Introduction

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Allosteric modulation by sodium ions is a hallmark of many G protein-coupled receptors (GPCRs), including most of the aminergic GPCRs. The molecular explanation for this is that the sodium binding site is formed by a strictly conserved aspartate residue situated approximately three helical turns from the intracellular side of the second transmembrane-spanning domain (TMS2) (Horstman et al., 1990; Neve et al., 1991a; Donnelly et al., 1994). Sodium generally decreases the agonist binding affinity while increasing the binding for some classes of antagonists (Motulsky and Insel, 1983; Neve, 1991b). In contrast, antagonist binding to some GPCRs has been shown to be allosterically displaced by 5-amino-substituted amiloride derivatives, and in the case of the _2A-adrenergic receptor and the D₂ dopamine receptor, it has been postulated that sodium and substituted amiloride derivatives bind to distinct sites (Wilson et al., 1990; Strange, 1997). Notably, the 5-amino-substituted amiloride derivatives such as methylisobutylamiloride (MIA) allosterically decrease antagonist binding to D₂ dopamine receptors (Hoare and Strange, 1996) in a manner similar to zinc (Schetz and Sibley, 1997; Schetz et al., 1999). However, zinc but not MIA binding affinity for the D₂ dopamine receptor is sodium-dependent (Schetz et al., 1999; S. R. J. Hoare, personal communication). Importantly, all three of these allosteric modulators have been shown to act directly on the GPCR and, although each of them is chemically distinct, their molecular mechanisms appear to be related. A major question, however, is whether their shared or interrelated effects are due to interaction at a shared site or at three physically distinct sites. The D₄ dopamine receptor was chosen as the model system for this investigation to simplify the interpretation of allosteric-allosteric interaction experiments, since zinc inhibition of antagonist binding to the D₄ dopamine receptor subtype is noncompetitive (Schetz et al., 1999). In addition, [³H]methylspiperone was chosen as the primary radioligand with which to study allosteric interactions, because it is an antagonist and its binding to dopamine receptors is relatively sodium-insensitive. In this report, we demonstrate that sodium, zinc, and MIA occupy three physically distinct allosteric binding sites on the D₄ dopamine receptor protein.

	Materials and Methods

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Reagents. All drugs were purchased from Research Biochemicals International (Natick, MA). Analytical grade binding and wash buffer reagents were from Sigma Chemical Co. (St. Louis, MO) and Fluka Chemical Corp. (Ronkonkoma, NY) and cell culture supplies were purchased from Life Technologies (Gaithersburg, MD). Ultrapure zinc chloride was purchased from Aldrich Chemical (Milwaukee, WI). The [³H]methylspiperone (NET856, 85.5 Ci/mmol) was purchased from DuPont-New England Nuclear (Boston, MA).

Site-Directed Mutagenesis. Mutations in the rat D₄ dopamine receptor were created using QwikChange, a DpnI-based site-directed mutagenesis kit (Stratagene, La Jolla, CA), and verified by ³³P dideoxy-nucleotide sequencing using Sequenase (Amersham, Piscataway, NJ). The naming convention for the mutant receptors begins with the name of the wild-type receptor background followed by the single-lettered code for the amino acid to be mutated and its position, and then ending with the corresponding amino acid substitution. For example, the D₄-D77N mutant has a D₄ background that has been mutated from an aspartate at position 77 to an asparagine.

Transfection of Wild-Type and Mutant DNA. The pcDNA3 plasmid constructs containing either the wild-type or a mutant dopamine receptor were transfected into COS-7 or CHO cells using CaPO₄ precipitation (Invitrogen, Carlsbad, CA). Specifically, 20 µg of plasmid DNA was mixed with a final volume of 1 ml of CaPO₄/HEPES solution and the resulting precipitate was added dropwise to 20 to 30% confluent cells on a 150-cm² plate in a total media volume of 20 ml. The following day, the media were removed by aspiration and replaced with fresh media. COS-7 cells were then grown to confluence and harvested (transient transfection system), whereas CHO cell were first selected with geneticin (Life Technologies) and single colonies were expanded before harvesting (stable transfection system).

