Introduction

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The phenomenon of agonist-mediated activation of a G protein-coupled receptor (i.e., seven transmembrane domain, or 7TM, receptors) can be described conveniently through the formalism of a two-state allosteric transition. The receptor exists in equilibrium between the active and inactive conformations, R and R*. In the absence of a bound agonist, constraints (that are structural in nature, perhaps) maintain the equilibrium shifted toward the inactive form, thus, little or no ligand-independent signaling occurs. Agonist binding, but also, remarkably, some mutations that cause constitutive activity (Kjelsberg et al., 1992; Parma et al., 1993; Samama et al., 1993; Shenker et al., 1993; Scheer et al., 1996, 1997), remove such constraints and make the active receptor form R* predominant. Consequently, the free energy change that we measure for the agonist-binding process (e.g., as equilibrium affinity) must reflect at least two contributions. One is the total sum of energies related to the intermolecular forces that hold ligands and receptors together. The other results from the perturbation of intramolecular forces that the bound receptor endures in the transition to the bioactive form. Both make up experimentally measured ligand binding constants. Mutations that alter ligand affinity, therefore, can do so by changing either component, or both. How to dissect or measure each contribution?

We sought to present this question to the case of ₂-adrenergic receptors (₂-AR), where a number of sites that are crucial for agonist binding were identified early in a series of ingenious mutagenesis studies (Strader et al., 1987, 1988, 1989). One fundamental interaction in the binding of catecholamine agonists (Strader et al., 1989) is considered to be hydrogen bonding between catecholic hydroxyl groups and two serine residues (S204 and S207 for human ₂-AR) located in the fifth putative transmembrane domain (TM5). The position of such residues, especially that of S207, is highly conserved among all members of the catecholamine receptor family, and their modification by site-directed mutagenesis decreases agonist-, but not antagonist-, binding affinity in all types of catecholamine receptors (Wang et al., 1991; Link et al., 1992; Cavalli et al., 1996; Hwa and Perez, 1996).

The magnitude of binding energy that is lost to elimination of each serine residue was consistent with what may be expected for the deletion of one hydrogen bond (Strader et al., 1989). However, the modification of S204 and S207 in -adrenergic receptors also causes impairment of agonist-mediated signal transduction (Strader et al., 1989), suggesting that the interaction between the catechol ring and TM5 is involved in the process of coupling ligand recognition to the emergence of the bioactive conformation in the receptor. Therefore, part of that lost binding energy may also reflect the reduced tendency of the receptor to interconvert into R* form.

In this study, we exploit the principle of energy conservation among simultaneous perturbations applied to a macromolecule binding process (Horovitz, 1987; Horovitz and Fersht, 1990; Hidalgo and MacKinnon, 1995; Faiman and Horovitz, 1996) to analyze the loss of agonist binding energy that follows the double deletion of S204 and S207 in the human ₂-AR.

Using this approach, we estimate that a fraction of the mutation-induced change of binding energy that follows the removal of hydroxyl groups is attributable to a shift of the intramolecular equilibrium of the receptor toward the inactive state. Consistent with such measurements, we also find that the mutant receptor displays a significant reduction of ligand-independent activity compared with the wild type, indicating that serine deletion can cause some degree of constitutive inactivation of the receptor.

These data demonstrate that a contact region that is involved in agonist-induced activation of the receptor also controls the equilibrium between active and inactive receptor forms.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Receptor Mutagenesis. The cDNA encoding the human ₂-AR subcloned in pTZ (Pharmacia, Uppsala, Sweden) was mutated by polymerase chain reaction, using Taq DNA polymerase. Recombinant clones were isolated and sequenced. The mutated DNA fragment was digested with NcoI and BglII and cloned into the expression vector pBC12BI containing the cDNA encoding the wild-type ₂-AR or its constitutively active mutant (cam) (Samama et al., 1993).

