Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

₂-Adrenoceptors (₂-AR) are implicated in the control of noradrenergic neurotransmission in the central nervous system and modulate several physiological processes peripherally (Timmermans et al., 1990; Szabadi and Bradshaw, 1996). There are now three characterized ₂-AR subtypes: _2A, _2B, and _2C; these are G protein-coupled receptors that are predominantly coupled to the G_i/o signaling system, inhibiting and/or stimulating the activity of adenylate cyclase, inhibiting the opening of voltage-gated Ca²⁺ channels, and activating K⁺ channels (see Hein and Kobilka, 1997). The ₂-AR may also couple to other intracellular pathways involving Na⁺/H⁺ exchange and the activation of phospholipase A₂ and C (Limbird, 1988; Cotecchia et al., 1990; Kukkonen et al., 1998). The ₂-AR subtypes are distributed differentially in cells and tissues, endowing these receptors with different physiological functions and pharmacological activity profiles. However, available ligands have only marginal subtype selectivity.

A widely accepted model used to describe agonist activation of G protein-coupled receptors is the ternary complex model, which accounts for the cooperative interactions among receptor, G protein, and agonist (De Lean et al., 1980). This model has recently been extended to accommodate the observation that many receptors can activate G proteins in the absence of agonist, and that mutations in different structural domains of the receptors can enhance the agonist-independent (constitutive) activity (Samama et al., 1993). The extended ternary complex model also accounts for the effects of different types of receptor ligands [full agonists, partial agonists, silent ligands (neutral antagonists), and inverse agonists (also defined as negative antagonists)] on receptor signaling (Gether and Kobilka, 1998). However, the pharmacological distinction between a neutral antagonist and an inverse agonist is often difficult to observe. One possible explanation is that the magnitude of inverse agonism is determined by constitutive receptor activation of specific G protein subtypes. Consequently, ligands may demonstrate distinct pharmacological effects (i.e., neutral antagonism or inverse agonism) depending on which receptor/G protein/effector pathway is involved. Perez et al. (1996) reported on a Cys¹²⁸Phe mutation in the _1B-AR, resulting in G protein coupling in the absence of agonist and constitutive activation of the phospholipase C, but not of the phospholipase A₂ pathway. A similar mutation (Cys¹¹⁶Phe) in the ₂-AR causes selective constitutive activation of Na⁺/H⁺ exchange through a pathway not involving cAMP (Zuscik et al., 1998). These data suggest that multiple and distinct activation states exist for a receptor, and that the pharmacological profile of a single receptor subtype may be codetermined by the effector pathway that is being considered.

The carboxy-terminal portion of the third intracellular loop (ICL) has been suggested (Kjelsberg et al., 1992) to be involved in constraining G protein-coupled receptors in the inactive (G protein-uncoupled) conformation. Mutagenesis studies of the BBXXB motif (where B represents a basic residue and X a nonbasic residue) in the third ICL of _2A-AR demonstrated a constitutively active mutant (i.e., Thr³⁷³Lys; Ren et al., 1993) by analogy with mutations affecting the same region in _1B-AR and ₂-AR, and more recently in 5-HT_2A and 5-HT_2C receptors (Egan et al., 1998; Herrick-Davis et al., 1997). The constitutively active mutant Thr³⁷³Lys _2A-AR demonstrates apparently disparate results (i.e., positive efficacy) for ligands that so far have been characterized as _2A-AR antagonists, by measuring the positive coupling of this mutant _2A-AR to the formation of inositol phosphates in the presence of a G₁₅ protein (Wurch et al., 1999). This led us to suggest that the pharmacology of constitutively active receptors may be more complex than is commonly assumed. These data also suggest that pure neutral antagonists at _2A-AR may be relatively uncommon. It has yet to be determined whether constitutive activity of _2A-AR can be inhibited by inverse agonists, and whether this type of ligand activity can be reversed by neutral antagonists. Tian et al. (1994) demonstrated that some ₂-AR antagonists display inverse agonist activity at precoupled wild-type (wt) rat _2D-AR in recombinant PC₁₂ cells.

In this study, the intrinsic activity of ₂-AR ligands was analyzed at the wt and mutant Thr³⁷³Lys _2A-AR on activation by a rat G_o protein. A Bordetella pertussis toxin (PTX)-resistant mutant G_o Cys³⁵¹Ile protein (Dupuis et al., 1999) was used to avoid potential coupling of the _2A-AR to endogenous G_i/o proteins of CHO-K1 cells. The activation of _2A-AR by either endogenous G proteins or a recombinant G_o protein was estimated by measuring agonist-independent and -dependent PTX-resistant binding of the stable GTP analog [³⁵S]GTPS. The process of constitutive activation of the mutant _2A-AR is considerably favored by coexpression of a G_o Cys³⁵¹Ile protein and can only be fully blocked by a minority of the putative ₂-AR antagonists that were investigated.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Cloning of wt and Mutant Human _2A-AR and Rat G Protein Genes. Wild-type genes were cloned by polymerase chain reaction (PCR) using primers designed at the start and stop codons of the respective nucleotide sequences. Site-directed mutagenesis of the human _2A-AR gene (Fraser et al., 1989; Genbank: M 23533) was performed using a modified overlap extension technique based on PCR (Wurch et al., 1998) using appropriate complementary primers carrying the Thr³⁷³ (ACG codon) to Lys (AAA codon) mutation. The mutant rat G_o Cys³⁵¹Ile protein gene (Jones and Reed, 1987; Genebank: M 17526) was amplified in the same way with primers carrying the Cys (TGT codon) to Ile (ATT codon) mutation. The Cys³⁵¹Ile mutants of either the G_i1 (M 17527) and G_i3 (M 20713) proteins, and the Cys³⁵²Ile mutant of the G_i2 protein (M 17528) were constructed in a similar way. The respective PCR products were separated by agarose gel electrophoresis, purified using a Geneclean II kit, and cloned into 50 ng of a pCR3.1 vector. The recombinant genes are expressed under the transcriptional control of the human cytomegalovirus immediate-early gene promoter and enhancer sequence, which permit efficient and high expression (Boshart et al., 1985). Sequencing, performed automatically on an ABI Prism 310 Genetic Analyser using a dichlororhodamine terminator cycle sequencing kit, confirmed the respective nucleotide sequences.

