The precise determination of partial agonist efficacy constitutes a major problem since efficacy is influenced by numerous variables such as the expression level of GPCR and the availability of G-protein and effector system (Hoyer and Boddeke 1993; Kenakin 1996a, 1997). The fusion protein approach offers a unique possibility to assess agonist efficacy in an expression level-independent manner by measuring steady-state GTPase activity (Seifert et al., 1999; Milligan, 2000). At ₂ARG_s, ligands activated GTP hydrolysis in the order of efficacy ()-ISO ~ SAL  (+)-ISO > DOB > EPH > DCI (Figs. 5 and 6; Table 2). In accordance with data obtained for effector system activation by nonfused ₂AR and ₂AR_CAM (Samama et al., 1993), the potencies of agonists for GTPase activation at ₂AR_CAMG_s were higher than at ₂ARG_s. There was a small but significant increase in the efficacy of the partial agonists DOB, EPH, and DCI at activating GTPase in membranes expressing ₂AR_CAMG_s compared with membranes expressing ₂ARG_s. For SAL and (+)-ISO, the increase in efficacy at ₂AR_CAMG_s versus ₂ARG_s was not significant.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Concentration-response curves for the stimulatory effects of ()-ISO, SAL, DOB, EPH, and DCI on steady-state GTPase activity in membranes expressing 2ARGs and 2ARCAMGs. GTPase activity in membranes expressing 2ARGs (3.3-7.5 pmol/mg) (A) or 2ARCAMGs (2.5-4.9 pmol/mg) (B) was determined as described under Experimental Procedures. Reaction mixtures contained ligands at the concentrations indicated on the abscissa. For each construct, the stimulatory effect of ()-ISO (10 µM) on GTP hydrolysis was set 100%, and all data points were referred to this stimulation. Data shown are the means ± S.D. of three to six independent experiments performed in duplicate.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Concentration-response curves for the stimulatory effects of ()-ISO and (+)-ISO on steady-state GTPase activity in membranes expressing 2ARGs and 2ARCAMGs. GTPase activity in membranes expressing 2ARGs (3.3-7.5 pmol/mg) (A) or 2ARCAMGs (2.5-4.9 pmol/mg) (B) was determined as described under Experimental Procedures. Reaction mixtures contained ligands at the concentrations indicated on the abscissa. For each construct, the stimulatory effect of ()-ISO (10 µM) on GTP hydrolysis was set 100%, and all data points were referred to this stimulation. Data shown are the means ± S.D. of three to six independent experiments performed in duplicate.

View this table: [in this window] [in a new window]   TABLE 2 Efficacies and potencies of 2AR ligands at the GTPase of 2ARGs and 2ARCAMGs GTPase activity was measured as described under Experimental Procedures in Sf9 membranes expressing various 2ARGs and 2ARCAMGs. Reaction mixtures contained 2AR ligands at 0.1 nM-1mM as appropriate to obtain saturated concentration-response curves. Ligand efficacies and potencies (EC50 values) were obtained by nonlinear regression analysis of the concentration-response curves shown in Figs. 3 and 4. The efficacy of ()-ISO was set 1.00, and the effects of other ligands are referred to this effect. Data shown are the means ± S.D. of three to seven independent experiments performed in duplicate. The data shown for 2ARGs were previously reported in Seifert et al. (1999a). Data for 2ARGs were compared versus 2ARCAMGs using the t test.

	Discussion

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Sf9 Cell Membranes Expressing GPCR-G Fusion Proteins as Model System for the Analysis of Ligand-Receptor Interactions. In the present study, we expressed a wild-type GPCR and a CAM-GPCR fused to G in Sf9 cells to study ligand-receptor interactions. Compared with conventional coexpression systems, the fusion protein approach offers several advantages. Specifically, the defined 1:1 stoichiometry of GPCR and G eliminates bias in the analysis of ligand potencies and efficacies because of varying GPCR/G ratio (Seifert et al., 199b; Milligan, 2000). In addition, the physical proximity of GPCR and G ensures efficient interaction of the proteins. Fusion proteins allow for the sensitive analysis of ligand potencies and efficacies in an expression level-independent manner directly at the G-protein level by measuring steady-state GTP hydrolysis. This is a very important point because nonfused ₂AR_CAM expresses at considerably lower levels than ₂AR (Samama et al., 1993; Gether et al., 1997), resulting in different GPCR/G-protein ratios. Finally, for GPCRs mediating adenylyl cyclase activation such as the ₂AR, the number of effector molecules limits signal output (Alousi et al., 1991), introducing additional bias into the analysis of ligand potencies and efficacies.

Can the extended ternary complex model explain the functional properties of ₂ARG_s and ₂AR_CAMG_s? Several of our results are in agreement with the extended ternary complex model, which proposes that GPCRs exist in an equilibrium between an inactive R state and an active R* state (Lefkowitz et al., 1993; Gether and Kobilka 1998). The model proposes that in CAM-GPCRs, R to R* isomerization occurs more readily than in wild-type GPCRs, but there is no fundamental difference in the R and R* states of CAM-GPCRs. Thus, the model predicts that the relative inhibitory effects of inverse agonists at CAM-GPCRs are higher than at wild type-GPCRs. The increased relative inhibitory effect of ICI at ₂AR_CAMG_s compared with ₂ARG_s is in agreement with this model (Fig. 1). The extended ternary complex model also predicts that the inverse agonist affinity and potency at CAM-GPCR is lower than at wild-type GPCR (Samama et al., 1994). Again, our findings with fusion proteins are in agreement with the model (Figs. 1 and 3). Another postulate of the extended ternary complex model is that guanine nucleotides increase inverse agonist affinity of GPCR, presumably by uncoupling of GPCR from G-protein and, thereby, stabilizing the R state (Barker et al., 1994; Leeb-Lundberg et al., 1994). In agreement with this postulate, we found an increase in inverse agonist-affinity of ₂AR_CAMG_s by GTPS (Fig. 3; Table 1).

