Introduction

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In most cells, G protein-coupled receptors interact with multiple distinct G protein oligomers, and the overall biological response to the agonist-activated receptor results from the concerted regulation of multiple G protein-dependent effector systems (Gudermann et al., 1996a). The ability of a receptor to activate multiple G proteins is specified by discrete, poorly conserved regions in the intracellular loops that connect the transmembrane helices and, in some cases, within the carboxyl terminus of the receptor (Gudermann et al., 1996b). Based on the observation that there is more than one active conformation of rhodopsin (Arnis et al., 1993, 1994), it has been proposed that other G protein-coupled receptors also may adopt several active conformations that interact with distinct G proteins; these may be selected by "biased" agonists, which will favor a conformation that preferentially interacts with one type of G protein (Gudermann et al., 1996a; Kenakin, 1996). This hypothesis is supported by two lines of experimental evidence. (1) Point mutations in a given receptor can elicit distinct effects on its coupling properties; for example, substitution of Ile486 by phenylalanine in the human thyroid-stimulating hormone receptor produces a receptor that constitutively activates both the adenylyl cyclase and phospholipase C cascade; in contrast, substitution of Phe631 by isoleucine only raises cAMP (Parma et al., 1995). (2) The PACAP receptor I can be stimulated by PACAP-27 and PACAP-38; however, on heterologous expression of the receptor, PACAP-27 activates adenylyl cyclase more potently than PACAP-38, whereas the reverse is true for stimulation of inositol trisphosphate formation (Spengler et al., 1993). Other arguments and additional experimental evidence in support of the hypothesis that multiple R* conformations exist have been reviewed recently (Gudermann et al., 1996a; Kenakin, 1996). A corollary of this concept is the assumption that compounds that block the interaction of R with G may be selective for specific R/G tandems. If compared with receptor antagonists, compounds that block the interaction of receptors and G proteins over receptor antagonists offer the advantage that they should provide for an additional level of selectivity in inhibiting signal transduction; provided that inhibitors with high selectivity can be found, they will block signaling of the activated receptor via one G protein-regulated pathway but will not perturb other receptor-generated signals within the cell.

Earlier work showed that suramin acted as an inhibitor of receptor/G protein coupling (Butler et al., 1988; Huang et al., 1990); circumstantial evidence for selective disruption of specific receptor/G protein tandems was provided by the observation that suramin inhibited the activation of pertussis toxin-substrate G proteins by -opioid receptors in membranes from NG 108-15 cells, whereas the stimulation of the guanine nucleotide exchange reaction of these G_o/G_i proteins by serum factors, which acted on an unidentified receptor, was not blocked (Huang et al., 1990). In addition, we recently reported that the didemethylated suramin derivative NF037 discriminated between A₁ adenosine receptor/G protein tandems in the human cerebral cortex and D₂ dopamine receptor/G protein tandems in the rat striatum (Beindl et al., 1996). However, in rat cerebral cortex, the A₁ adenosine receptor is resistant to the uncoupling effect of suramin unless the membranes are extracted with detergent to remove an inhibitory constraint imposed by an ancillary protein (Nanoff et al., 1997). Thus, the distinct activity profile of suramin and NF037 on human A₁ adenosine and rat D₂ dopamine receptor may have been due to species differences or may have arisen from the expression of the receptors in distinct cell types and/or in distinct microcompartments of the plasma membrane. Here, we eliminated these confounding variables by expressing the human and rat receptors in the same cell line; furthermore, the receptors were forced to interact with the same G protein  subunit. The results show that NF037 is selective for D₂ dopamine receptor/G protein tandems even if the receptors couple to the same G_i subtype. In addition, the potency of suramin and NF037 in uncoupling receptor/G protein complexes is inversely correlated to the affinity of the receptor for the G protein.

