G-protein activation by putative antagonists at mutant Thr³⁷³Lys α_2A adrenergic receptors

Abstract

1. Replacement of a threonine by a lysine at position 373 in the C-terminal portion of the third intracellular loop of the human α_2A-adrenergic receptor (α_2A AR) has been reported to generate a constitutively active mutant receptor in analogy with similar mutations in the α_1B and β₂ AR (Ren et al., 1993). In the present study, the mutant Thr³⁷³Lys α_2A AR receptor was investigated by measuring the formation of inositol phosphates in either the absence or presence of mouse G_α15 protein in Cos-7 cells.

2. Increased affinity, potency and/or efficacy for the agonists [(−)-adrenaline, UK 14304, clonidine, guanabenz and oxymetazoline] was observed, consistent with a precoupled mutant α_2A AR: G-protein state. The basal inositol phosphates response was similar at the wild-type (wt) and mutant α_2A AR, but was enhanced at the mutant α_2A AR upon co-expression with the mouse G_α15 protein. This enhanced response could not be attenuated in the presence of any of the tested α₂ AR antagonists (10 μM), suggesting that inverse agonist activity did not occur at the mutant α_2A AR.

3. Ligands that so far have been identified as antagonists at the wt α_2A AR demonstrated either no intrinsic activity (MK 912, WB 4101, RS 15385, RX 811059 and RX 821002) or positive efficacy [E_max, % vs. 1 μM UK 14304: dexefaroxan (27±7), idazoxan (34±9), atipamezole (27±4), BRL 44408 (59±5) and SKF 86466 (54±9)] at the mutant α_2A AR, but only in the presence of the mouse G_α15 protein. The ligand potencies corresponded with their respective pK_i values at the mutant α_2A AR receptor.

4. The partial agonist effect of SKF 86466 was resistant to pertussis toxin treatment (100 ng ml⁻¹) and not affected by co-expression of the rat G_αi1 protein. It was virtually absent in the presence of 10 μM RS 15385. SKF 86466 was without intrinsic activity upon co-expression of the mouse G_αq protein.

5. Some putative α₂ AR antagonists exerted a partial agonist activity that was highly dependent on the presence of specific G-protein α-subunits, suggesting that these ligands cause selective G-protein activation at the mutant α_2A AR.

Introduction

α₂-Adrenergic receptors (α₂ AR) are implicated in the control of noradrenergic neurotransmission in the brain and modulate a number of physiological processes peripherally, including aggregation of platelets, release of insulin from the pancreas, and inhibition of lipolysis (Timmermans et al., 1990; Szabadi & Bradshaw, 1996). Pharmacological results including ligand-binding data suggest that α₂ AR are not a homogeneous class of receptors. At least three distinct genes encoding α₂ AR subtypes (α_2C10/A AR, α_2C2/B AR, α_2C4/C AR) have been isolated in man and characterized in heterologous expression systems (Regan et al., 1988; Fraser et al., 1989; Lomasney et al., 1990). The α₂ AR belong to the superfamily of G-protein-coupled receptors, mediating their effects through the activation of heterotrimeric G-proteins. The α_2A AR, via its interaction with the pertussis toxin-sensitive G_i/G_o subclass of G-proteins, modulates multiple effector systems, including inhibition of adenylate cyclase, opening of Ca²⁺ channels and activation of K⁺ channels (Lakhlani et al., 1996). Eason et al. (1992) and Federman et al. (1992) have demonstrated paradoxical α₂ AR-mediated increases in cyclic AMP level via coupling to G_s. The degree of G_s coupling depends initially on the agonist's structural features, and ligands that act as full agonists for G_i coupling are not necessarily full agonists for G_s coupling (Eason et al., 1994). The α₂ AR may also couple to other intracellular pathways involving Na⁺/H⁺ exchange, phospholipase A₂ and phospholipase C activation (Limbird, 1988; Cotecchia et al., 1990).