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View larger version (32K): [in this window] [in a new window]   Fig. 7.   Relation between the efficacy of agonists at stabilizing the ternary complex and the ligand efficacy at activating GTPase in Sf9 membranes expressing 2ARGs and 2ARCAMGs. The efficacies of ligands at stabilizing the ternary complex in membranes expressing 2ARGs and 2ARCAMGs (Figs. 2-4; Table 1) were plotted against the respective efficacies of these ligands at activating GTPase (Figs. 5 and 6; Table 2). Data were analyzed by linear regression analysis. In A and B, ternary complex formation is expressed as the percentage of receptors displaying high agonist affinity (Rh, %). A, r2 = 0.71; p = 0.035. B, r2 = 0.08; p, = 0.586 (slope not significantly different from zero). In C and D, ternary complex formation is expressed as the ratio KlGTPS/Kh. For EPH and DCI, distinct Kh values at 2ARGs could not be determined. Therefore, we used the values listed under Kl in Table 1 to calculate the ratio. C, r2 = 0.86; p = 0.007. D, r2 = 0.04; p = 0.716 (slope not significantly different from zero). The dotted lines indicate the 95% confidence interval of the regression lines.

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Mechanisms of Action of Full and Partial Agonists. Of interest, the ratio of EC₅₀ for GTPase activation to K_h for agonists at ₂AR_CAMG_s shows a significant correlation with the efficacy of agonists at activating GTPase (Fig. 8). The data obtained for ₂ARG_s show a similar trend as for ₂AR_CAMG_s but do not reach significance. It is surprising that the ratio of EC₅₀ to K_h is significantly higher for full agonists than partial agonists. This suggests that occupancy of a GPCR by a full agonist is less likely to lead to a complete G-protein activation/deactivation cycle than is GPCR occupancy by a partial agonist. A possible mechanism for this hypothesis is illustrated in Fig. 9. This model proposes that the ternary complex for partial agonists is more stable, relative to the other GPCR and G-protein states (PR, G, G_GDP, G_GTP), than is the ternary complex for full agonists, relative to the other GPCR and G-protein states (FR, G, G_GDP, G_GTP). Thus, rate constants k₁ and k₂ for full and partial agonists may be similar. However, if the rate constants k₁, k₂, and k₃ are all smaller for partial agonists, compared with these constants for full agonists, the relative amount of the ternary complex (partial agonists) will be greater and the number of total G-protein hydrolysis cycles will be smaller, compared with full agonists. The model shown in Fig. 9 also proposes that the ternary complex for full agonists is more likely to dissociate without binding GTP, thus k₁ and k₂ are greater for full agonists than for partial agonists. This could explain the larger EC₅₀ to K_h ratio for full agonists. A larger receptor occupancy would be required to compensate for larger k₁ and k₂ rates for full agonists as compared to partial agonists.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Relation between the ratio EC50/Kh and efficacy of agonists at activating GTPase in Sf9 membranes expressing 2ARGs and 2ARCAMGs. The ratios of the EC50 for GTPase activation (Figs. 5 and 6; Table 2) and Kh values (Figs. 2 and 3; Table 1) for various agonists at 2ARGs and 2ARCAMGs were plotted against the respective efficacies of these ligands at activating GTPase (Figs. 5 and 6; Table 2). Data were analyzed by linear regression analysis. A, r2 = 0.653; p = 0.192. B, r2 = 0.82; p = 0.014. The dotted lines indicate the 95% confidence interval of the regression line.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Model of the different effects of full and partial agonists on the G-protein activation/deactivation cycle. The arrows symbolize the relative rate constants for interconversion of the various GPCR and G-protein states. G, guanine nucleotide-free G-protein; GGDP, GDP-liganded G-protein; GGTP, GTP-liganded G-protein; F, full agonist; P, partial agonist; R, G-protein-coupled receptor.

Conclusions. Our present data and an increasing body of data obtained with wild-type GPCRs (Chidiac et al., 1994; Chiu et al., 1996; Keith et al., 1996; Blake et al., 1997; Bouaboula et al., 1997) and constitutively active GPCR mutants (Zuscik et al., 1998; Thomas et al., 2000) and theoretical considerations (Kenakin 1996a,b; Tucek 1997) suggest that ligands stabilize distinct GPCR conformations that differ from each other in their ability to interact with G-proteins. Thus, the conformations stabilized by ()-ISO, (+)-ISO, and SAL in both ₂AR and ₂AR_CAM are functionally distinguishable from those induced by agonists with lower efficacies, i.e., DOB, EPH, and DCI. Additionally, our results suggest that some GPCR ligands act as partial agonists because the GPCR conformation that they stabilize promotes a more stable ternary complex than the conformation stabilized by full agonists. By stabilizing the guanine nucleotide-free ternary complex these partial agonists reduce G-protein turnover.