Transient Expression of Human _2A-AR and Rat G Proteins in CHO-K1 Cells. The CHO-K1 cell line (American Type Culture Collection, CCL 61) was cultured in Petri dishes (50 cm²) with nutrient mixture Ham's F12 supplemented with 10% heat-inactivated fetal calf serum. Cells grown to 60 to 80% confluence were used for transfection using a LipofectAMINE plus kit. Three micrograms of pCR3.1 plasmid containing either the wt or mutant Thr³⁷³Lys _2A-AR gene supplemented with 3 µg of empty pCR3.1 plasmid, or 3 µg of wt or mutant Thr³⁷³Lys _2A-AR gene and 3 µg (unless otherwise indicated) of either wt or mutant G protein gene were mixed with 10 µl of LipofectAMINE plus reagent in 0.2 ml of Opti-Mem and incubated at room temperature for 15 min. Subsequently, 20 µl of LipofectAMINE reagent diluted in 0.2 ml of Opti-Mem was added for 15 min and exposed with 5 ml of Opti-Mem to CHO-K1 cells for 3 h at 37°C. Thereafter, cells were incubated with 10 ml of complete growth medium and harvested 48 h after transfection. Treatment with PTX (20 ng/ml) was performed overnight before membranes were prepared.

Membrane Preparation and Radioligand Binding Experiments. Membrane preparation steps were performed at 4°C. Cells were washed twice with PBS and stored at 80°C. Cells were then scraped mechanically in 10 mM Tris-HCl supplemented with 0.1 mM EDTA (pH 7.5) and centrifuged for 10 min at 45,000g. The pellet was homogenized in the same buffer using a Polytron and recentrifuged. The final pellet was distributed in aliquots of 0.5 ml of Tris-EDTA buffer (0.5 to 1.5 mg/ml of protein), and stored at 80°C until used. Membrane preparations were diluted in 50 mM Tris-HCl (pH 7.7) containing 4mM CaCl₂, 10 µM pargyline, and 0.1% ascorbic acid, and used for [³H]2-(2-methoxy-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole ([³H]RX 821002, 2 nM) binding experiments as described (Wurch et al., 1999). Ten micromolar phentolamine was used to determine nonspecific radioligand binding.

[³⁵S]GTPS Binding Responses. Agonist-independent (basal) and -dependent [³⁵S]GTPS binding responses (Pauwels et al., 1997) were performed to the above described membrane preparation in 20 mM HEPES (pH 7.4) supplemented with 30 µM GDP, 100 mM NaCl, 3 mM MgCl₂, and 0.2 mM ascorbic acid. [³⁵S]GTPS binding responses were systematically performed in the presence of 10 µM 2-(2-ethoxy-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole (RX 811059) to correct for enhanced basal [³⁵S]GTPS binding. Maximal stimulation of [³⁵S]GTPS binding was defined in the presence of 10 µM ()-adrenaline and calculated versus basal [³⁵S]GTPS binding, unless otherwise indicated. pEC₅₀ values were defined as the concentration of the ligand at which 50% of its own maximal stimulation of [³⁵S]GTPS binding was obtained. pIC₅₀ values represent the concentration of the ligand that showed 50% inhibition of its own maximal inhibition of basal [³⁵S]GTPS binding. In antagonist experiments, atipamezole was coincubated with the indicated ligand. pK_B values were calculated as K_B = (B)/(A'/A)  1, where B is the concentration of the antagonist, and A and A' are the EC₅₀ or IC₅₀ values of ligand measured in either the absence or presence of antagonist, respectively. Saturation [³⁵S]GTPS binding responses were performed as described previously (Pauwels et al., 1998). Statistical analysis was performed on E_max values using one- way (either without or with repeated measures) ANOVA, followed by all pairwise multiple comparison procedures (Tukey's test).