	Experimental Procedures

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Materials. [³⁵S]GTPS, [¹²⁵I]OH-PIPAT [(+)-trans-7-hydroxy-2[N-propyl-N-3-[¹²⁵I]iodo-2'-propenyl)aminotetralin] and [¹²⁵I] were purchased from New England Nuclear Research Products (Boston, MA). [¹²⁵I]HPIA [()N⁶-3[¹²⁵I](iodo-4-hydroxyphenyl-isopropyl)adenosine] was synthesized according to Linden (1984). Guanine nucleotides and adenosine deaminase were from Boehringer-Mannheim Biochemica (Mannheim, Germany). 1-O-n-Octyl--D-glucopyranoside (octylglucoside), CHAPS, and HEPES were from BIOMOL (Munich, FRG). Suramin, sulpiride, and XAC were obtained from Research Biochemicals (Natick, MA). The materials required for SDS-polyacrylamide gel electrophoresis were from BioRad (Richmond, CA). Fetal calf serum was from PAA Laboratories (Linz, Austria). Dulbecco's modified Eagle medium, nonessential amino acids, -mercaptoethanol, and G418 (geneticin) were obtained from GIBCO-BRL (Grand Island, NY). CPA, pertussis toxin, L-glutamine, penicillin G, and streptomycin were purchased from Sigma Chemical (St. Louis, MO). Buffers and salts were from Merck (Darmstadt, FRG). The cDNA coding for the rat A₁ adenosine receptor in the plasmid vector pBC-A1R (Freund et al., 1994) and the HEK 293 cell clone expressing the human A₁ adenosine receptor were kindly provided by M. J. Lohse (University of Würzburg). The human D_{2 (short isoform)} plasmid vector and NF037 were generous gifts of C. Pifl (Institute of Biochemical Pharmacology, Vienna University) and of P. Nickel (Institut of Pharmaceutical Chemistry, University of Bonn), respectively. The vectors pEGFP-C1 and pRc-CMV were obtained from Clontech (Palo Alto, CA).

Generation of transient and stable cell lines. COS-7 (African green monkey kidney fibroblasts) cells were plated at a density of 3 × 10⁶ cells/10-cm dish and transiently transfected with 5 µg of the cDNAs pBC-A1dhfr containing the rat A₁ adenosine receptor cDNA insert (Freund et al., 1994) and pCMV5 plasmid vector containing the D_2short receptor cDNA using the calcium phosphate precipitation method (Chen and Okayama, 1988). The cells were harvested 48 hr after transfection; plasma membranes were prepared and used for radioligand binding assays. HEK 293 cells were plated at a density of 2.5 × 10⁶ cells/10-cm dish and transfected with 7.5 µg of the plasmid pBC-A1dhfr (encoding the rat A₁ adenosine receptor) and 0.75 µg of the resistance marker plasmid pRc-CMV carrying the neomycin phosphotransferase gene. Similarly, the plasmid encoding the short splice variant of the human D₂ dopamine receptor was cotransfected with either pRc-CMV or pEGFP-C1, a vector carrying a red-shifted variant of wild-type green fluorescent protein cDNA from the jellyfish Aequoria victoria and a neomycin resistance cassette. The cells were grown in Dulbecco's modified Eagle medium containing 10% fetal calf serum, 2 mM L-glutamine, -mercaptoethanol, nonessential amino acids, 100 units/ml penicillin G, and 100 µg/ml streptomycin at 5% CO₂ and 37° for 16 hr. Thereafter, the medium was removed, and the cells were subjected to an osmotic shock by adding 15% glycerol in phosphate-buffered saline for a few seconds. Cells were grown for another 24 hr and subsequently selected by adding G418 (0.8 mg/ml) to the medium for 4-6 days. pEGFP-positive clones were identified by fluorescence microscopy. Positive clones appeared in bright green and were subjected to further selection to obtain clones with different expression levels. Three clones were selected that differed in D₂ dopamine receptor density (ranging from ~0.3 to 4 pmol/mg membrane protein).

Membrane preparation and protein purification. Cells were grown to confluency in 10-cm tissue culture dishes, washed once with ice-cold phosphate-buffered saline, and scraped off their plastic support in HME buffer (25 mM HEPES·NaOH, pH 7.5, 2 mM MgCl₂, 1 mM EDTA). After centrifugation at 20,000 × g for 10 min, the cell pellet was resuspended in HME, subjected to a freeze/thaw cycle with liquid nitrogen, and further homogenized by sonication. Membranes were sedimented by centrifugation (38,000 × g for 10 min) and resuspended in HME at a protein concentration of 8-10 mg/ml and stored in aliquots at 80°. Recombinant (R) G_i-1 and rG_i-2 were expressed in Escherichia coli BL21DE3 harboring a plasmid-encoding yeast myristoyl-CoA transferase and purified from bacterial lysates (Mumby and Linder, 1994). Oligomeric G proteins were purified from bovine or porcine brain, and free dimers were chromatographically resolved from the  subunits (Casey et al., 1989).