Cell Culture and Transfections. COS-7 cells were grown in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal calf serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate, in a humidified atmosphere of 5% CO₂ at 37°C. Cells were plated in 80- or 25-cm² flasks and transfected transiently with plasmids (pBC12BI or pcDNA3) harboring either wild-type or mutant receptor cDNA using the DEAE-dextrane/chloroquine procedure (Cotecchia et al., 1990). Gradual levels of receptor expression were obtained by transfecting varying amounts of receptor cDNA with the total mass of input DNA (0.2 µg/cm²/0.1 ml) maintained constant through the addition of empty vector. Cells were harvested 48 h after transfection for the binding assay or plated 24 h after transfection in 24-well plates, and then grown for an additional 24 h before the determination of cAMP levels. For the generation of stably expressing clonal lines, Chinese hamster ovary (CHO) cells were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium. Cells were transfected using Lipofectin (Life Technologies, Paisley, Scotland) according to the manufacturer's instructions. Clones (30-40 for each transfected plasmid) resistant to Geneticin (400 µg/ml of active drug; Life Technologies) were isolated and tested for their ability to bind ¹²⁵I- pindolol (NEN Life Science Products, Boston, MA).

cAMP Determination in Intact Cells. For determination of basal levels of cAMP, COS-7 cells were seeded in 24-well plates. After aspiration of the medium, the cells were incubated in a buffer containing: 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 20 mM NaHEPES, 2 mM CaCl₂, 1.2 mM MgSO₄ , 1 mM EGTA, 11.1 mM D-glucose, 0.01 mM Rolipram, and 0.05% BSA, pH 7.4. Incubations lasted 20 min at 37°C and were arrested by the removal of the buffer and the addition of 0.5 ml of ice-cold 0.1 N HCl to each well. Plates were placed on ice, and an aliquot of the HCl extract was removed for the determination of cAMP concentration using radioimmunoassay, as described (Vachon et al., 1987).

Adenylate Cyclase Assays. Membranes from frozen transfected cells were prepared as described previously (Vachon et al., 1987) and stored at 80°C (protein concentration 1-2 mg/ml) until used. The adenylyl cyclase reaction mix included 50 mM Tris/HEPES, 10 mM MgSO₄, 0.5 mM ATP, 100 µM GTP, 5 mM phosphocreatine, 25 mM creatine phosphokinase, 150 mM NaCl (pH 7.5), and 10 µM Rolipram, in a final volume of 100 µl. Reactions were started by the addition of the membrane suspensions (2-5 µg of protein) and arrested after 10 min at 37°C by the addition of 0.1 ml of ice-cold 0.2 M HCl. The determination of cAMP formed was performed using radioimmunoassay.

Binding Assay in Membrane Preparations and Intact Cells. The binding of ¹²⁵I-pindolol was measured in 1 ml of 50 mM Tris-HCl and 0.1 mM EGTA (pH 7.4) for 90 min at room temperature using 0.1-10 µg of membrane proteins. The concentration of radiotracer was maintained constant at 10 pM in the presence of increasing concentration of unlabeled ligands. Reactions were terminated by rapid filtration onto GF/B glass fiber filtering microplates (Filtermate 196; Packard Instruments, Meriden, CT). Filters were washed three times in 1 ml of ice-cold 50 mM Tris-HCl pH 7.4 and allowed to dry for a few hours. The plates were counted in a Top Count (Packard Instruments) after the addition (25 µl) of Microscint 20 (Packard) to each well. To measure binding in intact cells, transfected COS-7 cells were seeded 24 h after transfection in opaque culture plates (Packard Instruments) and incubated the next day in a reaction buffer with a composition identical with that used for determination of cAMP in intact cells. The reaction mixture (1 ml) contained either ¹²⁵I-pindolol (20 pM) or [³H]CGP 12177 (0.5 nM; NEN Life Science Products) and increasing concentration of cold ligands. The incubation lasted 90 min at 4°C and was terminated by rapid aspiration of the incubation buffer, followed by washing the monolayer twice in ice-cold PBS. After draining plates overnight onto filter paper, 250 µl of Microscint 20 was added to each well and the plates were counted in a Top Count.