Immunological G Protein Detection. Membrane preparations of CHO-K1 cells transfected with the mutant Thr³⁷³Lys _2A-AR in either the absence or presence of Cys³⁵¹Ile G_o, Cys³⁵¹Ile G_i1, Cys³⁵²Ile G_i2, and Cys³⁵¹Ile G_i3 protein were prepared as described above. Total proteins were separated by denaturing SDS-polyacrylamide gel electrophoresis (PAGE) (12.5%, w/v) as described (Laemmli, 1970). Thereafter, the proteins were blotted onto a nylon membrane by semidry electrotransfer (23 V, 45 min) in 192 mM glycine, methanol 20% (v/v), and 25 mM Tris-HCl buffer (pH 8.3). Proteins were probed using either a selective, monoclonal anti-G_o antibody raised against a synthetic peptide corresponding to the amino acids 13 to 88 of the rat G_o protein, or a nonselective polyclonal anti-G subunit antibody raised against a synthetic peptide corresponding to the amino acids 40 to 54 of the rat G_z protein. The incubation was performed in PBS buffer containing 0.1% Tween 20 (w/v), 5% dried nonfat milk, and the indicated antibody at a dilution of 1:1000. Proteins were visualized with an anti-rabbit or an anti-mouse IgG antibody coupled to alkaline phosphatase using a colorimetric reaction (0.12 mM 4-nitroblue tetrazolium chloride monohydrate, 0.12 mM 5-bromo 4-chloro 3-indolylphosphate p-toluidine salt, 5 mM MgCl₂ in 100 mM diethanolamine, pH 9.6). The computer-based image analysis system Imagena 2000 was used for quantification of the G protein signals.

Protein Content. Membrane protein levels were estimated with a dye-binding assay using a Bio-Rad kit; BSA was used as a standard (Bradford, 1976).

Materials. The ABI Prism 310 Genetic Analyser and the dichlororhodamine terminator cycle sequencing kit were obtained from Perkin-Elmer (Foster City, CA). The pCR3.1 expression vector was purchased from Invitrogen (San Diego, CA). The Geneclean II kit was obtained from Bio 101 Inc. (La Jolla, CA). CHO-K1 cells were obtained from American Type Culture Collection (Rockville, MD). [³H]RX 821002 (50 Ci/mmol) and [³⁵S]GTPS (1035-1163 Ci/mmol) were obtained from Amersham (Les Ulis, France). The LipofectAMINE plus kit, cell culture media, fetal calf serum, culture plates, and PTX (50 µg/ml) were obtained from Gibco Biocult Laboratories (Paisley, UK). The Emulsifier-Safe was obtained from Packard (Warrenville, PA). The anti-G_o, anti-G, and anti-rabbit IgG antibodies were purchased from Calbiochem Corp. (La Jolla, CA). The anti-mouse IgG antibody was obtained from NEN (Boston, MA). The Imagena 2000 quantification system was purchased from Biocom (Les Ulis, France). Clonidine, ()-adrenaline, yohimbine, oxymetazoline, and substrates for the colorimetric immunodetection were obtained from Sigma (St. Louis, MO). (±)-2[(4,5-Dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole (BRL 44408) and 2-(2,6-dimethoxyphenoxyethyl)aminoethyl-1,4-benzodioxane hydrochloride (WB 4101) were obtained from Research Biochemicals, Inc. (Natick, MA). d-Medetomidine was purchased from SmithKline Beecham (Ploufragan, France). 5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline tartrate (UK 14304), dexefaroxan, atipamezole, (+)-(8aR,12aS,13aS)-3-methoxy-12-(methylsulphonyl)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-6H-isoquino[2',1-g] [1,6]naphthyridine (RS 15385), and racemic, (+)-, and ()-RX 811059 were prepared intramuros. Idazoxan and RX 821002 were purchased from Reckitt and Colman (Kingston-upon-Hill, UK). 6-Allyl-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin-2-ylamine (BHT 920) was a gift from Boehringer Ingelheim (Biberach an der Riss, Germany). 6-Chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine (SKF 86466) was obtained from SmithKline Beecham (Herts, UK). Fluparoxan was obtained from Glaxo (Hertfordshire, UK). Stock solutions of ligands were prepared at 10³ M. Serial dilutions were made in the respective incubation buffers.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