Mutagenesis studies of several G-protein-coupled receptors indicate that the coupling to specific G-proteins is predominantly mediated by the intracellular loops (Dalman and Neubig, 1991). The second and third intracellular loops are clearly involved in α_2A AR:G-protein interactions (Dalman & Neubig, 1991; Näsman et al., 1997). Replacement of a threonine by a lysine or a cysteine at position 373 in the BBXXB motif (where B represents a basic residue and X a non-basic residue) of the third intracellular loop of the human α_2a AR, results in a constitutively active mutant receptor in analogy with mutations affecting the same region in the α_1B AR and β₂ AR (Ren et al., 1993). The mutant α_2A AR demonstrates an increased affinity for the agonist UK 14304 associated with an agonist-independent inhibition of adenylyl cyclase in HEK 293 cells. The functional consequences of this mutant receptor have also been studied in a stably transfected 3T3F442A preadipose cell line (Bétuing et al., 1997). Similar (in the absence of agonist) and amplified (in the presence of agonist) cellular responses were measured at the preadipocyte stage indicating constitutive activation of the mutant receptor. However, the α_2A AR-dependent antilipolysis at the adipocyte stage was neither reproduced nor amplified by the mutant receptor. Conversely, down-regulation of G_αi-proteins and an increase in the maximal response to lipolytic agents have been observed in cell lines expressing the mutant α_2A AR (Bétuing et al., 1997). Other than the agonist UK 14304, very few α₂ AR ligands have been evaluated at this mutant α₂ AR. Also, it has yet to be determined whether the inhibition of constitutive activity by α₂ AR inverse agonists can be reversed. Tian et al. (1994) demonstrated that some α₂ AR antagonists display inverse agonist activity at precoupled wild-type (wt) rat α_2D AR in recombinant PC12 cells; the rank order of maximal effectiveness differed from that of receptor binding affinities.

Our studies of constitutively active mutant α_2A AR provide apparently opposite results for ligands that so far have been characterized as α₂ AR antagonists. By measuring the positive coupling of the mutant receptor to the formation of inositol phosphates, partial agonist instead of either neutral antagonist or inverse agonist activity was observed for a series of α₂ AR ligands at the mutant Thr³⁷³Lys α_2A AR in the presence of mouse G_α15 protein. Experiments were performed in parallel with the wt α_2A AR, and were compared to receptor activity as measured by inhibition of stimulated cyclic AMP formation in stably transfected C6-glial cells. We found an increased affinity of the mutant α_2A AR receptor for diverse agonists, whereas no functional evidence could be observed for inverse agonist activity. In contrast, a number of putative α₂ AR antagonists displayed positive efficacy and behaved as partial agonists at the mutant α_2A AR in the presence of mouse G_α15 protein. The partial agonist activity is discussed with regard to the receptor binding properties, G-protein coupling and partial agonist activity of antagonists at other G-protein-coupled receptor mutants.

Methods

Cloning of human wt and mutant Thr³⁷³Lys α_2A-adrenergic receptors, rat G_αi protein, and mouse G_α15 and G_q protein genes

Genes were cloned by PCR using primers designed at the start and stop codons of the respective nucleotide sequences (Fraser et al., 1989; Jones & Reed, 1987; Strathmann & Simon, 1990; Wilkie et al., 1991). The PCR mixture (50 μl) consisted of 250 ng of reverse-transcribed poly(A⁺) RNA from either human, mouse or rat total brain, or 1 μg of mouse genomic DNA, dNTP (350 μM), primers (400 nM), DMSO (10% for the α_2A AR gene only) and 1 μl of Expand long template DNA polymerase mix in PCR buffer [mM: (NH₄)₂SO₄, 16; MgCl₂, 1.75; Tris-HCl, 50 (pH 9.2)]. The PCR program consisted of 30 repetitive cycles with a strand separation step at 96°C for 30 s, an annealing step at 60°C for 1 min and an elongation step at 68°C for 1.5 min. The amplification cycle was preceded by a hot start step at 96°C for 5 min in which the DNA polymerase mix was omitted. PCR fragments were purified by 1% agarose gel electrophoresis, purified using a Geneclean II kit and subsequently cloned into 50 ng of a pCR3.1 vector. Sequencing was performed automatically on an ABI Prism 310 Genetic analyser using a dichloro-rhodamine terminator cycle sequencing kit. Nucleotide sequences were identical to the published sequences. Site-directed mutagenesis of the α_2A AR was performed using a modified overlap extension technique based on PCR (Wurch et al., 1998) using appropriate complementary primers carrying the Thr (ACG codon) to Lys (AAA codon) mutation. The obtained PCR fragment contained the presence of the mutated codon: Thr³⁷³Lys.

Transient co-transfection of α_2A-adrenergic receptors and various G-protein genes in Cos-7 cells

Ten micrograms of plasmid containing either wt α_2A AR or mutant Thr₃₇₃Lys α_2A AR and 10 μg of the indicated G-protein plasmid were co-transfected with 5×10⁶ exponentially-growing Cos-7 cells using a Bio-Rad electroporator (250 mV, 250 μF) in DMEM supplemented with 1% DMSO. Cultures were grown in 24-well tissue culture plates with DMEM supplemented with 10% foetal calf serum, washed and treated after 24 h with 2% dialysed foetal calf serum and [2-[³H](N)] myoinositol (2 μCi per well) for a period of 24–48 h before functional receptor analysis. For radioligand experiments, cells were grown for 48 h in DMEM supplemented with 10% foetal calf serum.