Data Analysis and Calculations. Equilibrium dissociation (K_d) and association (K_eq = 1/K_d) constants were calculated by nonlinear fitting of the binding curves to a four-parameter logistic equation using the program ALLFIT (DeLean et al., 1978). When required, the binding affinity was also estimated with the computer program LIGAND (Munson and Rodbard, 1980). Free energy changes, except when indicated otherwise, are given in RT units (R, gas constant, T, absolute temperature) i.e.: G = ln(1/K_d). Energy calculations (i.e., differences from wild type and sums of single mutations) were computed experiment by experiment before taking their averages, thus their variances reflect mostly true experimental variance and not accumulating errors that propagate when multiple calculations are applied to averaged affinity values. Similarly, statistics for G and free energy coupling values (G) were computed for the repeated measurements after converting data into free energy in each experiment. The variation of free energy attributable to mutation (G) is the difference in binding energy between mutant and wild-type receptor, i.e., G = G(mut)  G(wt). For a double-mutant cycle consisting of single mutations 1 and 2, and the double mutation (1,2), the free energy coupling can be computed as: G_1,2 = G(1,2) + G(wt)  [G(1) + G(2)]. The theoretical background for these calculations is given below.

Theoretical Background. First we recall the meaning of apparent binding energy as derived from an experimentally measured equilibrium binding constant in an allosteric protein. Next, we will examine its implications in the analysis of multiple mutations cycles.

Apparent Binding Energy for an Allosteric Receptor. Let us assume that a receptor (R) can exists in a large number (n) of interconvertible conformational states (Onaran and Costa, 1997). The transitions among all of them at equilibrium can be expressed with respect to an arbitrarily chosen reference state (s₀). The concentration of each individual state ([s_i]), (with i  0), is then given by a first-order equilibrium constant (j), such that j_i = [s_i]/[s₀]. Thus, [R] = [s₀](1 + _i=1ⁿ j_i). If the receptor binds a ligand (H), the stability of each individual state in the bound receptor form will be perturbed by a factor (b), such that b_i = [Hs_i][s₀]/[Hs₀][s_i]. Hence, the concentration of bound receptor is [HR] = [Hs₀](1 + _i=1ⁿ b_ij_i), and the apparent second-order equilibrium affinity constant (Onaran and Costa, 1997) can be expressed as:

(1)

Here, k₀ is the microscopic second-order binding constant that would be measured if all the molecules of the receptor could exist "frozen" in only one of the possible conformational states (s₀), and the fractional term is the rearrangement of the first-order state transitions equilibria as the receptor passes from the unbound to the bound form. The corresponding experimentally measured binding energy, [i.e., RT ln(K_app)], thus can be approximated as the sum of two components:

(2)

where B can be interpreted as the binding contribution resulting from the intermolecular interactions between ligand and receptor contact sites (k_o), and Cb  Cf is the conformational contribution attributable to "displacement" of intramolecular equilibria of the receptor, i.e., the difference in intramolecular interactions between bound and free receptor forms (the fractional term in eq. 4). We might obviously derive a similar relation from the ligand side, but if the ligand is a small molecule, its conformational space will be vastly limited compared with that of the receptor and can be ignored in many cases.

Free Energy Conservation for Alchemical Reaction Paths. Whether additivity principles (i.e., the idea that several subconstituents of a system contribute linearly to its macroscopic behavior) can be used to dissect the components of free energy changes in proteins is a matter of heated theoretical debate (Boresch and Karplus, 1994; Mark and van Gusteren, 1994) and unresolved questions (Dill, 1997). However, the existence of free energy conservation among chemical (Weber, 1972, 1973) or alchemical (Horovitz and Fersht, 1990) perturbations applied to a macromolecule stands as a valid principle to analyze the presence or the lack of additivity in biomolecular interactions. The use of this strategy to study the effect of chemically or genetically engineered mutations was explained and discussed elsewhere (Horovitz, 1987; Horovitz and Fersht, 1990; Hidalgo and MacKinnon, 1995; Faiman and Horovitz, 1996). For convenience, we will summarize only briefly the key principles.

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(3)

(4)

(5)

Free Energy Coupling between Alchemical Changes in an Allosteric Receptor. We examined how this strategy can be used to analyze apparent free energy changes at allosteric receptors. Let's consider two specific examples that are relevant to the work described in this study.