On transient expression in CHO-K1 cells, wt _2A-AR displayed a weak ()-adrenaline-dependent [³⁵S]GTPS binding response, which could be strongly enhanced by coexpression with the G_o protein (Fig. 1). In the absence of recombinant G proteins, the basal [³⁵S]GTPS binding response was enhanced by 47 ± 15% with ()-adrenaline (10 µM), whereas the basal response was not affected by the ₂-AR antagonist RX 811059 (10 µM). PTX treatment (20 ng/ml) fully abolished the ()-adrenaline-mediated [³⁵S]- GTPS binding response (Fig. 1B). Coexpression with a wt G_o protein enhanced the ()-adrenaline response by 367 ± 31%, whereas RX 811059 produced some inhibition (15 ± 2%, P < .05) of the basal [³⁵S]GTPS response (Fig. 1C). Both effects were absent on treatment by PTX (20 ng/ml) (Fig. 1D). In contrast, coexpression with a PTX-resistant mutant G_o Cys³⁵¹Ile protein in the presence of PTX (20 ng/ml) yielded an ()-adrenaline response (504 ± 78%) and RX 811059-mediated inhibition (19 ± 3%, P < .05) of the basal [³⁵S]GTPS binding response (Fig. 1E). Nonetheless, the ()-adrenaline-mediated [³⁵S]GTPS binding response was easily measurable in the transfected CHO-K1 cells, the basal [³⁵S]GTPS response was only poorly enhanced by coexpression with the G_o protein. Therefore, a similar set of experiments was performed with a mutant Thr³⁷³Lys _2A-AR that previously has been shown to be constitutively active (Ren et al., 1993). Coexpression of the mutant Thr³⁷³Lys ₂-AR with either a wt G_o or mutant G_o Cys³⁵¹Ile protein clearly enhanced the basal [³⁵S]GTPS response. Both basal responses were attenuated by RX 811059 (10 µM). This effect was largest (54 ± 3%) with the G_o Cys³⁵¹Ile protein and resistant to PTX treatment (20 ng/ml; Fig. 1J). The enhanced constitutive activity of mutant Thr³⁷³Lys _2A-AR was highly dependent on the amount of G_o Cys³⁵¹Ile plasmid expression in CHO-K1 cells as illustrated in Fig. 2. In contrast to transfection with empty plasmid, dose-dependent G_o protein expression was observed in CHO-K1 cells on transfection with 0.3 to 3 µg of G_o Cys³⁵¹Ile plasmid (Fig. 2B). Under these conditions, constitutive Thr³⁷³Lys _2A-AR activity was enhanced by 46 to 114%. Figure 2C illustrates RX 811059 (10 µM)-mediated inhibition of the enhanced basal [³⁵S]GTPS binding response from a receptor amount of 100 fmol/mg protein onward.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   The effects of coexpression of wt 2A-AR and mutant Thr373Lys 2A-AR with rat Go protein and PTX-treatment on [35S]GTPS binding responses in CHO-K1 cells. [35S]GTPS binding responses were performed with 0.5 nM [35S]GTPS either in the absence (basal, ), or presence of 10 µM ()-adrenaline () or 10 µM RX 811059 () as described in Experimental Procedures. A, 2A-AR; B, 2A-AR + PTX (20 ng/ml); C, 2A-AR + wt Go Cys351; D, 2A-AR + wt Go Cys351 + PTX (20 ng/ml); E, 2A-AR + mutant Go Cys351Ile + PTX (20 ng/ml); F, mutant Thr373Lys 2A-AR; G, Thr373Lys 2A-AR + PTX (20 ng/ml); H, Thr373Lys 2A-AR + wt Go Cys351; I, Thr373Lys 2A-AR + wt Go Cys351 + PTX (20 ng/ml); J, Thr373Lys 2A-AR + mutant Go Cys351Ile + PTX (20 ng/ml). The corresponding expression levels of the wt 2A-AR and mutant Thr373Lys 2A-AR were from 5.3 to 6.1 and from 1.9 to 2.8 pmol/mg protein as estimated by [3H]RX 821002 binding. Bar graphs are constructed using mean values ± S.E. of three to eight independent transfection experiments, each one performed in duplicate. The ratio of the RX 811059 versus basal mediated [35S]GTPS binding response was significantly (P < .05) different between A and C or E, and between F and H or J. One-way ANOVA, followed by all pairwise multiple comparison procedures (Tukey's test).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Magnitude of constitutive activity of mutant Thr373Lys 2A-AR by various amounts of mutant receptor and Go Cys351Ile protein expression in CHO-K1 cells. A, the mutant Thr373Lys 2A-AR (3 µg/plate) was coexpressed with the indicated amounts of plasmid containing the mutant Go Cys351Ile gene. Cultures were treated overnight with PTX (20 ng/ml) except those in the absence of the Go Cys351Ile plasmid. [35S]GTPS responses were performed with 0.5 nM [35S]GTPS either in the absence (basal, ), or presence of 10 µM ()-adrenaline (), or 10 µM RX 811059 () as described in Experimental Procedures. Bar graphs are constructed using mean values ± S.E. of three independent transfection experiments, each one performed in duplicate. B, immunological detection of Go protein expression in CHO-K1 cells transfected with either 3 µg empty plasmid (0) or 3, 1 and 0.3 µg of Go Cys351Ile plasmid and treated with PTX (20 ng/ml). Total membrane proteins (200 µg) were analyzed by SDS-PAGE, and immunodetection was performed with a selective Go protein antibody as described in Experimental Procedures. Data are shown from a representative experiment of three independent experiments. Molecular weight markers are indicated in the left margin. C, various amounts of Thr373Lys 2A-AR plasmid (0.01 to 3 µg/plate) were coexpressed with Go Cys351Ile plasmid (0.3 to 3 µg/plate). Cultures were treated with PTX (20 ng/ml) and analyzed for specific [3H]RX 821002 binding and RX 811059 (10 µM)-mediated inhibition of basal [35S]GTPS binding as described in Experimental Procedures. RX 811059 [35S]GTPS binding data were normalized to maximal inhibition (100%) as obtained with 3 µg of Thr373Lys 2A-AR plasmid and 3 µg of Go Cys351Ile plasmid. Hyperbolic curve was constructed (y = yo + ab/G + x) using individual mean values of eight independent experiments.