Stable transfection of wt α_2A-adrenergic receptor gene in C6-glial cells

C6-glial cells were transfected as described above with 10 μg pCR3.1/human α_2A AR and were subcultured after 48 h in DMEM in the presence of 1.25 mg geneticin ml⁻¹. After about 10 days, individual clones were isolated and subcultured as described (Pauwels et al., 1996). Intact cells were assayed for [³H] (1,4-[6,7(n)-[³H]-benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride (RX 821002) binding and inhibition of forskolin-induced cyclic AMP formation. Results presented in this study were obtained with a clone that expresses 1.3 pmol mg⁻¹ protein of [³H] RX 821002 binding sites.

Receptor binding assay for α_2A-adrenergic receptors

Membrane preparations were prepared in Tris-HCl pH 7.6 (50 mM) as described (Pauwels et al., 1996). Binding assays were performed with [³H] RX 821002 (2 nM). Incubation mixtures consisted of 0.4 ml of cell membranes (6 μg protein), 0.05 ml of radioligand and 0.05 ml of displacing compound or phentolamine (10 μM) to determine nonspecific binding. K_i values were calculated according to the equation K_i=IC₅₀/(1 + C/K_d) with C representing the concentration and K_d the equilibrium dissociation constant of the [³H] ligand. pK_d values were obtained from saturation binding studies performed as previously described (Pauwels et al., 1996).

α_2A-adrenergic receptor-mediated inhibition of forskolin-induced cyclic AMP formation

Inhibition of forskolin (100 μM)-stimulated cyclic AMP formation in the C6-glial cell line stably expressing the wt α_2A AR was measured as previously described (Pauwels et al., 1996). The cellular cyclic AMP content was assayed using a radioimmunoassay kit. Inhibition of forskolin-induced cyclic AMP formation was expressed as the percentage of that obtained with UK 14304 (1 μM). EC₅₀ values (concentrations of the compounds producing 50% inhibition of their own maximal inhibition) were derived. Antagonists were added 15 min prior to agonists. 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 values of agonist concentration measured in the absence and presence of antagonist, respectively.

α_2A-adrenergic receptor-mediated phosphatidyl inositol phosphates hydrolysis

Cos-7 cells were washed with 1.0 ml CSS, and were incubated for 1 h at 37°C with 1.0 ml CSS containing LiCl (10 mM) either in the presence or absence of agonist. The reaction was stopped by the addition of 0.1 ml 1 N HClO₄ and neutralized prior to the addition of 0.25 ml of sample buffer (mM: Na₂B₄O₇, 30; EDTA, 3). The fraction of total [³H]-inositol phosphates produced after agonist stimulation was separated from free [³H]-myoinositol and [³H]-glycerophosphoinositol by chromatography on a strong anion exchange AG1-X8 resin equilibrated in water. A 1.3 ml aliquot of the cell lysate was loaded on a column as described (Pauwels et al., 1990). After two washes with 10.0 ml water, the glycerophosphoinositol was washed out in 15.0 ml of [mM: Na₂B₄O₇, 5; CH₅NO₂, 60]. The total [³H]-inositol phosphates were eluted with 2.0 ml of [M: CH₅NO₂, 2; HCOOH, 0.1]. The radioactivity was extracted in 16 ml Ultima Gold XR and counted with a Tri-Carb 2500 liquid scintillation counter. Maximal stimulation of the total production of inositol phosphates was defined in the presence of UK 14304 (1 μM). EC₅₀ values were defined as the concentrations of compounds producing 50% of their own maximal response. Antagonist studies were performed by pre-incubating the cells with the antagonist for 10 min before the addition of the agonist. pK_B values were calculated as described above. In the case of pertussis toxin treatment (100 ng ml⁻¹), cells were pretreated for 16 h before inositol phosphates were measured.

Statistical analysis

Statistical comparison of ligand binding affinities, agonist and antagonist potencies, and basal and UK 14304-mediated responses between wt α_2A and mutant Thr³⁷³Lys α_2A AR were made using the two-tailed Student's t-test.