Deletions of Chemically Complementary Groups from the Ligand and the Receptor. A set of complementary chemical groups are deleted from both the ligand and the receptor. Even if the two deletions produce similar or identical losses of binding energy, we cannot conclude that the two groups interact with each other in the ligand-receptor complex. In fact, the ligand group may interact elsewhere in the receptor, whereas the receptor group could be engaged in a network of intramolecular interactions that are crucial to the binding process. In terms of eq. 2, the decrease of binding energy attributable to the ligand deletion results from a primary effect on B, whereas that attributable to the receptor deletion comes from conformational contributions. Thus, the losses of binding energy can have the same magnitude even if the deleted groups do not interact with one another.

Indirect Interactions. A receptor mutation that does not hit a docking site can decrease binding energy by turning into a destabilizing contribution the intramolecular equilibria that control bound and free receptor forms. If so, the effect of perturbation 1 is: G(1) = C'b + C'f. When the mutation in the ligand instead suppresses a contact group, both docking and conformational contributions that are attributable to that group are affected. Thus, G(2) = B' + C''b. If the receptor change does not disturb the conformational contribution of the ligand change (i.e., C'b and C''b are independent), the total difference is: G(T) = B' + C'b + C'f + C''b, and G_1,2  0, (eq. 5). The effects are truly additive. However, if the conformational change caused by the receptor mutation mimics or cancels that from the loss of the ligand bond, ligand and receptor mutations may share a conformational contribution. Thus: G(1) = C'b + C'f + C''b. The resulting G_1,2 will not be zero, but C''b, and the analysis reveals that the effects are not perfectly additive, but show a small degree of interaction. How small should G_1,2 be to decide that the effects are predominantly additive? Because it is likely that C''b G(1) and C''b < G(2), then the magnitude of G_1,2 should be smaller than either changes attributable to each individual perturbation. Therefore, we may state in general that if free energy coupling is close to zero or significantly smaller than the individual changes produced by each single perturbation, we should discard the hypothesis that the groups deleted from the receptor and from the ligand form a direct bond.

Direct Interaction. When mutations in both ligand and receptor suppress groups that bind to one another, the perturbations will share an identical "loss" of docking contribution and most likely also a similar conformational contribution that each group has on binding energy. If so, G(1)  G(2) = B' + C'b; G(1|2)  G(2|1)  0 and G_1,2  (B' + C'b), which means strong interaction and total lack of additivity.

Direct Interaction with Additive Components. The situation is identical with that of case 2, but the mutation of the receptor also affects the intramolecular equilibria of the "vacant" protein. In this case, G(1) = B'+ C'b + C'f; G(2) = B' + C'b; G(T) = B' + C'b + C'f; and G_1,2 = (B' + C'b), which is exactly the same result of case 2. In fact, the contribution of the additive component will cancel out and does not disturb the detection of the strong interaction caused by the removal of complementary groups. A significant difference between G(1) and G(2) is the sole, but very important, clue to suspect a role of the intramolecular equilibria of the empty receptor. In general, a strong interaction evidenced by G_1,2 0, with G(1) > G(2) suggests that the mutation in the receptor not only suppress the docking point for the ligand, but also changes binding energy by affecting intramolecular equilibria in the receptor itself.

Mutations in Two Separated Domains of the Receptor. The principle of analysis is identical with that detailed above, but the implications of presence of interaction or lack of interaction are quite different. If two mutations applied to distant sites of the same receptor affect the energy of binding for a ligand in a nonadditive manner, that means that the two mutated sites are coupled through propagated intramolecular interactions in regulating the reactivity of the receptor binding site for that ligand (Horovitz, 1987; Horowitz and Fersht, 1990). Using this strategy thus is possible to map conformational "sensitive" residues in the ligand binding site. For example, suppose we know that a residue of the receptor is located in the ligand binding pocket and that mutation of a second far-apart residue of the molecule (which we can assume is not accessible to the ligand) affects ligand affinity, presumably via a conformational change. Let's call 1 and 2 the mutations of the first and second residue, respectively. If, by constructing a double-mutant thermodynamic cycle, the effect of the two mutations are additive (not coupled), then the change of binding energy induced by mutating the binding residue 1 does not involve any conformational contribution common to that induced by the mutation of the second residue. On the contrary, if there is interaction (lack of additivity), it means that the two mutations share a common conformational mechanism in changing binding affinity.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Lack of Additivity between Single and Double Substitutions of S204 and S207. Strader et al. (1989) proposed that serines 204 and 207 in TM5 of ₂-AR may act as hydrogen bond donors for the attraction of the two hydroxyl groups of the catechol moiety of adrenergic agonists. In their study (Strader et al., 1989), as well as others (Kikkawa et al., 1997, 1998), site-directed mutagenesis studies, single-substitutions of either S204 or S207 were examined. Each single replacement caused roughly similar decreases of agonist binding energy, which supports the idea that two hydrogen bonds may be formed between meta and para hydroxyl groups of the ligand and the serine side chains of the receptor (Strader et al., 1989). It was not known, however, whether the effects of the two deletions were additive, as it may have been expected if the diminution of binding energy would primarily reflect the loss of stabilizing effect attributable to the individual bonds removed by each mutation.