A comparison between the ₂-AR agonist's maximal [³⁵S]GTPS binding responses as mediated by wt and mutant Thr³⁷³Lys _2A-AR in either the absence or presence of G_o protein is summarized in Fig. 3. Besides UK 14304, none of these ligands attained at the wt _2A-AR protein a maximal [³⁵S]GTPS binding response similar to that of ()-adrenaline. The maximal response of UK 14304 was not significantly different from that of ()-adrenaline, whereas the agonists d-medetomidine, BHT 920, oxymetazoline, and clonidine displayed E_max values between 28 and 39%. SKF 86466 and BRL 44408 were virtually inactive at micromolar concentrations as agonists. The coexpression with a wt G_o protein did not much affect this agonist's pattern of [³⁵S]GTPS binding responses (Fig. 3B). However, after coexpression with a mutant G_o Cys³⁵¹Ile protein, the otherwise partial agonists BHT 920, d-medetomidine, and clonidine acted as agonists with an efficacy similar to that of ()-adrenaline (Fig. 3C). Some intrinsic activity [13 and 8% versus ()-adrenaline] was also obtained with SKF 86466 and BRL 44408. Both ligands displayed even more intrinsic activity at the mutant Thr³⁷³Lys _2A-AR, in particular, on coexpression with a G_o Cys³⁵¹Ile protein [56 and 51% versus ()-adrenaline]. Otherwise, BHT 920, d-medetomidine, oxymetazoline, and clonidine did apparently display an intrinsic activity slightly (P < .05) exceeding that of ()-adrenaline (Fig. 3F). Each of these [³⁵S]GTPS binding responses was dose-dependent; E_max and pEC₅₀ values for the Thr³⁷³Lys _2A-AR with coexpression of a G_o Cys³⁵¹Ile protein are summarized in Table 1.

View larger version (69K): [in this window] [in a new window]   Fig. 3.   Maximal [35S]GTPS responses as mediated by 2-AR agonists at wt 2A-AR and mutant Thr373Lys 2A-AR in either the absence or presence of rat Go protein. [35S]GTPS binding responses were performed with 0.5 nM [35S]GTPS in the presence of ligands' concentration, which displayed a maximal response (1 µM UK 14304, 10 µM BHT 920, 1 µM d-medetomidine, 10 µM oxymetazoline, 10 µM clonidine, 10 µM SKF 86466, and 1 µM BRL 44408). These responses are expressed in percentage versus the respective response obtained with 10 µM ()-adrenaline on subtraction of its basal [35S]GTPS response. A, 2A-AR; B, 2A-AR + wt Go Cys351; C, 2A-AR + mutant Go Cys351Ile + PTX (20 ng/ml); D, mutant Thr373Lys 2A-AR; E, Thr373Lys 2A-AR + wt Go Cys351; F, Thr373Lys 2A-AR + mutant Go Cys351Ile + PTX (20 ng/ml). Bar graphs are constructed using mean values ± S.E. of three to eight independent transfection experiments, each one performed in duplicate. Absolute basal and ()-adrenaline-mediated [35S]GTPS binding values for each of these conditions are shown in Fig. 1.

View this table: [in this window] [in a new window]   TABLE 1 Emax and pEC50 values of 2-AR agonists' [35S]GTPS binding responses mediated by Thr373Lys 2A-AR and Go Cys351Ile protein in CHO-K1 cells Cells were transfected and treated with PTX (20 ng/ml) as described in Experimental Procedures. [35S]GTPS binding responses were performed with 0.5 nM [35S]GTPS. Emax values are expressed in percentage versus the maximal stimulation as obtained with 10 µM ()-adrenaline (188 ± 14%) and correspond to mean values ± S.E. of four to twelve independent experiments. Mean pEC50 values ± S.E. were derived from dose-response curves that were at least three times independently performed.

Analysis of a series putative ₂-AR antagonists at the constitutively active Thr³⁷³Lys _2A-AR on coexpression with a rat G_o Cys³⁵¹Ile protein by concentration [³⁵S]GTPS binding response curves is shown in Fig. 4. The corresponding E_max, pIC₅₀, or pEC₅₀ values are summarized in Table 2 and compared with their respective pK_i values. RX 811059 potently inhibited basal [³⁵S]GTPS binding by 54%; the activity resided in the (+)-enantiomer, which was 2 times more potent, whereas the ()-enantiomer was almost inactive (Fig. 4A). (+)-RX 821002, yohimbine, and RS 15385 yielded maximal inhibition of the basal [³⁵S]GTPS binding with a magnitude almost similar to that of (+)-RX 811059. Fluparoxan and WB 4101 displayed partial inhibition of basal [³⁵S]GTPS binding, whereas dexefaroxan and atipamezole were almost inactive at 10 micromolar on basal [³⁵S]GTPS binding. In contrast, idazoxan yielded stimulation of [³⁵S]GTPS binding, like SKF 86466, with a potency in agreement with its pK_i value for the mutant Thr³⁷³Lys _2A-AR (Fig. 4C). The inhibition of basal [³⁵S]GTPS binding as mediated by (+)-RX 811059 could be blocked by atipamezole (1 µM) in a competitive manner. The antagonist potency (pK_B: 8.73 ± 0.07) of atipamezole fitted with that observed for the antagonism of the UK 14304-mediated [³⁵S]GTPS binding response (pK_B: 8.55 ± 0.04; Fig. 5).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent [35S]GTPS binding responses of putative 2-AR antagonists at the mutant Thr373Lys 2A-AR coexpressed with a rat Go Cys351Ile protein. Cultures were treated overnight with PTX (20 ng/ml) and assayed for [35S]GTPS binding responses as indicated in Experimental Procedures. A, inhibition of basal [35S]GTPS binding by (+)-RX 811059 (), (±)-RX 811059 (), and ()-RX 811059 (); B, inhibition of basal [35S]GTPS binding by (+)-RX 821002 (), RS 15385 (), yohimbine (), WB 4101 (), and fluparoxan (); C, effects of idazoxan (), SKF 86466 (), atipamezole (), and dexefaroxan () on [35S]GTPS binding. Data are presented in percentage versus basal [35S]GTPS binding. Concentration binding curves are constructed using mean values ± S.E. from three to four independent experiments, each one performed in duplicate. Emax, pIC50, or pEC50 values of ligands are summarized in Table 2.