Radioligand binding experiments. Equilibrium binding with the A₁ adenosine receptor agonist [¹²⁵I]HPIA and with the dopaminergic D₂ agonist [¹²⁵I]OH-PIPAT were carried out in a final volume of 40 µl containing 50 mM Tris·HCl, pH 8, 1 mM EDTA, 5 mM MgCl₂, 1 mM ascorbic acid, 8 µg/ml adenosine deaminase, 10 µg of membrane protein, and the concentrations of suramin and NF037 as indicated (ascorbic acid and adenosine deaminase are not required for determining binding to A₁ adenosine and D₂ dopamine receptors, respectively, but were present in all incubations to obtain identical incubation conditions). The binding reaction was carried out for 90 min at 25° and terminated by filtration over glassfiber filters using a cell harvester (Skatron, Lier, Norway). Nonspecific binding was determined in the presence of 1 µM XAC (for A₁ adenosine receptors) or 10 µM sulpiride (for D₂ dopamine receptors) and amounted to ~5-10% of total binding in the K_D concentration range. In experiments using membranes from clones with a high receptor expression level (1.5-3.9 pmol/mg of membrane protein) or low radioligand concentrations, the amount of membrane protein added and the assay volume was adjusted (up to 250 µl) to avoid depletion of the radioligand (bound <10% of total). Specific binding of agonist or antagonist radioligands ([³H]DPCPX and [¹²⁵I]epideprid for A₁ adenosine and D₂ dopamine receptors, respectively) was not detectable in membranes prepared from untransfected HEK 293 cells. Radioligand binding to membranes from transfected cells was displaced by unlabeled receptor ligands with the appropriate pharmacological specificity, and specific binding for both agonists and antagonist radioligands was saturable; B_max values for antagonist binding were ~1.2-fold higher than those for agonist radioligands, indicating that the majority of the receptors were capable of interacting with G proteins endogenous to the HEK 293 membranes (not shown).

Receptor-mediated [³⁵S]GTPS binding. Receptor-promoted binding of [³⁵S]GTPS was determined essentially as described previously (Nanoff et al., 1995). In brief, membranes from HEK 293 cells (~10 µg) were suspended in 40 µl of buffer containing 25 mM HEPES·NaOH, pH 7.5, 1.5 mM MgCl₂, 150 mM NaCl, 1 mM EDTA, 0.01 mM GDP, and the concentrations of dopamine, CPA, and suramin analogues indicated in the respective figures. After a preincubation of 10 min at 25°, the assay was initiated by adding 10 µl of buffer containing [³⁵S]GTPS to yield a final concentration of 1 nM (specific activity, 2000 cpm/fmol). The assay was terminated after 10 min by adding 0.5 ml of ice-cold stop buffer containing 10 mM Tris·HCl, pH 8.0, 100 mM NaCl, 20 mM MgCl₂ and 0.1 mM GTP. Bound and free nucleotides were separated by filtration over glassfiber filters.

Determination of adenylyl cyclase activity. Adenylyl cyclase activity in HEK 293 membranes expressing the recombinant receptors was assayed in 0.1 ml containing 50 mM HEPES·NaOH, pH 8.0, 0.05 mM [-³²P]ATP (~200 cpm/pmol), 5 mM MgCl₂, 0.1 mM rolipram, 10 mM creatine phosphate, membrane protein (25 µg), 1 mg/ml creatin kinase, 8 µg/ml adenosine desaminase, and 1% bovine serum albumin. Inhibitory regulation of adenylyl cyclase by the D₂ dopamine and A₁ adenosine receptor agonists was determined in the presence of 1 µM prostaglandin E₁ and 10 nM GTPS. The reaction was carried out for 20 min at 25°; cAMP was separated from ATP by sequential chromatography on Dowex and Alumina (Johnson and Salomon, 1991).