Materials

Cos-7 and C6-glial cells were obtained from ATCC (Rockville, U.S.A.). The ABI Prism 310 Genetic analyzer and the dichloro-rhodamine terminator cycle sequencing kit were from Perkin Elmer (Forster City, U.S.A.). The pCR3.1 vector was from InVitrogen (San Diego, U.S.A.). The Geneclean II kit was purchased from Bio 101 Inc. (La Jolla, U.S.A.). The Expand long template PCR kit was from Boehringer Mannheim (Mannheim, Germany). The AG1-X8 resin was from Bio-Rad Laboratories (Hercules, U.S.A.). [³H] RX 821002 (50.6 Ci mmol⁻¹) and [2-[³H](N)]-myoinositol (20 Ci mmol⁻¹) were obtained respectively from Amersham (Les Ulis, France) and New England Nuclear (Les Ulis, France). 2-(2,6-dimethoxyphenoxyethyl)aminoethyl-1,4-benzodioxane hydrochloride (WB 4101) was from RBI (Natick, U.S.A.). Clonidine, (−)-adrenaline, oxymetazoline and guanabenz were from Sigma. (±)-2[(4,5-dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole (BRL 44408) and 6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine (SKF 86466) were from Smith Kline Beecham (Herts, U.K.). (2S,12bS) 1′,3′-dimethylspiro (1,3,4,5′,6,6′,7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinolizine)-2,4′-pyrimidin-2′-one (MK 912) was from Merck (Essex, U.K.). 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline tartrate (UK 14304), dexefaroxan, (−)-efaroxan, 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 2-[2-(2-ethoxy-1,4-benzodioxan-2-yl)-1-imidazoline (RX 811059) were prepared intra-muros. Idazoxan and (1,4-benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride (RX 821002) were from Reckitt and Colman (Kingston-upon-Hull, U.K.).

Results

Binding properties of α₂-adrenergic ligands at wt α_2A and mutant Thr³⁷³Lys α_2A-adrenergic receptors

Saturation binding experiments with [³H] RX 821002 in membrane preparations of Cos-7 cells transfected with either a wt α_2A or mutant Thr³⁷³Lys α_2A AR gene indicated the presence of a single high affinity binding site with similar affinity for [³H] RX 821002 (pK_d: 8.98±0.05 and 8.84±0.02 for wt α_2A and mutant α_2A AR receptors, respectively). The maximal binding capacity for [³H] RX 821002 in these transfected cells was similar (4.0±0.4 and 4.5±1.0 pmol mg⁻¹ protein, for the wt and mutant α_2A AR, respectively). A series of eleven α₂-adrenergic antagonists and five agonists were characterized for inhibition of [³H] RX 821002 binding to wt α_2A and mutant α_2A AR (Table 1). The mutation significantly decreased (1.8–2.9 fold) the affinity of RS 15385, RX 811059 and WB 4101, and left unchanged that of the other antagonists. In contrast, each of the agonists appeared to be significantly more potent at the mutant α_2A AR; the following rank order of increased affinity was obtained (fold-increase): (−)-adrenaline (20) > UK 14304 (8.7) > clonidine (3.5) = guanabenz (3.2) > oxymetazoline (1.9) (Table 1). Co-expression of either the wt α_2A or mutant Thr³⁷³Lys α_2A AR with the mouse G_α15 protein enhanced the maximal binding capacity for [³H] RX 821002 without affecting the affinity constant for [³H] RX 821002. Binding affinities of the ligands tested were not affected upon co-expression with mouse G_α15 protein or co-incubation with GTP (100 μM) (not shown).

Table 1.

pK_i values of α₂-adrenergic ligands at human wt α_2A and mutant Thr³⁷³Lys α_2A AR in transfected Cos-7 cells

Intrinsic activity of α₂-adrenergic ligands at wt α_2A and mutant Thr³⁷³Lys α_2A-adrenergic receptors

In contrast to nontransfected Cos-7 cells, UK 14304 produced a concentration-dependent stimulation of inositol phosphates formation in cells transfected with either wt α_2A or mutant Thr³⁷³Lys α_2A AR. The basal activity as well as the maximal response to UK 14304 were the highest with the mutant Thr³⁷³Lys α_2A AR in the presence of mouse G_α15 protein (Table 2). Otherwise, for the wt α_2A AR, the basal and maximal responses to UK 14304 were similar in the presence and absence of mouse G_α15 protein. The UK 14304-mediated responses are likely to occur via different G-protein subclasses as they could be differentially blocked by pretreatment of the cells with pertussis toxin. The responses at wt α_2A and mutant Thr³⁷³Lys α_2A AR were respectively blocked by 84 and 67% in the presence of pertussis toxin but in the absence of mouse G_α15 protein. In contrast, the UK 14304 responses were less affected upon pertussis toxin treatment in the presence of mouse G_α15 protein, although this effect was variable between experiments (Table 2). Further studies were conducted unless otherwise specified in the presence of mouse G_α15 protein. UK 14304 and clonidine stimulated the production of inositol phosphates to the same magnitude as (−)-adrenaline at the wt α_2A AR, whereas guanabenz and oxymetazoline were less active (73 and 51%, respectively; Figure 1). The corresponding potencies and E_max values are summarized in Table 3, and are compared with the respective agonist potencies as measured by inhibition of 100 μM forskolin-stimulated cyclic AMP formation in C6-glial cells stably transfected with the wt α_2A AR, and stimulation of the production of inositol phosphates at mutant Thr³⁷³Lys α_2A AR in Cos-7 cells. Agonist potencies were systematically lower in the cyclic AMP assay, in particular for (−)-adrenaline (21 fold) in the presence of propranolol (1 μM) (to block β AR). This may be due to the lower levels of receptor expression in C6-glial cells (1.3 pmol mg⁻¹ protein) versus Cos-7 cells (4.0–4.5 pmol mg⁻¹ protein). The cyclic AMP assay with the C6-glial cell line appeared to be also less sensitive in differentiating between the maximal responses of the agonists. Nonetheless, the E_max values of guanabenz and oxymetazoline were 24 and 20% inferior to that of UK 14304. Potencies for each of the agonists in the inositol phosphates assay for the mutant Thr³⁷³Lys α_2A were significantly greater than at wt α_2A AR, and their maximal responses were similar to that of UK 14304 (Figure 1 and Table 3).