Preparation of Membranes and Radioligand Binding. Membranes from transient or stable cell lines expressing wild-type or mutant D₄ dopamine receptors were isolated and used in radioligand binding assays as described previously (Schetz et al., 1999). In the case of saturation isotherm binding, membranes were equilibrated with increasing concentrations of [³H]methylspiperone (0.03-3 nM). For competition experiments, membranes were equilibrated with a fixed concentration of [³H]methylspiperone (ca. 500 pM) and increasing concentrations of the competing ligand. Nonspecific binding was defined by 5 µM (+)-butaclamol.

Calculations and Data Analysis. All experiments were performed in triplicate and repeated three to four times. All averaged values are reported as a geometric mean with a standard deviation. The error bars in all the figures are standard error bars. The equilibrium dissociation constant (K_D) of the primary radioligand was measured by saturation isotherm analysis. Inhibition constant (K_i) values for dopamine and MIA were calculated from IC₅₀ values using the Cheng-Prusoff equation K_i = IC₅₀/(1 + [ligand]/K_D). This form of the equation assumes a purely competitive interaction and a pseudo Hill slope of 1. Even though MIA is an allosteric modulator of [³H]methylspiperone binding to D₄ dopamine receptors, its K_i affinity value derived from the competitive form of the equation is a good approximation because its binding to the D₄ dopamine receptor is highly cooperative (Hoare et al., 2000). In the case of dopamine, the best-fit curve has a pseudo Hill slope significantly less than unity, and consequently, the K_i affinity values are K_0.5 value approximations. Since zinc is a noncompetitive allosteric inhibitor of methylspiperone binding to D₄ receptors, its K_i binding affinity value was taken to be approximately equal to its IC₅₀ value, i.e., the noncompetitive form of the Cheng-Prusoff equation (Cheng and Prusoff, 1973). A 95% confidence interval was used for all curve-fitting procedures and for comparing different curve-fitting models using GraphPad Prism, version 2.0. The statistical measures of fit were the F test, the run test, and a correlation coefficient.

	Results

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Sodium ions modulate a variety of different G protein-coupled receptors via electrostatic interaction with a highly conserved aspartic acid located approximately three helical turns from the intracellular side of the second transmembrane-spanning domain. Consequently, our strategy for determining whether the allosteric modulators sodium, zinc, and MIA bind to separate or related sites on the D₄ dopamine receptor was to first create a sodium-insensitive D₄ mutant by neutralizing the sodium binding site. This neutralization was achieved by mutating the corresponding negatively charged aspartic acid (D) at position 77 in TMS2 of the D₄ dopamine receptor to a neutral asparagine (N). For comparison, another mutant D₄ dopamine receptor was constructed by replacing the D₄ subtype-specific methionine in the first turn of TMS3 with the corresponding valine residue, which is present in all four other dopamine receptor subtypes. To ensure that the mutant receptors were properly folded and expressed in COS cells, the resulting D₄-D77N and D₄-M107V mutant dopamine receptors were assayed for their ability to specifically bind with high affinity to the D₂/D₃/D₄-selective radioligand antagonist [³H]methylspiperone. Direct determination of [³H]methylspiperone equilibrium dissociation constants (K_D) by saturation isotherm analysis yielded a K_D = 294 ± 30, 84 ± 29, and 138 ± 22 pM for the wild-type D₄, the D₄-D77N mutant, and the D₄-M107V mutant dopamine receptors, respectively (n = 3). Since the D₄-D77N and D₄-M107V mutants bind [³H]methylspiperone with high affinity and [³H]methylspiperone binding to wild-type dopamine receptors is displaced by zinc and MIA but not by sodium ions, [³H]methylspiperone was selected as a suitable primary radioligand for measuring sodium, zinc, and MIA interactions by competition binding.