Pertussis toxin treatment and reconstitution of HEK 293 cell membranes with rG_i-1. HEK 293 cells expressing the rat A₁ adenosine, human A₁ adenosine, or human D₂ dopamine receptors were incubated with 100 ng/ml pertussis toxin for 24 hr, and membranes were prepared as described. To insert exogenously added G protein into the membranes, the stable reconstitution protocol (Freissmuth et al., 1991a) was adapted as follows: PTX-treated membranes were incubated with 4.5 ng pf rG_i-1/µg membrane protein in HME containing 1% octylglucoside. After 1 hr on ice, membranes were diluted 1:10 in detergent-free buffer and centrifuged at 38,000 × g for 12 min. Pellets were resuspended in HME and stored in aliquots at a concentration of ~10 mg/ml at 80°. The amount of rG_i-1 incorporated into the membranes was determined by immunoblotting. To assess the potency of rG_i-1 to restore high affinity agonist binding, rG_i-1 was combined with a 4-fold molar excess of purified dimers in 1% octylglucoside (or 10 mM CHAPS); appropriate dilutions were added to the membranes to give 0.5% octylglucoside (or 5 mM CHAPS) and preincubated on ice for 15 min. Subsequently, radioligand binding assays were carried out after diluting the detergent 2-fold.

Immunoblots. Membrane proteins (~25 µg/lane) were separated on SDS-polyacrylamide gels (10% acrylamide, 0.13% bisacrylamide) and transferred to nitrocellulose membranes that were probed with AS7, an antiserum recognizing G_i-1 and G_i-2 (McClue et al., 1992) or with the G_i-1-specific antiserum I1C (Selzer et al., 1993). The immunostained bands were visualized by enhanced chemoluminescence using an anti-rabbit IgG antibody conjugated to horseradish peroxidase (Amersham, Arlington Heights, IL). Purified recombinant G protein  subunits were used as standards. To verify that comparable amounts of membrane proteins had been applied in individual lanes, blots also were probed with a rabbit antiserum directed against the G protein  subunit (Hohenegger et al., 1995).

	Results

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Uncoupling of A₁ adenosine and D₂ dopamine receptors after heterologous expression. High affinity binding of agonists to G protein-coupled receptors depends on the formation of a ternary complex of agonist, receptor, and G protein (Hepler and Gilman, 1992).After stable expression of the human D₂ dopamine and the rat and human A₁ adenosine receptors in HEK 293 cells, the coupling of the receptors with G proteins in the membrane was assessed by using agonist radioligands. Suramin and NF037 did not block binding of appropriate antagonist radioligands to the receptors (not shown; see Beindl et al., 1996) but inhibited equilibrium binding of the A₁-selective agonist [¹²⁵I]HPIA and the D₂-dopaminergic agonist [¹²⁵I]OH-PIPAT (Fig. 1); suramin ( in Fig. 1) was >10-fold more potent than NF037 ( in Fig. 1) in suppressing ternary complex formation of the rat (Fig. 1A) and human (Fig. 1B) A₁ adenosine receptor. In contrast, the compounds were equipotent in inhibiting binding of the D₂ dopamine receptor agonist [¹²⁵I]OH-PIPAT (Fig. 1C). The same difference was seen if the rat A₁ adenosine receptor and human D₂ dopamine receptor were transiently expressed in a cell line of nonhuman origin, namely, COS-7 cells (not shown). In addition, the apparent affinity of suramin and of NF037 was highest for human D₂ receptor/G protein complexes and lowest for human A₁ receptor/G protein complexes (Table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Inhibition of agonist radioligand binding to rat A1 (A), human A1 adenosine (B), and human D2 dopamine receptors (C) stably expressed in HEK 293 membranes by suramin and NF037. The binding reaction was carried out in 40 µl containing membranes (6-12 µg of protein), and [125I]HPIA (final concentration, 1.5 nM, A and B) or [125I]7-OH-PIPAT (0.7 nM, C) and increasing amounts of suramin () and NF037 () for 90 min at 25° as outlined in Experimental Procedures. The membranes used in C were from the cell clone expressing D2 dopamine receptors at 0.3 pmol/mg. Nonspecific binding was determined in the presence of 1 µM XAC or 10 µM sulpiride. Specific binding in the absence of any analogue (1.5-2 fmol ligand bound) was set 100%. Data are mean ± standard error values from three separate experiments carried out in duplicate.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Inhibition of [35S]GTPS binding to HEK 293 membranes expressing the rat (A and B) and human A1 adenosine receptor (C) and the human D2 dopamine receptor (D) by suramin and NF037. A and B, [35S]GTPS binding to HEK 293 membranes (10 µg) expressing the rat A1 adenosine receptor was determined in the presence of 0.3 µM CPA (, ) or of 1 µM XAC (, ) and of increasing concentrations of suramin (, ) and NF037 (, ). The binding reaction was initiated by the addition of 1 nM [35S]GTPS and carried out for 10 min at 25° as outlined in Experimental Procedures. B, Basal binding of [35S]GTPS was subtracted from CPA-stimulated binding. C, [35S]GTPS binding was determined as in A using membranes expressing the human A1 adenosine receptor. Shown is the agonist-stimulated binding (i.e., the difference between total binding in the presence of CPA and basal binding in the presence of XAC). D, [35S]GTPS binding was determined as in A using membranes expressing the human D2 dopamine receptor at a level of 3.9 pmol/mg, dopamine (1 µM) as the agonist, and sulpiride (10 µM) as the antagonist. Shown is the agonist-stimulated binding (i.e., the difference between total binding in the presence of dopamine and basal binding in the presence of sulpiride). Total [35S]GTPS binding in the presence of CPA (human A1, 167 ± 33 fmol/mg; rat A1, 168 ± 12 fmol/mg) or dopamine (153 ± 13 fmol/mg) was set at 100%. Data are mean ± standard error from three experiments carried out in duplicate.