Table 2.

Comparison between inositol phosphates formation in Cos-7 cells transfected with either wt α_2A or mutant Thr³⁷³Lys α₂ AR in the absence or presence of mouse G_α15 protein and blockade by pertussis toxin

Figure 1.

Stimulation of inositol phosphates formation and inhibition of forskolin-induced cyclic AMP formation by α₂ AR agonists in transfected cells expressing either wt α_2A AR or mutant Thr³⁷³Lys α_2A AR. Stimulation of agonist-stimulated inositol phosphates formation in Cos-7 cells expressing either wt α_2A AR or mutant Thr³⁷³Lys α_2A AR upon co-expression of mouse G_α15, and agonist-mediated inhibition of 100 μM forskolin-induced cyclic AMP formation in C6-glial cells expressing wt α_2A AR were measured as described in Methods. Representative dose-response curves are shown from 3–4 independent experiments. pEC₅₀ and E_max values are summarized in Table 3.

Table 3.

pEC₅₀ and E_max values of α-adrenergic agonists for stimulating inositol phosphates formation in the presence of mouse G_α15 protein, or for inhibiting forskolin-induced cyclic AMP formation in transfected cells expressing either wt α_2A or mutant Thr³⁷³Lys α_2A AR

Table 4 summarizes the intrinsic activities of the α₂-adrenergic antagonists as measured at wt α_2A and mutant Thr³⁷³Lys α_2A AR. Two distinct subgroups were revealed within the antagonists by measuring their stimulation of inositol phosphates at the mutant Thr³⁷³Lys α_2A AR. One group consisted of RS 15385, RX 811059, RX 821002 and WB 4101 which displayed neither positive nor negative efficacy at mutant and wt α_2A AR. The other group consisted of atipamezole, dexefaroxan, SKF 86466, idazoxan and BRL 44408 which demonstrated between +27 to +59% stimulation of inositol phosphates (versus 1 μM of UK 14304) at the mutant α_2A AR. These ligands were apparently free of intrinsic activity (−1.3 to +13.0% versus 1 μM of UK 14304) at the wt α_2A AR receptor. The observed potencies for the partial agonist activities of these ligands corresponded with their respective pK_i values at the mutant α_2A AR (Tables 1 and 4). The partial intrinsic activity of these ligands was attenuated or absent in the presence of RS 15385 (10 μM, Figure 2). The partial agonist effect was also resistant to pertussis toxin treatment and virtually absent without expression of the mouse G_α15 protein. This is shown for SKF 86466 when compared to RS 15385 in Table 5.

Table 4.

Intrinsic activity of antagonists at wt α_2A and mutant Thr³⁷³Lys α_2A AR in the presence of mouse G_α15 protein in Cos-7 cells

Figure 2.

Intrinsic activity of putative α_2A AR antagonists at mutant THr³⁷³Lys α_2A AR upon co-expression of mouse G_α15 protein. Stimulation of inositol phosphates formation by putative α_2A AR antagonists in Cos-7 cells expressing mutant Thr³⁷³Lys α_2A AR and mouse G_α15 protein, in either the absence or presence of RS 15385 (10 μM), was measured as described in Methods. Dose-response curves were constructed using mean±s.e.mean of five independent experiments. pEC₅₀ values are summarized in Table 4.

Table 5.

Effects of pertussis toxin treatment and mouse G_α15 protein on the intrinsic activity of SKF 86466 and RS 15385 at mutant Thr³⁷³Lys α_2A AR in Cos-7 cells

In order to demonstrate that the partial agonist effect of SKF 86466 is selective for the mouse G_α15 protein, further experiments were performed by co-expression of the mutant α_2A AR with either mouse G_αq protein or mouse G_α15 plus rat G_αi1 protein. The data summarized in Table 6 illustrate that SKF 86466 (1 μM) was without intrinsic activity upon co-expression of the mouse G_αq protein. The partial agonist effect of SKF 86466 was not affected by co-expression of the rat G_αi1 and the mouse G_α15 proteins. SKF 86466 and RS 15385 did not display intrinsic activity at the wt α_2A AR by co-expression of mouse G_αq or mouse G_α15 + rat G_αi1 proteins (not shown).