Zinc ions inhibit [³H]methylspiperone binding to the wild-type D₄ dopamine receptor expressed in COS cells with an 8-fold lower affinity in the presence of 120 mM sodium than in the absence of sodium (Fig. 1A). This sodium-sensitive decrease in zinc affinity observed for the wild-type D₄ receptor was abolished in the D₄-D77N mutant, even though the D₄-D77N mutant retains its D₄ wild-type binding affinity for zinc (Fig. 1A). Like zinc, dopamine displayed sodium-sensitive binding to the wild-type D₄ receptor, which was lost in the D₄-D77N mutant receptor (Fig. 1B). However, dopamine binding affinity for the D₄-D77N mutant also increased slightly (i.e., 2.5-fold after applying the Cheng-Prusoff equation for a competitive inhibitor) (Fig. 1B). In contrast to dopamine and zinc, MIA binding affinity for the wild-type D₄ dopamine receptor was not significantly affected by sodium ions (Fig. 1C). In further contrast to dopamine and zinc, MIA binds to the D₄-D77N mutant dopamine receptor with a 4-fold lower apparent affinity than it does to the wild-type D₄ dopamine receptor (Fig. 1C).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Zinc, MIA, and dopamine inhibition of [3H]methylspiperone binding to wild-type D4 and mutant D4-D77N dopamine receptors. Membranes were isolated from COS-7 cells transiently expressing mutant and wild-type dopamine receptors and assayed as described under Materials and Methods. A through C, binding to the wild-type D4 receptor in the absence () and presence of 120 mM NaCl (), and binding to the mutant D4-D77N receptor in the absence () and presence of 120 mM NaCl (). A, corresponding affinity values for zinc binding to the wild-type D4 and mutant D4-D77N dopamine receptors are Ki = 21 ± 4 and 13 ± 2 µM in the absence of sodium and Ki = 171 ± 36 and 19 ± 6 µM in the presence of 120 mM sodium chloride, respectively. The Ki value for zinc binding to the wild-type D4 receptor in the presence of NaCl is significantly different (P  0.05) from all the other Ki values for zinc binding. The corresponding pseudo Hill slopes for zinc binding to the wild-type D4 and mutant D4-D77N dopamine receptors are nH = 1.23 ± 0.16 and 1.19 ± 0.11 in the absence of sodium and nH = 1.47 ± 0.05 and 1.06 ± 0.10 in the presence of 120 mM sodium chloride, respectively. The nH value for zinc binding to the wild-type D4 receptor in the presence of NaCl is significantly greater (P  0.05) than all the other nH values for zinc binding. B, corresponding affinity values for dopamine binding to the wild-type D4 and mutant D4-D77N dopamine receptors are Ki = 17.8 ± 8.5 and 7.0 ± 1.9 µM in the absence of sodium and Ki = 166 ± 66 and 6.4 ± 2.3 µM in the presence of 120 mM sodium chloride, respectively. The Ki values for dopamine binding to the wild-type D4 receptor in the presence of NaCl are significantly different (P  0.05) from all the other Ki values for dopaminebinding. The corresponding pseudo Hill slopes for dopamine binding to the wild-type D4 and mutant D4-D77N dopamine receptors are nH = 0.62 ± 0.06 and 0.77 ± 0.12 in the absence of sodium and nH = 0.60 ± 0.11 and 0.81 ± 0.11 in the presence of 120 mM sodium chloride, respectively. The only significant differences in nH values are between dopamine binding to the wild-type D4 and the mutant D4-D77N dopamine receptors (P  0.05). C, corresponding affinity values for MIA binding to the wild-type D4 and mutant D4-D77N dopamine receptors are Ki = 341 ± 200 and 1322 ± 348 nM in the absence of sodium and Ki = 567 ± 178 nM and 1517 ± 353 µM in the presence of 120 mM sodium chloride, respectively. The Ki values for MIA binding to the mutant D4-D77N receptor are significantly greater (P  0.05) than for MIA binding to the wild-type D4 receptor. The corresponding pseudo Hill slopes for MIA binding to the wild-type D4 and mutant D4-D77N dopamine receptors are nH = 1.12 ± 0.24 and 1.47 ± 0.5 in the absence of sodium and nH = 1.05 ± 0.08 and 1.3 ± 0.29 in the presence of 120 mM sodium chloride, respectively. None of the nH values for MIA binding are significantly different (P > 0.05).