Stable reconstitution of high affinity agonist binding to membranes from pertussis toxin-treated cells by rG_i-1. The different affinity of suramin and NF037 for ternary complexes formed by the A₁ adenosine and D₂ dopamine receptor may have been due to an interaction of the receptors with distinct G proteins. This possibility is substantiated by the following observations: both A₁ adenosine and D₂ dopamine receptors physiologically recruit adenylyl cyclase inhibition as one of the signaling pathways to elicit a biological response, and a marked (>50%) inhibition of prostaglandin E₁-stimulated cAMP formation was observed for both receptor types in intact transfected HEK 293 cells (Roka F and Nanoff C, unpublished observations). However, attenuation of adenylyl cyclase activity was observed only on activation of the D₂ dopamine receptor in HEK 293 membranes, whereas A₁ adenosine receptors were inactive (data not shown). We therefore determined the expression of G_i in the transfected cells. Two forms of G_i were detected in HEK 293 membranes (Fig. 3A). The top band commigrates with purified rG_i-1 and is recognized by I1C, an antiserum specific for G_i-1 (see Fig. 3B). The bottom band is detected only by the antiserum that reacts with G_i-1 and G_i-2 but not by antiserum I1C and commigrates with rG_i-2. The levels of G_i-2 and of the G protein  subunits (migrating as a 35/36 doublet) were comparable in all cell lines. In contrast, there was some variability in the level of G_i-1; the membranes from the cell line that expressed the A₁ adenosine receptor (Fig. 3A, lanes 4 and 5) had more G_i-1 than the control cells (Fig. 3A, lane 2), whereas those harboring the D₂ dopamine receptor contained less G_i-1 (Fig. 3A, lane 3). However, this is most likely not related to an effect of receptor expression per se; other cell clones that expressed more D₂ receptors than that shown in Fig. 2A had G_i-1 levels comparable to those of untransfected control cells (not shown). This suggests that the variation in the amount of G_i-1 may be due to clonal selection.