Table 6.

Effects of various G-protein subunits on the intrinsic activity of SKF 86466 and RS 15385 at mutant Thr³⁷³Lys α_2A AR in Cos-7 cells

Blockade of UK 14304-mediated responses by α₂-adrenergic antagonists at wt α_2A and mutant Thr³⁷³Lys α_2A-adrenergic receptors

UK 14304 was selected as a highly efficacious agonist to characterize the potencies of the antagonists at either wt α_2A and mutant Thr³⁷³Lys α_2A AR as measured both by stimulation of inositol phosphates in the presence of mouse G_α15 protein (Figure 3), and by inhibition of stimulated cyclic AMP formation at wt α_2A AR (Table 7). In both assay systems, antagonists (1 μM) inhibited the UK 14304-mediated response at the wt α_2A AR. (−)-Efaroxan (1 μM) did not affect the UK 14304-mediated response. The calculated pK_B values (Table 7) for antagonism of the UK 14304-mediated stimulation of inositol phosphates were similar to their corresponding pK_i values. The pK_B values obtained in the cyclic AMP assay were inferior compared to their pK_i values and the pK_B values in the inositol phosphates assay. Partial blockade of the UK 14304-mediated response at the mutant Thr³⁷³Lys α_2A AR was obtained with atipamezole, dexefaroxan, SKF 86466, idazoxan and BRL 44408 (Figure 3). In contrast, RS 15385, RX 811059, RX 821002 and WB 4101 antagonized the UK 14304-mediated inositol phosphates response at the mutant α_2A AR in a manner that appeared competitive (Figure 3) and consistent with their lack of intrinsic activity. The antagonist potencies were inferior (2–7 fold) to their respective pK_i values, and the pK_B values (5–13 fold) at the wt α_2A AR (Table 7).

Figure 3.

Antagonism of UK 14304-mediated stimulation of inositol phosphates formation by α_2A AR antagonists in transfected Cos-7 cells expressing either wt α_2A AR or mutant Thr³⁷³Lys α_2A AR and mouse G_α15 protein. Stimulation of UK 14304-mediated inositol phosphates formation in Cos-7 cells upon co-expression of mouse G_α15 was measured in either the absence and presence of 1 μM antagonist as described in Methods. Representative curves are shown from 3–5 independent experiments. pK_B values are summarized in Table 6. In each panel, the dotted lines represent the UK 14304-mediated dose-responses for stimulation of inositol phosphates at wt α_2A AR and mutant Thr³⁷³Lys α_2A AR, as shown in the upper left-hand panel for RS 15385.

Table 7.

Antagonist potencies of α₂-adrenergic ligands against UK 14304-mediated stimulation of inositol phosphates formation in the presence of mouse G_α15 protein or inhibition of forskolin-induced cyclic AMP formation in transfected cells expressing either wt α_2A or mutant Thr³⁷³Lys α_2A AR

Discussion

The present study compares the intrinsic activities of α₂ AR ligands at the wt α_2A and mutant Thr³⁷³Lys α_2A AR. This receptor mutant has previously been reported to be constitutively active in HEK 293 cells as well as in a 3T3F442A preadipose cell line (Ren et al., 1993; Bétuing et al., 1997). We also found evidence for constitutive activity at the mutated α_2A AR. Enhanced affinity, potency and/or efficacy for the agonists was observed in line with a precoupled mutant α_2A AR:G-protein state. The basal inositol phosphates response was also enhanced but only upon co-expression of the mutant α_2A AR with the mouse G_α15 protein. However, this response could not be attenuated in the presence of any of the tested antagonists. Indeed, ligands previously characterized as antagonists at the wt α_2A AR demonstrated either no intrinsic activity or positive efficacy at the mutant α_2A AR upon co-expression with the mouse G_α15 protein. This observation suggests that some of these ligands may actually possess a partial agonist activity at the wt α_2A AR receptor, which is augmented by the facilitating Thr³⁷³Lys mutation. Importantly, this partial agonist effect is dependent on the presence of specific G-protein α-subunits. In particular, SKF 86466 and BRL 84408 displayed in the presence of mouse G_α15 protein, an intrinsic activity with maximal responses similar to that of oxymetazoline at the wt α_2A AR. The partial agonist effect was not attenuated by the co-expression of the rat G_αi1 protein, and was not observed upon co-expression of the mouse G_αq protein.

The partial agonists did not display modifications in their binding affinities for the mutant α_2A AR. The ligands characterized as antagonists at both the wt α_2A and mutant α_2A AR showed less affinity at the mutant α_2A AR, in line with what would be expected for inverse agonists (Westphal & Sanders-Bush, 1994). However, no direct functional evidence was obtained for the occurrence of such inverse agonist activity. The α₂ AR antagonists appear under the present experimental conditions to behave as either neutral antagonists or partial agonists at the mutant α_2A AR, depending on the co-expressed G-protein α-subunit.