Having established the interaction of sodium ions with the conserved aspartic acid in TMS2, we next sought to determine whether the apparent similarity in the molecular mechanisms for zinc and MIA modulation of antagonist binding is a result of coupling between their binding sites. We reasoned that if zinc and MIA bind to wild-type D₄ dopamine receptors at the same site (competitive) or structurally linked (allosterically coupled) sites, then the affinity of either of these modulators should be influenced by the presence of the other modulator. Remarkably, the apparent affinity values for MIA-[³H]methylspiperone inhibition curves performed in the absence of zinc were not significantly different from those done in the presence of a concentration of zinc that produces approximately a half-maximal effect (Fig. 2A). However, in the reverse experiment with zinc-[³H]methylspiperone inhibition curves, the apparent affinity for zinc is significantly increased in the presence of a concentration of MIA that produces about a half-maximal effect compared with in the absence of MIA, although the effect was relatively small, i.e., about 3-fold (Fig. 2B). The combined zinc-MIA experiments allowed us to rule out any competitive allosteric interactions between zinc and MIA binding at two separate sites. A purely competitive binding of zinc and MIA for an identical site could also be ruled out, since the apparent affinity for zinc measured in the presence of an IC₅₀ concentration of a perfectly competitive ligand would be significantly decreased.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Inhibition of [3H]methylspiperone binding to wild-type D4 dopamine receptors by either one of the allosteric modulators, zinc or MIA, in the presence or absence of the other allosteric modulator. Membranes were isolated from CHO cells stably expressing the wild-type dopamine receptor and assayed as outlined under Materials and Methods. A and B, dose-response curves for one modulator in the absence () or presence () of approximately an IC50 concentration of the other modulator. A) corresponding affinity values for MIA binding in the absence and presence of 50 µM zinc are Ki = 299 ± 66 and 208 ± 64 µM, respectively. These Ki values are not significantly different (P > 0.05). The corresponding pseudo Hill slopes for MIA binding to the wild-type D4 dopamine receptor in the absence and presence of 50 µM zinc are nH = 1.04 ± 0.05 and 1.21 ± 0.16, respectively. These nH values are not significantly different (P > 0.05). B, corresponding affinity values for zinc binding in the absence and presence of 600 nM MIA are Ki = 21 ± 3.5 and 6.7 ± 2.5 µM, respectively. The Ki value for zinc binding in the presence of 600 nM MIA is significantly less (P  0.05) than in the absence of MIA. The corresponding pseudo Hill slopes for MIA binding to the wild-type D4 dopamine receptor in the absence and presence of 600 nM MIA are nH = 1.10 ± 0.14 and 1.15 ± 0.07, respectively. These nH values are not significantly different (P > 0.05).