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Immunodetection of Gia subunits in native HEK 293 cell membranes (A) and after pertussis toxin treatment and stable reconstitution (B). A, Membrane proteins (25 µg/lane) from untransfected control cells (lane 2), HEK 293 cells expressing the human D2 dopamine receptors (lane 3), as well as the rat (lane 4) and human A1 adenosine receptor (lane 5) were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose; the blot was immunostained with AS7 (an antiserum recognizing Gi-1 and Gi-2). Lane 1, rGi-1 (10 ng). Lane 7, rGi-2 (5 ng). Lane 6, combination of rGi-1 (10 ng) and rGi-2 (5 ng). To rule out differences in the amount of protein loaded in each lane, the blot was also probed with a G protein -subunit antiserum (arrow). B, Confluent cultures of HEK 293 cells expressing the recombinant receptors were treated with vehicle (Con) or 100 ng/ml pertussis toxin (PTX) for 24 hr; membranes prepared from the latter cells were also stably reconstituted with recombinant rGi-1 (Rec) as outlined in Experimental Procedures. Membrane proteins (~25 µg) from HEK 293 cells expressing the D2 dopamine receptor (lane 1-3) and the human A1 adenosine receptor (lane 5-7) were separated on a 10% polyacrylamide gel and transferred to nitrocellulose; the blot was analyzed using the Gi-1-specific antiserum I1C. Lane 4,immunostaining of 5 ng Gi-1. Two additional experiments gave comparable results.

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View larger version (18K): [in this window] [in a new window]   Fig. 4.   Agonist radioligand binding to the rat (A) and human A1 adenosine (B) and human D2 dopamine receptors (C) stably expressed in HEK 293 cell membranes. Membranes were prepared from control () and pertussis toxin-treated () HEK 293 cells expressing the recombinant rat (A) and human (B) A1 adenosine receptors and the human D2 dopamine receptors (C). Membranes prepared from pertussis toxin-treated cells were also stably reconstituted with Gi-1 (4.5 ng/mg; ). Saturation isotherms were generated with the agonist radioligands [125I]HPIA for rat (A) and human A1 adenosine receptors (B) or [125I]OH-PIPAT for the human D2 dopamine receptor (C). The binding reaction was carried out in 40 µl containing membranes (2-5 µg) and the indicated concentrations of the radioligands for 90 min at 25°. Nonspecific binding was determined in the presence of 1 µM XAC (A, B) or 10 µM sulpiride (C) and amounted to ~5% of total binding. Insets (A and C), saturation isotherms of the stably reconstituted membranes () with the y-axis range (fmol/mg of radioligand bound) scaled down. Data are mean values of duplicate determination; two additional experiments gave similar results.

Uncoupling of A₁ adenosine and D₂ dopamine receptors after stable reconstitution. The reconstituted membranes in which the receptors were forced to interact with identical G protein  subunits were used to evaluate the ability of suramin and NF037 to inhibit receptor agonist binding (Fig. 5). The selectivity of the two compounds toward the individual receptor/G protein tandems was essentially unchanged (compare Figs. 1 and 5). In addition, the IC₅₀ estimates obtained for inhibition of [¹²⁵I]OH-PIPAT binding to the D₂ dopamine and of [¹²⁵I]HPIA binding to the human A₁ adenosine receptor were identical for suramin and NF037 in native and reconstituted membranes (Table 1). Only after reconstitution of the rat A₁ adenosine receptors with exogenous rG_i-1 complement was the inhibitory potency of both compounds moderately shifted to higher IC₅₀ values.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Suramin- and NF037-mediated inhibition of agonist radioligand binding to membranes prepared from pertussis toxin-treated rat A1 (A), human A1 adenosine (B), and human D2 dopamine receptors (C) in HEK 293 membranes that had been reconstituted with rGi-1. Membranes prepared from pertussis toxin-treated cells were stably reconstituted with rGi-1, and the binding reaction was carried out in 40 µl containing membrane protein (~10 µg), [125I]HPIA (final concentration, 1.5 nM in A and B), or [125I]OH-PIPAT (0.7 nM, C) and increasing amounts of suramin () and NF037 () for 90 min at 25° as outlined in Experimental Procedures. The reconstituted membranes used in C were from the cell clone expressing D2 dopamine receptors at 3.9 pmol/mg. Specific binding in the absence of any analogue (~1 fmol ligand bound) was set 100%. Data are mean ± standard error from three independent experiments carried out in duplicate.