View this table: [in this window] [in a new window]   TABLE 2 Emax, pIC50, or pEC50 values of putative 2-AR antagonists' [35S]GTPS binding responses mediated by Thr373Lys 2A-AR and Go Cys351Ile protein in CHO-K1 cells, and their corresponding pKi values Cells were transfected and treated with PTX (20 ng/ml) as described in Experimental Procedures. [35S]GTPS binding responses were performed with 0.5 nM [35S]GTPS. Emax values are expressed in percentage versus either basal [35S]GTPS binding response (313 ± 37 fmol/mg protein), the maximal inhibition as obtained with (+)-RX 811059 (10 µM, 146 ± 16 fmol/mg protein), or the maximal positive response as obtained with ()-adrenaline (10 µM, 923 ± 134 fmol/mg protein). Data correspond to mean values ± S.E. of a minimum of four independent transfection experiments. pKi values were taken from either Wurch et al. (1999) or performed as described in Experimental Procedures.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Antagonism of UK 14304- and (+)-RX 811059-mediated [35S]GTPS binding responses by atipamezole at the mutant Thr373Lys 2A-AR coexpressed with a rat Go Cys351Ile protein. Cultures were treated overnight with PTX (20 ng/ml) and assayed for [35S]GTPS binding responses as indicated in Experimental Procedures. A, antagonism of UK 14304-mediated [35S]GTPS binding response by 1 µM atipamezole (pKB: 8.73 ± 0.07); B, antagonism of (+)-RX 811059-mediated [35S]GTPS binding response by 1 µM atipamezole (pKB: 8.55 ± 0.04). Responses are expressed versus that obtained with 10 µM ()-adrenaline (A) or 10 µM (+)-RX 811059-mediated inhibition of basal [35S]GTPS binding (B). Concentration binding curves are constructed using mean values ± S.E. from three independent transfection experiments, each one performed in duplicate.

Another set of experiments with (+)-RX 811059 was performed by coexpression of the mutant Thr³⁷³Lys _2A-AR with the PTX-resistant Cys³⁵¹Ile mutants of the G_i1 and G_i3 proteins, and Cys³⁵²Ile mutant of the G_i2 protein instead of a G_o Cys³⁵¹Ile protein. Membrane preparations for [³⁵S]GTPS binding responses as mediated by these various G proteins were selected on the basis of a similar amount of G protein expression as shown in Fig. 6. Analysis of ()-adrenaline-specific saturation [³⁵S]GTPS binding indicated a single class of high-affinity [³⁵S]GTPS binding sites for each of the investigated G proteins, with a slightly higher affinity for the G_o Cys³⁵¹Ile protein. The maximal adrenaline-mediated [³⁵S]GTPS binding capacity for each of these G proteins was in the same range; it varied between 0.67 and 1.73 pmol/mg protein (Fig. 7). Whereas basal [³⁵S]GTPS binding in the presence of each of the G_i proteins was virtually not affected by (+)-RX 811059, it attenuated basal [³⁵S]GTPS binding in case of a G_o Cys³⁵¹Ile protein. Consequently, the maximal [³⁵S]GTPS binding capacity of ()-adrenaline to the G_o Cys³⁵¹Ile protein was enhanced (Fig. 7D). Table 3 summarizes similar (+)-RX 811059 data as mediated by these G proteins for four independent experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Immunological detection of G protein expression in CHO-K1 cells transfected with the Thr373Lys 2A-AR and PTX-resistant forms of Gi/o proteins. CHO-K1 cells were transfected with either 3 µg of empty pCR3.1 plasmid (A), Gi1 Cys351Ile (B), Gi2 Cys352Ile (C), Gi3 Cys351Ile (D), or 0.3 µg of Go Cys351Ile protein (E), and treated with PTX (20 ng/ml). Total membrane proteins (200 µg) were analyzed by SDS-PAGE and immunodetection was performed with a nonselective anti-G protein antibody as described in Experimental Procedures. Molecular weight markers are indicated in the left margin. Quantification of G protein expression over empty plasmid-transfected CHO-K1 cells yielded in gray level units: 4.1, 9.6, 5.1, and 3.6 for, respectively, lanes B, C, D, and E. These data represent one representative experiment of three independent experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Homologous displacement and analysis of saturation [35S]GTPS binding to CHO-K1 cell membranes transfected with the Thr373Lys 2A-AR and PTX-resistant forms of Gi/o proteins. Cells were transfected with either 3 µg of Gi1 Cys351Ile (A), Gi2 Cys352Ile (B), Gi3 Cys351Ile (C), or 0.3 µg of Go Cys351Ile plasmid (D), and treated with PTX (20 ng/ml). Membranes were incubated with 0.5 nM [35S]GTPS, 30 µM GDP, and either without or with 0.1 nM to 3 µM unlabeled GTPS in the absence () or presence () of 10 µM ()-adrenaline. Inset: analysis of ()-adrenaline-specific saturation [35S]GTPS binding () is shown for a representative experiment of three independent experiments. A similar analysis was performed with 10 µM (+)-RX 811059 (/) in D. This was not shown for A, B, and C as (+)-RX 811059-mediated [35S]GTPS binding values were virtually similar to basal [35S]GTPS binding values. High-affinity [35S]GTPS binding constants (pKd) for ()-adrenaline are indicated in the insets.