Specificity of the α_2A AR-mediated inositol phosphates responses in Cos-7 cells

The following findings suggest that the observed inositol phosphates responses in transfected Cos-7 cells are mediated by stimulation of a human α_2A AR: (i) the absence of a UK 14304-mediated inositol phosphates response in the non-transfected cell type together with a lack of specific [³H] RX 821002 binding; (ii) the observed agonist stimulation of inositol phosphates at wt α_2A AR which is consistent with the agonist data on inhibition of forskolin-stimulated cyclic AMP formation in C6-glial cells; (iii) the competitive blockade of UK 14304-mediated responses by the various antagonists at the wt α_2A AR, and in particular the rank order of potency of dexefaroxan and its inactive enantiomer (−)-efaroxan, at concentrations similar to their binding affinities; and (iv) the correlation of the binding affinities for UK 14304 at the wt α_2A and mutant Thr³⁷³Lys α_2A AR in membrane preparations of Cos-7 cells with those measured in transfected HEK 293 cells (Ren et al., 1993). This further extends the observation that α_2A AR may mediate multiple second messenger pathways, including the stimulation of polyphosphoinositide hydrolysis (Cotecchia et al., 1990). The ligand binding characteristics at the wt α_2A AR in either the absence or the presence of mouse G_α15 protein were similar, thus excluding a spontaneous coupling of the wt α_2A receptor to this particular G-protein. Inositol phosphates responses at wt α_2A AR could be more readily measured in the presence of mouse G_α15; the intrinsic activity of the various ligands was apparently not affected by this G-protein. However, the partial agonist effect for some of the antagonists at the mutant α_2A AR was only observed upon co-expression of mouse G_α15. Therefore, inositol phosphates responses mediated by the coupling of mutant α_2A AR to pertussis-toxin insensitive mouse G_α15 protein (Milligan et al., 1996; Table 2) are distinct from the responses evoked by mutant α_2A AR- pertussis-toxin sensitive G protein coupling in Cos-7 cells. Specific coupling to multiple second messenger pathways by synthetic agonists has previously been observed for α_2A, α_2B and α_2C AR (Eason et al., 1994). Differential G-protein activation by agonists has been suggested for a splice variant of the pituitary adenylate cyclase activating polypeptide (PACAP) receptor (Spengler et al., 1993) and the Drosophila octopamine-tyramine receptor (Robb et al., 1994). In the present study, G_α15 protein activation by putative α₂ AR antagonists was observed. The concept that G-protein-coupled receptors can assume different conformations that can selectively and differentially permit their coupling to specific second messenger pathways also receives support from mutagenesis studies. Conversion of Asp79 to asparagine in the second transmembrane domain of the α_2A AR results in a selective uncoupling of the receptor from K⁺ currents while retaining the receptor's ability to inhibit cyclic AMP production and open voltage-sensitive Ca²⁺ channels (Lakhlani et al., 1996). Ligands may differentially affect multiple active receptor states. As a consequence, multiple receptor:G-protein interactions, each of them specific for a particular G-protein/effector pathway, can be induced and lead to different, maybe distinct, receptor-mediated responses. Therefore, the possi-bility exists for the development of new ligands for these receptors which could selectively activate a desired second messenger pathway and reduce collateral side effects mediated via other undesired pathways.

Partial agonist activity of antagonists at mutant G-protein-coupled receptors

Partial agonist activity of antagonists has been reported (Cho et al., 1996; Groblewski et al., 1997) at constitutively active mutant human dopamine D₁ and rat AT_1A angiotensin II receptors. R(+)SCH 23390, a classical antagonist at the wt D₁ receptor, behaves as a partial agonist at the mutant Leu²⁸⁶Ala D₁ receptor, with an efficacy reaching 40% of the maximal stimulation attained with the full D₁ agonist (±)SKF 82958. However, like (±)SKF 82958, R(+) SCH 23390 failed to demonstrate increased affinity at the mutated D₁ receptor, similar to our observations for the partial agonists at the mutant α_2A AR. In a similar way, the (Sar¹, Ile⁸)AII and (Sar¹, Ala⁸)AII derivatives, which are peptidic antagonists of the wt rat AT_1A angiotensin II receptor, display maximal activities of 52 and 27%, respectively, with unchanged affinities for the mutant Asn¹¹¹Ala AT_1A angiotensin II receptor. Otherwise, induction of partial agonist properties of the AT₂-specific peptide ligand CGP 42112A [27% of the response to the agonist (Sar¹)AII] is associated with an increased affinity for the mutant AT_1A angiotensin II receptor (Groblewski et al., 1997). Thus, with the exception of CGP 42112A, partial agonist activity was not accompanied by an increased binding affinity at this latter receptor. However, Noda et al. (1996) described a large increase in affinity with the antagonist (Sar¹, Ile⁸) A II for the mutant AT₁ angiotensin receptor. The apparent absence of increased binding affinity may be related to a lack of sensitivity in the models being investigated.