View this table: [in this window] [in a new window]   TABLE 1 Comparison of agonist binding affinity for single and double mutations of serines 204 and 207 in TM5 of 2AR Binding parameters for isoproterenol in competition for 125I-pindolol were estimated using LIGAND (Munson and Rodbard, 1980). In all cases, curves were fitted satisfactorily assuming a single class of binding sites. Mutation change is the net change of agonist binding energy caused by the mutations, calculated as G = Gwt  Gmut. The data are the means of four independent experiments in each of which the binding to the wild-type receptor and all six mutants was measured in parallel. Statistics on free energy data were computed after calculating for each experiment the mutation changes and the expected sum of the single mutations. The significance of the differences between the sum expected from single mutants (col. 5) and the energy measured for double mutants (col. 4) was evaluated using t-statistics (paired two-tailed t-tests).

Thermodynamic Analysis for the Deletion of Both Hydroxyl Groups from Ligand and Receptor. We first chose to determine the overall component of agonist binding energy that can be attributed to hydroxyl group interactions by computing the free energy couplings for concomitant mutations applied to each and both interacting partners of a ligand binding process (see "Theoretical Background" in Methods and Methods).

View this table: [in this window] [in a new window]   TABLE 2 Dissociation constant of adrenergic ligands for the binding to wild type and mutant adrenergic receptors Binding isotherms for adrenergic ligands in competition for monoiodinated 125I-pindolol were generated as described in Materials and Methods in membranes prepared from CHO cells expressing the indicated mutant or wild-type receptors. Data were analyzed with the computer program ALLFIT (DeLean et al, 1978) and are presented as dissociation constants. Mean ± S.E. computed from three to seven independent experiments.

Effect of the Hydroxyl Groups' Deletion on Signal Transduction. It is not possible to compute the free energy coupling for the effect of the removal of hydroxyl groups on the conversion of the receptor into active form, because the signaling activity of the receptor cannot be described as a free energy change. However, we compared on a qualitative basis how dehydroxylation of either ligand and receptor affects signal transduction.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Adenylyl cyclase responses mediated by wild-type and (S204A, S207A) 2-AR. CHO lines expressing wild-type (SS) or (S204A, S207A) 2-AR (AA) were prepared as described in Materials and Methods. Top panels, concentration-response curves for epinephrine or mape-stimulated enzymatic activity (10 min) in membranes prepared from CHO cells expressing wild-type (A) or mutant (B) receptor. The points are averages of triplicate determinations and were divided for the Bmax values measured in the membrane of the two cell lines (12.5 and 11.2 pmol/mg in wild-type and mutant-expressing cells, respectively). Bottom panels, concentration-response curves as those shown in the top panels were generated in membranes prepared from CHO cells expressing different concentrations of wild-type and mutant receptors, as indicated. Maximal (Emax, C) and half-maximal (EC50, D) stimulations were estimated using ALLFIT (DeLean et al., 1978) and are plotted as a function of receptor concentration measured in the corresponding membranes.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of GTP on the binding isotherms of adrenergic agonists in wild-type and [S204A, S207A]2-AR. The binding of the indicated agonists was studied as competition for the sites labeled by 125I-pindolol in membranes of CHO cells expressing wild-type or mutant receptors, either in the presence or absence of GTP (100 µM). The points are means of data generated in three independent experiments, each performed as duplicate determinations.