View this table: [in this window] [in a new window]   TABLE 3 [35S]GTPS binding responses by mutant Thr373Lys 2A-AR co-expressed with PTX-resistant forms of either Gi1, Gi2, Gi3, or Go proteins in CHO-K1 cells Cells were transfected with 3 µg of Thr373Lys 2A-AR and either 3 µg of empty plasmid, 3 µg of Gi1 Cys351Ile, Gi2 Cys352Ile, or Gi3 Cys351Ile plasmid, or 0.3 µg of Go Cys351Ile plasmid to yield a similar amount of G protein expression (see Fig. 6). Cells were treated with PTX (20 ng/ml), except for cells in the absence of recombinant G proteins, as described in Experimental Procedures. The receptor density was estimated with [3H]RX 821002 as described in Experimental Procedures. [35S]GTPS binding responses were performed with 0.5 nM [35S]GTPS. Data represent mean values ± S.E. of four independent transfection experiments, each one performed in duplicate. [35S]GTPS binding data as obtained with (+)-RX 811059 were expressed either in femtomoles per milligram protein or in percentage of the basal [35S]GTPS binding response.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The present study demonstrates that the _2A-AR can be transformed into a constitutively activated form by coexpression with a rat G_o protein. This process could be enhanced by a single amino acid mutation (Thr³⁷³Lys) in the distal part of the third ICL of the _2A-AR. By measuring the agonist-independent activation of a PTX-resistant G_o Cys³⁵¹Ile protein by this mutant _2A-AR, the constitutive activity represented about 21% of the maximal receptor activation as obtained with ()-adrenaline. This mutant receptor has previously been shown to be constitutively active (Ren et al., 1993; Wurch et al., 1999). However, no evidence was provided for blockade of the constitutive activity by inverse agonists, and whether this type of ligand activity can be reversed by neutral antagonists. The present study illustrates that the enhanced basal activity is yet present at a low receptor amount (100 fmol/mg protein) and can be fully blocked by several ligands previously characterized as presumably antagonists. The ligands RX 811059, RX 821002, RS 15385, and yohimbine acted as efficacious inverse agonists with potencies that corresponded with their binding affinities for the _2A-AR. This type of ligand activity could be competitively reversed by a neutral antagonist, such as atipamezole, with properties similar to its reversal of agonist-mediated (positive) responses. Moreover, the described model system demonstrates a spectrum of ligand-mediated intrinsic activities that allow a clear distinction between various degrees of either inverse agonism or positive agonism, and silent neutral antagonists.

Several features were apparent by coexpression of the _2A-AR with a G_o protein. The magnitude of the ()-adrenaline [³⁵S]GTPS binding response was enhanced about 10-fold by coexpression with a wt G_o protein, and this magnitude was not differently affected by a mutant G_o Cys³⁵¹Ile protein and/or Thr³⁷³Lys _2A-AR. Besides UK 14304, maximal responses of the agonists investigated at wt _2A-AR were different from that of ()-adrenaline. Similar data have been reported at wt _2A-AR in recombinant HEK 293 and CHO cells using [³⁵S]GTPS and Ca²⁺ responses (Jasper et al., 1998; Kukkonen et al., 1998). Less differentiation between the agonists' maximal responses was found by measuring ₂-AR agonist modulation of [³⁵S]GTPS binding to G proteins in human platelet membranes (Gessi et al., 1999). In the present study, the partial agonists BHT 920, d-medetomidine, and clonidine were converted at the wt _2A-AR into highly efficacious agonists by the presence of a mutant G_o Cys³⁵¹Ile protein. This observation underlines the role of the amino acid at position 351 in the G_o protein. This amino acid position has been shown to be involved in the magnitude of agonist-mediated responses in addition to its key role in PTX resistance (Dupuis et al., 1999). The hydrophobic amino acid isoleucine instead of a cysteine at the position 351 of the G_o protein increases the magnitude of responses mediated by partial agonists. Similar data have also been reported for the G_i1 protein and porcine _2A-AR in Cos-7 cells (Bahia et al., 1998; Jackson et al., 1999). An increase in the magnitude of the intrinsic activity of partial agonists was also apparent at the mutant Thr³⁷³Lys _2A-AR; this effect was enhanced by coexpression with a G_o protein and more importantly with a mutant G_o Cys³⁵¹Ile protein. Wurch et al. (1999) also observed an increased efficacy for partial agonists by following the mutant Thr³⁷³Lys _2A-AR by measuring the stimulation of inositol phosphates in the presence of a G₁₅ protein. The wt as well as the mutant Thr³⁷³Lys _2A-AR exhibited (in our experimental conditions in the absence of recombinant G proteins) no measurable constitutive activity although they were responsive to ()-adrenaline stimulation. This contrasts with data obtained in transfected HEK 293 cells (Ren et al., 1993); their basal activity represented 8 and 82% of UK 14304-dependent inhibition of stimulated cAMP formation for the wt and Thr³⁷³Lys _2A-AR, respectively. The basal activity of the wt ₂-AR in CHO-K1 cells was slightly increased to 3% of maximal agonist-dependent receptor activation by coexpression with either a wt G_o or mutant G_o Cys³⁵¹Ile protein. Furthermore, the increase of the basal activity at the mutant Thr³⁷³Lys _2A-AR was highly dependent on the coexpression with G_o proteins; 6 and 21% of maximal receptor activation was observed with a wt G_o and mutant G_o Cys³⁵¹Ile protein, respectively. Constitutive _2A-AR activation in our study was observed mainly with a G_o Cys³⁵¹Ile protein. It is not clear why the mutant Cys³⁵²Ile and Cys³⁵¹Ile forms of, respectively, the G_i2 and G_i3 protein did not yield constitutive _2A-AR activity. The possibility that coupling of the _2A-AR to different G proteins in different cells would be causing distinct pharmacological properties (i.e., silent antagonism versus inverse agonism) for a given ligand cannot be excluded.