The apparent distinction between zinc and MIA binding sites was investigated further by comparing zinc and MIA binding properties for a different mutant D₄ dopamine receptor than the sodium-insensitive D₄-D77N mutant. In contrast to the D₄-D77N mutant, which binds MIA with a 4-fold lower affinity (Fig. 3A), the TMS3 mutant D₄-M107V dopamine receptor had a 30-fold higher affinity for MIA (Fig. 3A). The pronounced and opposing effects of these two point mutants on MIA binding were not mimicked by zinc. In fact, zinc binding to both mutant D₄ receptors was similar to the wild-type D₄ dopamine receptor background from which they were derived (Fig. 3B). Furthermore, the molecular mechanisms of allosteric modulation for zinc and MIA at the wild-type D₄ dopamine receptor are distinct: the primary effect on [³H]methylspiperone binding for zinc is a change in the maximum number of binding sites (Schetz et al., 1999), whereas for MIA it is a change in affinity (Hoare et al., 2000). Thus, the combined data from experiments using wild-type and sodium-sensitive, and sodium-insensitive mutant D₄ dopamine receptors demonstrates at several levels that sodium, zinc, and MIA each bind a distinct site on D₄ dopamine receptors.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Zinc and MIA inhibition of [3H]methylspiperone binding to wild-type D4, mutant D4-D77N and mutant D4-M107V dopamine receptors. Membranes were isolated from COS-7 cells transiently expressing mutant and wild-type dopamine receptors, and then assayed as described under Materials and Methods. A and B, binding to the wild-type D4 (), the D4-D77N mutant (), and the D4-M107V () mutant dopamine receptors. A, corresponding affinity values for MIA binding to the wild-type D4, the mutant D4-D77N, and the mutant D4-M107V dopamine receptors are Ki = 341 ± 200, 1322 ± 348, and 11.2 ± 4.2 nM, respectively. The Ki values for MIA binding to the wild-type D4, the mutant D4-D77N, and the mutant D4-M107V receptors are all significantly different from one another (P  0.05). The corresponding pseudo Hill slopes for MIA binding to the wild-type D4 and mutant D4-D77N and D4-M107V dopamine receptors are nH = 1.12 ± 0.24, 1.47 ± 0.5, and 0.72 ± 0.2, respectively. These nH values are not significantly different (P > 0.05). B, corresponding affinity values for zinc binding to the wild-type D4, the mutant D4-D77N, and the mutant D4-M107V dopamine receptors are Ki = 26.2 ± 12.8, 13.2 ± 2, and 18.2 ± 2.5 µM, respectively. The Ki values for zinc binding to the wild-type D4 receptor, the mutant D4-D77N, and D4-M107V receptors are not significantly different from one another (P > 0.05). The corresponding pseudo Hill slopes for zinc binding to the wild-type D4 receptor and the mutant D4-D77N and D4-M107V dopamine receptors are nH = 1.23 ± 0.16, 1.19 ± 0.11, and 1.23 ± 0.15, respectively. These nH values are not significantly different (P > 0.05).

	Discussion

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A growing appreciation for the role of receptor conformation in defining GPCR receptor pharmacology has resulted in a renewed interest in allosteric modulation of GPCR proteins. The term allosteric modulation is frequently synonymous with conformational change, because allosteric modulators are defined as occupying a different site than the "primary" site of ligand binding. Consequently, the conformational effect of the allosteric site on the primary site must somehow be propagated through the protein structure, when the allosteric site is occupied by other proteins (Kramer and Karpen, 1998), drugs (Hoare and Strange, 1996), or ions (Neve, 1991b; Schetz and Sibley, 1997). Allosteric modulators also offer a potentially important means for modulating therapeutic responses to drugs (Ehlert, 1986; Birdsall et al., 1995; Lazareno and Birdsall, 1995; Strange, 1997; Schetz et al., 1999). Moreover, some endogenous modulators appear to have a physiological role in modulating ligand-induced receptor function. For example, millimolar concentrations of sodium ions acting from the intracellular side accelerate the dissociation of agonist from the GPCR. The same sodium-induced changes in the receptor that result in a decrease in agonist affinity also increase the binding for some classes of antagonists (Motulsky and Insel, 1983; Neve, 1991b). These results suggest that the binding of sodium ions to an allosteric site on the receptor changes the shape of the receptor's ligand binding pocket such that the favored conformation is the one associated with a less active state of the receptor. Sodium has been shown to have this effect on dopamine receptors (Neve, 1991b), and more recently, it was found that zinc and derivatives of amiloride allosterically decrease antagonist binding to dopamine receptors (Hoare and Strange, 1996; Schetz and Sibley, 1997). The question that arises is whether the interrelated pharmacological properties for sodium, zinc, and MIA are a result of differential allosteric interaction of these three modulators at the same site or at two or three distinct sites on the D₄ dopamine receptor protein.