Interaction between Hydroxyl Group Deletion and Constitutive Activation. The data presented suggest that the interactions between catechol hydroxyl groups of the ligand and TM5 serines of the receptor contribute to both agonist affinity and agonist-induced conversion of the receptor into R*. In addition, the double mutant analysis of this interaction suggests that the loss of binding free energy attributable to the removal of serine residues from the receptor may involve a conformational shift. To verify this hypothesis we used a second double-mutant cycle. We measured the possible interaction between two receptor mutations that affect binding energy through different mechanisms. One is the double-alanine substitution (AA) investigated above. The second is a mutation enhancing the constitutive activity of the receptor. As described previously, mutagenesis of residues located in the C-terminal portion of the third intracellular loop of the receptor enhance ligand-independent activity but also increase agonist affinity in a manner related to agonist efficacy (Samama et al., 1993). This increase in affinity is mediated allosterically because the targeted residues cannot directly contact the ligand, and it can be explained if we assume that such mutation shifts the equilibrium of the receptor toward the active form R* (Samama et al., 1993).

View this table: [in this window] [in a new window]   TABLE 4 Thermodynamic cycle for the effects on ligand affinity of mutations applied to the agonist (SS  AA) or to the G protein side (SS  Cam) of the receptor Computations and data presentation as in Table 3.

Serine Deletion Diminishes the Constitutive Activity of the Receptor. We first endeavored to test such prediction using stably transfected CHO lines exhibiting a suitable range of receptor expression. But the relation between basal adenylyl cyclase activity and receptor density failed to show any consistent degree of constitutive activity for wild-type receptors (Fig. 3A), which makes consequently impossible to determine a potential inhibitory effect of the AA mutation on such parameter. Enhancement of constitutive activity was readily detectable instead in clones transfected with the cam mutation, despite the much lower level of receptor expression (Fig. 3B). Through the comparison of cells expressing cam or camAA mutant receptors we examined the effect of superimposing the OH deletion on the constitutive activated mutation. There is a definite trend for a decrease of constitutive activity in the receptor carrying both modifications compared with cam alone (Fig. 3B). However, given the very modest range of expression of the camAA mutant, such result provides only presumptive evidence that OH groups deletion may lower the constitutive activity induced by mutagenesis.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Basal and agonist-stimulated adenylyl cyclase activity as a function of receptor concentration. Adenylyl cyclase activity was measured in membranes from CHO cells expressing wild-type and mutant 2 AR. The enzymatic activity was determined using three time-points in duplicate (2, 4, and 8 min) either in the absence (BAS) or presence of 100 µM ()isoproterenol (ISO) and was calculated from the slope of the linear regressions. The data are plotted as a function of the receptor density measured in the same membranes by 125I-pindolol binding curves, and are means ± S.E. of three independent experiments. A, comparison between cells expressing wild-type (SS) and [S204A, S207A] mutant (AA) receptor. Note that no receptor-dependent increase of basal activity can be detected. B, comparison between cells expressing cam and receptors carrying both mutations (cam-AA).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Receptor density as a function of the concentration of coding cDNA. COS-7 cells were transfected with cDNA coding for wild-type or mutant receptors as indicated. Different ratios of empty and coding pBC vectors (as indicated on the x-axis) were used to maintain the mass of transfected DNA constant. Receptor density was measured in membranes from the binding isotherms of 125I-pindolol.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Enhancement of cAMP levels by transient receptor expression. COS-7 cells were transfected in duplicate 25-cm2 flasks, using increasing concentrations of coding plasmids as described in Fig. 4. At 24 h post-transfection, cells from one of the duplicate flasks were harvested and seeded into 24-well plates. At 48 h post-transfection, cells in the second duplicate flask were harvested for membrane preparation and determination of receptor concentration, whereas the multiwell plated cells were used for the assessment of intracellular cAMP concentration as described in Materials and Methods. The actual range of receptor expression varied between experiments, despite the use of the same dilution range of coding plasmids. The plots are thus an overlay of three independent experiments in each of which wild-type and all the mutants were compared within the same transfection. Each point represents triplicate cAMP determinations and a Bmax computed by nonlinear fitting of a 12-dilution binding isotherm of radiolabeled Pindolol. To assess the significance of the difference between mutant and wild-type receptors, the data were fitted by linear regressions (solid and dotted lines). The calculated slopes (mole cAMP/mol receptor) with lower-upper 95% confidence limits (in parentheses) are as follows: SS, 0.53 (0.46-0.6); AA, 0.24 (0.20-0.28); cam, 5.5 (4.3-6.7); and camAA, 2.9 (2.2-3.6). The differences in slopes were tested by F statistics. For the comparison SS versus AA, F (1,33) = 20.3, P < .0001; For the comparison cam versus camAA, F (1,25) = 5.5, P = .027. The slope of the AA mutant curve is significantly different from zero, F (1,18) = 102, (P < .0001), indicating that the constitutive activity of this receptor is reduced compared with the wild-type, but is not abolished.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Comparison of the constitutive activity of wild-type and mutant 2-AR COS-7 cells were transfected with cDNA coding for mutants, wild-type receptors, and empty vector. Receptor density and cAMP levels were measured in parallel as explained in Fig. 5. Constitutive activity is computed as the ratio between net pmols of cAMP produced and net pmol of expressed receptor, after subtraction of the basal cAMP levels (range, 15-25 pmol/mg) and the basal concentration of endogenous 2-AR (range, 0.3-0.5 pmol/mg) measured in cells transfected with noncoding vector in parallel. These ratios are equivalent to the slopes of the regression lines measured in Fig. 5. The data are means ± S.D. of the number of independent experiments shown on top of each bar. In each experiment, cAMP levels were determined in quadruplicate wells, and Bmax was estimated from computer analyses of a 12-dilution binding isotherms of pindolol.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Constitutive activity of wild-type and mutant 2-AR determined in intact cell assays COS-7 cells were transfected and processed as described in Figs. 5 and 6. However, the concentration of expressed receptors was determined in intact cells (see Materials and Methods) using the hydrophilic adrenergic antagonist [3H]CGP 12177.