In contrast to assessment of _2A-AR antagonist efficacy with a GTPase assay at wt _2A-AR, where none of the investigated ₂ AR antagonists acted as an inverse agonist (Virolainen et al., 1997), we here did observe inverse agonist activity at _2A-AR. The magnitude of this activity was more pronounced at the mutant Thr³⁷³Lys _2A-AR. RX 811059, RX 821002, RS 15385, and yohimbine displayed maximal inhibition of constitutive activity at potencies relevant to their binding affinities. Fluparoxan and WB 4101 acted as partial inverse agonists. Tian et al. (1994) also reported on ₂-AR antagonists that reduced basal G protein activation by the recombinant _2D-AR in PC12 cells with the following rank order of maximal effectiveness: yohimbine = phentolamine > idazoxan = rauwolscine > WB 4101. Rauwolscine and atipamezole have also been shown to display inverse agonist activity at endogenous _2A-AR in human erythroleukemia cells (HEL 92.1.7) by following both Ca²⁺ elevation and cAMP production (Jansson et al., 1998). This effect was less marked for idazoxan (Jansson et al., 1998). In the present study, idazoxan and atipamezole did not display inverse agonist activity. Atipamezole as well as dexefaroxan acted as essentially neutral antagonists, whereas idazoxan, BRL 44408, and SKF 86466 displayed partial agonist properties. The stereoselective interaction of the (+)-enantiomer of RX 811059 compared with its inactive ()-enantiomer emphasizes the specificity of the blockade of constitutive activity by ₂-AR ligands in our study. The reversal of this effect by atipamezole in a competitive manner, and at a potency similar to that observed for its blockade of the agonist UK 14304, confirms that the interaction of the observed inverse agonists with the mutant Thr³⁷³Lys _2A-AR is indeed transduced by ₂-AR. The data on idazoxan, BRL 44408, and SKF 86466 are consistent with the finding (Wurch et al., 1999) that these ligands also acted as partial agonists at the mutant Thr³⁷³Lys _2A-AR in the presence of a G₁₅ protein. However, dexefaroxan and atipamezole, which appeared also partial agonists at the mutant _2A-AR in the presence of a G₁₅ protein (Wurch et al., 1999), seem to be neutral antagonists by coexpression with a G_o Cys³⁵¹Ile protein. It is possible, therefore, that certain pharmacological differences in intrinsic activity for some of these ligands may be due to _2A-AR interactions with selective G protein subunits. The ligands RS 15385, RX 811059, and WB 4101 characterized as inverse agonists at the Thr³⁷³Lys _2A-AR showed less binding affinity compared with the wt _2A-AR (Wurch et al., 1999) in line with what would be expected for inverse agonists (Westphal and Sanders-Bush, 1994). Although these binding shifts were significant, they were weak. The resolution of the herein described [³⁵S]GTPS binding responses is higher and therefore more suitable to monitor intrinsic activity of ₂-adrenergic ligands.

Although the physiological implications of a constitutively active mutant _2A-AR remain unclear, Neubig et al. (1988) concluded that approximately 30% of platelet ₂-AR exist in a precoupled state. The levels of G protein expression are regulated in vivo (Burstein et al., 1997) and can possibly operate to affect _2A-AR functioning. Up-regulation of G protein levels may provide increased sensitivity to signaling and/or enhance alternative signaling pathways (see Burstein et al., 1997). In the present study, enhanced sensitivity to [³⁵S]GTPS binding responses was observed by coexpression of both the wt _2A-AR and the activating mutant Thr³⁷³Lys _2A-AR with G_o proteins. The observed spectrum of ligands' intrinsic activities in this specified _2A-AR system suggests that several common antagonists behave as either partial agonists or (partial) inverse agonists. Ligands may probably demonstrate distinct pharmacological effects, depending on which G proteins and effector pathways are involved. Therefore, it cannot be excluded that a ligand that behaves as an inverse agonist at a mutant receptor with a specific G protein may as well behave as a neutral antagonist at native receptors. Future studies are needed to establish whether the constitutively active mutant Thr³⁷³Lys _2A-AR mimics a transient state of the native mechanism of _2A receptor activation.