Since all three allosteric modulators of D₄ dopamine receptors bind with relatively low affinity (10⁵-10² M), it is not possible to use radiolabeled derivatives (e.g., ⁶⁵Zn²⁺) to directly measure their binding using rapid filtration techniques. Instead, the binding of these allosteric modulators to D₄ dopamine receptors had to be measured indirectly by measuring their ability to inhibit the high-affinity binding of the D₂-like antagonist [³H]methylspiperone. Although some information concerning their respective binding sites can be obtained by indirectly measuring their binding properties in the presence of one another, the allosteric nature of their interactions coupled with the indirect method of measuring their binding precludes the definitive determination of whether sodium, zinc, and MIA bind to three distinct sites. For these reasons, we adopted a classical approach to studying physical chemistry phenomena, which is to perturb the system and then study the relative changes. The "perturbation" was achieved by first mutating the wild-type D₄ dopamine receptor, and then, relative changes in binding properties for the three allosteric modulators were measured either singly or in combination. By analogy with other catecholamine GPCRs (Donnelly et al., 1994), the site of sodium modulation corresponded to a conserved aspartic acid residue located approximately three helical turns from the intracellular side of the second transmembrane-spanning domain or aspartate at position 77 of the D₄ dopamine receptor. Mutation of this negatively charged aspartate 77 to the neutrally charged but similarly sized asparagine resulted in a total loss of sodium sensitivity of the D₄ dopamine receptors as judged by its loss of sodium-sensitive binding for dopamine and for zinc ions. The D₄-D77N mutant also bound MIA with approximately 4-fold lower affinity than the wild-type D₄ receptor, but had essentially no effect on zinc binding affinity. A different mutant located in the first helical loop of the extracellular side of TMS3 had an approximate 30-fold increase in MIA binding affinity, again with essentially no effect on zinc binding. Thus, in perturbed or mutant D₄ dopamine receptors the effect on the binding of MIA and zinc is clearly different.

Overall, several lines of evidence indicate that the allosteric modulators sodium, zinc, and MIA bind to three distinct sites on the D₄ dopamine receptor protein. First, the allosteric modulation of [³H]methylspiperone binding to wild-type D₄ dopamine receptors by zinc ions is sodium-sensitive, whereas allosteric modulation by MIA is insensitive to sodium ions. Second, the sodium sensitivity of zinc binding was eliminated in the D₄-D77N mutant, without affecting the apparent binding affinity for zinc. Thus, zinc does not act at the sodium binding site (aspartate 77) and sodium does not act at the zinc binding site, rather sodium is allosterically modulating the binding of zinc. Third, the allosteric effects produced by zinc and MIA appear to be mutually exclusive at the wild-type D₄ dopamine receptor. For example, the apparent affinity for either allosteric modulator is the same or slightly higher when the other allosteric modulator is present at its IC₅₀ concentration. However, this would not be the case if zinc and MIA bound to the same site, i.e., a purely competitive binding. Consequently, zinc and MIA do not appear to be exerting their macroscopically similar allosteric effects on [³H]methylspiperone binding by binding to a common site. Fourth, mutant D₄ dopamine receptors were identified that either increase or decrease MIA binding affinity but in either case zinc binding is essentially unaffected. Fifth, the molecular mechanisms of allosteric modulation for zinc (Schetz et al., 1999) and MIA (Hoare et al., 2000) are distinct. These last three results, in particular, suggest that zinc and MIA do not share a common binding site on the D₄ dopamine receptor and demonstrate that similar allosteric outcomes can result from binding to physically distinct allosteric sites.

To our knowledge, this report is the first demonstration of three distinct sites of allosteric coupling to the "binding site crevice" on a GPCR. This novel finding has several important implications with respect to conformational pharmacology. First, the presence of multiple sites of allosteric modulation should allow for the development of higher affinity modulators with increased selectivity using polymer-linked ligand dimers as has been recently shown for GTPases and cyclic nucleotide-gated channels (Kramer and Karpen, 1998). Second, these findings suggest that two different allosteric sites on the protein may induce similar conformational effects on the binding site crevice, and consequently, produce a similar pharmacological outcome. Third, as the number of allosteric sites of interaction on a given protein increases so will our understanding of which are the critical intra protein-protein interactions that produce a desired protein conformation. In other words, more tools are available for dynamically probing the conformational pharmacology of membrane-bound protein receptors.