View larger version (39K): [in this window] [in a new window]   Fig. 8.   Lack of antagonism between cam and AA mutant receptors for the transfection-induced accumulation of cAMP in COS-7. Cells were transfected with either individual plasmids coding for the cam (0.19 µg/cm2) or AA (0.01 µg/cm2) mutation or cotransfected with the mixture of both. The total mass of input cDNA (0.2 µg/cm2) in the individual transfections was equalized by the addition of empty vector. The levels of cAMP and receptor expression were measured as described in Fig. 6. A, comparison of cAMP levels in cells transfected with empty vector (pBC), individual mutants (AA or cam) or cotransfected with both (cam + AA). Receptor densities measured for the various conditions are presented in bold on top of each bar. The actual concentrations of cam and AA mutants expressed in the membranes of cotransfected cells were resolved by computer analysis (see C, below). Note that the relative levels of the two mutants expressed in the cotransfection cannot be predicted from the individual transfection, even if performed in parallel. B, the extent of activity attributable to each of the mutants expressed in the cotransfected cells was computed by multiplying the ratios: net cAMP/net Bmax (measured from the single transfection of each mutant in A, second and third bars) for the pmol of each mutant receptor estimated in the membranes of cotransfected cells by computer analysis. The data are then converted into fractions of the total activity determined in cotransfected cells and plotted in histogram form. It is clear that the total activity observed in the cotransfection is equal to the exact sum of the activities expected for the pmol of each mutant present in the mixture. Thus, even if present in stoichiometric excess, the AA mutant cannot antagonize the basal increase induced by the cam mutant. C, isoproterenol binding isotherms for inhibition of 125I-pindolol binding in membranes prepared from cotransfected cells. Because the difference in agonist affinity between cam and AA mutant is very large (Table 2), the agonist binding curve is clearly biphasic and show two high- and low-affinity components. When data were fitted according to mass action law models for multiple binding sites (LIGAND, Munson and Rodbard, 1980), a two-site model (solid line) provided a very significant improvement in the fit (P < .0001). The total binding capacity of the membranes (55 pmol/mg) was resolved into a high-affinity (Keq 2.6 ± 1.2 × 108 M1) component (i.e., the cam mutant) accounting for 13.9 ± 1.1 pmol/mg, and a low-affinity (Keq 8.9 ± 0.3 × 104 M1) component (i.e., the AA mutant) accounting for 41.1 ± 2.1 pmol/mg. The data illustrate the results of a single representative experiment. Two additional experiments, which resulted in different ratios of coexpressed AA and cam mutant (8.6:1 and 3.7:1, respectively), gave identical results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We used an approach based on mutant cycles to analyze the energetic contribution of the postulated interaction between OH groups of the catecholic moiety of a bound adrenergic agonist and serines 204 and 207 of the fifth transmembrane domain of the ₂-adrenergic receptor. We tested the closure of two nested thermodynamic reaction cycles. In one, the same deletions of OH groups is applied to distinct molecules, ligand, and receptor. In the other, two diverse perturbations, OH deletion and constitutive activation, are imposed onto separate functional domains of the same receptor. From this analysis we can draw two conclusions.