According to the extended ternary complex model of Lefkowitz and colleagues (Lefkowitz et al., 1993; Samama et al., 1993) and refined by Kenakin (1995), the receptor is capable of either spontaneous or ligand-facilitated isomerization to an activated state, which is then capable of coupling to G-proteins. It is hypothesized that the adoption of this activated conformation is favoured by certain mutations that display not only increases in basal activity but also increases in agonist activity, proportional to the intrinsic activity of the agonist. The model described by Lefkowitz and colleagues may hold for constitutively activating mutations that structurally mimic the ligand-activated state of the receptor throughout its entire tertiary structure. Different mutational substrates may very well result in different local effects on binding and G-protein-coupling geometries. It is conceivable that structural alterations near the ligand-binding pocket may modify the nature of ligand/receptor interactions altogether, such that some antagonists are capable of stabilizing the active conformation at the new binding interface generated by the mutation. The Leu²⁸⁶Ala mutation in transmembrane domain VI is within the ligand-binding transmembrane core of the D₁ receptor. The mutation Thr³⁷³Lys, located in the carboxyl-terminal region of the third intracellular loop of the α_2A AR, seems to favour the coupling of the mutated receptor to the mouse G_α15 protein. This receptor:G-protein interaction may modify the tertiary structure of the mutated receptor in such a way that some antagonists are capable of stabilizing the active receptor conformation, thereby generating positive efficacy. The Asn¹¹¹Ala mutation in transmembrane domain III of the AT_1A angiotensin II receptor is more difficult to interpret since peptidic ligands bind the receptor via both transmembrane residues and extracellular loops (Hjorth et al., 1994; Noda et al., 1995).

Partial agonist activity for certain antagonists has also been measured at mutated G-protein-coupled receptors which do not display constitutive activity. The inverse agonist 11-cis-retinal behaves as a partial agonist at the Gly¹²¹Leu mutation in transmembrane domain III of rhodopsin (Han et al., 1997). The single amino acid substitution Asp¹¹³Glu in the third transmembrane domain of the hamster β₂ AR promotes partial agonist activity for pindolol, oxyprenolol and alprenolol, ligands that act as antagonists at the wt β₂ AR (Strader et al., 1989). The other β AR antagonists being investigated did not demonstrate intrinsic activity at the mutant β₂ AR. However, the affinity of the agonist isoproterenol was also reduced 100 fold, suggesting that this mutant receptor has partially lost both affinity and specificity for activation by ligands. This contrasts with our binding data at the α_2A AR where both ligand affinity and specificity are conserved. Strader et al. (1989) explained their findings by suggesting the existence of overlapping binding sites for agonists and antagonists. Claude et al. (1996) hypothesize that antagonists with high structural homology to agonists are capable of agonist activity if the receptor is permissive. This was based on their observation that the mutation of conserved serine residues (Ser¹⁷⁷Leu or Ser¹⁹⁶Leu) in transmembrane domain IV of μ-opioid receptors confers full agonistic properties to peptide (TIPPϕ) and alkaloid antagonists (naloxone, naltrexone and naltriben). A similar explanation may also account for atipemazole and BRL 44408 in our study since their structures resemble the α₂ AR agonist medetomidine.

In conclusion, the results provided in this study imply that the pharmacology of constitutively active receptors is more complex than is commonly assumed. The mutant Thr³⁷³Lys α_2A AR facilitates detection of partial agonist activity for a subclass of putative antagonists. This further supports the hypothesis that pure neutral antagonists may be relatively uncommon. Targeting of these mutant receptors may open new possibilities to manipulate the α_2A AR receptor.

Abbreviations

α_2A AR

α_2A-adrenergic receptor

BRL 44408

(±)-2[(4,5-dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole

CSS

controlled salt solution

DMEM

Dulbecco's modified Eagle Medium

MK 912

(2S,12bS) 1′,3′-dimethylspiro (1,3,4,5′,6,6′,7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinolizine)-2,4′-pyrimidin-2′-one

RS 15385

(+)-(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

RX 811059

2-[2-(2-ethoxy-1,4-benzodioxan-2-yl)-1-imidazoline

[³H]RX 821002

[³H](1,4-[6,7(n)-[³H]-benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride

SKF 86466

6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine

UK 14304

5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline tartrate

WB 4101

2-(2,6-dimethoxyphenoxyethyl)aminoethyl-1,4-benzodioxane hydrochloride