First, we reconfirm the notion that the two sets of OH groups in the ligand and the receptor interact directly, probably via the formation of a pair of hydrogen bonds, as originally proposed (Strader et al., 1989).

Second, we uncover an additional and unexpected role for S204 and S207 of TM5. They seem to control the equilibrium between the active (R*) and inactive (R) forms of the receptor. This suggestion is verified by the finding that the ligand-independent basal activity of the double-alanine mutant is significantly smaller than that of the wild-type receptor. This means that dehydroxylation in that particular agonist-docking spot of ₂-AR also decreases its constitutive activity.

The results presented in this study bear a number of interesting implications. One concerns our understanding of the nature of the agonist-binding site. If serines 204 and 207 in TM5 provide an anchor site for the agonist and also regulate the setpoint for the equilibrium of the R* form, this suggests that several, if not all, of the subsites where the agonist binds to the receptor may also contribute key intramolecular interactions for the conversion of the molecule into active form. The agonist binding pocket is thus a multidimensional map of residues that take part in the molecular motion underlying the emergence of a bioactive conformation in the receptor. Although obvious on an intuitive basis, this notion has received little experimental evidence thus far. Our data show that a fraction of the agonist binding energy lost on removal of serine residues is associated with the shift of intramolecular equilibrium of the receptor toward inactive conformations. This not only supports the idea that experimentally observable binding energies of any ligand reflect both intermolecular and intramolecular forces, but also demonstrates that for an agonist, the intramolecular component must be necessarily related to the process that generates the biologically relevant conformation. It is likely that agonist-receptor complexes have been shaped through evolution to optimize the maximum number of contacts with conformationally sensitive points of the receptor rather than the formation of the most stable energy-minimized assembly. We may speculate that the key difference in the mode of binding between agonist and antagonist is more energetic than topographic. A fine-tuned energy balance between intermolecular stabilization and intramolecular perturbation within the configuration of the binding pocket may be the fundamental requirement for full agonism, far more than the selective interaction with a particular set of critical docking residues.

A second implication regards our views on the mechanism by which agonists induce receptor activation. It was thought that receptors were essentially "silent" molecules in the classical view and that agonist binding would endow them with the ability to generate biological signals. The discovery that certain residues of their sequence exert a very stringent role in suppressing ligand-independent activity (Kjelsberg et al., 1992; Scheer et al., 1993) has suggested an opposite paradigm. A receptor may have signaling activity by default that is clamped down by structural factors and released by agonist binding (Kjelsberg et al., 1992; Lefkowitz et al., 1993; Samama et al., 1993). The finding in this study that an agonist docking site can also control the level of receptor constitutive activity brings additional support to this notion. It is interesting to note that alanine mutation of serine residues inhibits in parallel receptor constitutive activity and agonist-induced maximal stimulation. Thus, the suppression of an agonist-docking site restricts biological activity by an essentially identical mechanism, regardless of whether the receptor is bound or not to the agonist. This suggests that the role of bound agonist is to amplify or catalyze a process that is intrinsically wired into the receptor, not to impart novel molecular properties. In other words, the bioactive receptor conformers that are "induced" by an agonist are improbable, but not impossible, in its absence.