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View larger version (25K): [in this window] [in a new window]   Fig. 6.   Reconstitution of high affinity agonist binding to A1 adenosine and D2 dopamine receptors in pertussis toxin-treated HEK 293 cell membranes by rGi-1. Membranes (~10 µg protein) prepared from pertussis toxin-treated cells expressing the rat A1 (), the human A1 adenosine (), or the human D2 dopamine receptors () were preincubated with increasing concentrations of a combination of rGi-1 and (molar ratio = 1:4) in the presence of 0.5% octylglucoside. The binding reaction included radioligand (~1.9 nM [125I]HPIA or ~0.6 nM [125I]PIPAT), rGi-1 at the indicated concentrations, and octylglucoside at a final concentration of 0.25%. Nonspecific binding was assayed in the presence of 1 µM XAC or 10 µM sulpiride. In the same experiment, specific binding was determined in untreated HEK 293 membranes and was set at 100%. These values were 158 ± 41, 447 ± 5, and 576 ± 87 fmol/mg for human and rat A1 adenosine and the human D2 dopamine receptor, respectively. Data are mean ± standard error from three experiments.

Effect of receptor occupancy on the apparent affinity of suramin and NF037. Suramin and NF037 do not compete for binding of antagonists to the A₁ adenosine and D₂ dopamine receptors, nor do they inhibit the binding of agonists in the absence of a productive interaction between receptor and G protein (Beindl et al., 1996). If the receptors were allowed to couple to G proteins, suramin inhibited agonist binding in a quasicompetitive manner (i.e., the IC₅₀ values increased at higher concentrations of the agonist radioligand; Beindl et al., 1996; see also below). This phenomenon may result from a competition of the agonist-liganded receptor with suramin for binding to the G protein or, alternatively, from the direct action of suramin on the receptor to prevent the agonist-promoted transition of the receptor to the active conformation R*. In this case, variations in the membrane concentration of the receptor should not affect the IC₅₀ values of suramin analogues. This was tested by using membranes from the three clones of HEK 293 cells expressing different D₂ dopamine receptor densities (0.3, 1.3, and 3.9 pmol/mg). The IC₅₀ value of suramin and NF037 was determined in the presence of the agonist radioligand [¹²⁵I]OH-PIPAT at a concentration close to the K_D value (0.5 nM). As shown in Fig. 7A for NF037, the IC₅₀ value was shifted to the right with increasing expression levels of the receptors. The same was true for suramin and the decrease in the apparent affinity of suramin and of NF037 was related in a linear manner to the amount of bound agonist (Fig. 7B, solid symbols). Control experiments were carried out with the D₂-dopaminergic antagonists sulpiride and haloperidol; as expected, receptor density did not affect IC₅₀ values of the receptor antagonists (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Correlation between receptor agonist occupancy and the IC50 values for suramin and NF037 in uncoupling the human D2 dopamine receptor. A, Inhibition of [125I]OH-PIPAT binding was performed with membranes derived from three different HEK 293 cell clones expressing the D2 dopamine receptor at various densities ( 0.3,  1.3,  3.9 pmol/mg). Concentration-dependent inhibition of agonist binding by NF037 was determined at a concentration of 0.5 nM [125I]OH-PIPAT with 1-10 µg of membrane protein such that the proportion of bound radioligand was <10% of the amount of radioligand added to the incubation volume. Inhibition curves were fitted by nonlinear least-squares regression analysis. B, The IC50 of NF037 () determined in Fig. 1A and those of suramin () determined in parallel were replotted as a function of [125I]OH-PIPAT bound. In addition, inhibition binding experiments with NF037 () and suramin () were carried out at logarithmically spaced concentrations of radioligand (0.1-1 nM) on membranes prepared from the clone with intermediate expression levels (Bmax = 1.3 pmol/mg) resulting in the indicated occupancy of the receptor by [125I]OH-PIPAT. Each point represents the IC50 ± standard error of the estimate from three independent experiments. Straight lines, drawn by calculating the linear regression through all data points. C, The IC50 value of suramin was determined in inhibition experiments with various concentrations of [125I]HPIA (0.15-7.5 nM) on HEK 293 membranes that carried the human A1 adenosine receptor (). These IC50 values were plotted as a function of relative receptor occupancy (i.e., Bmax was set at 1.0). The IC50 values of suramin in inhibiting [125I]OH-PIPAT binding to the membranes with intermediate expression levels (; Bmax = 1.3 pmol/mg) from B were replotted for the purpose of comparison and receptor occupancy was also expressed as a fraction of Bmax.

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	Discussion

Top Summary Introduction Procedures Results Discussion References

The current results unequivocally demonstrate that suramin and its didemethylated analogue NF037 discriminate among receptor/G protein tandems formed by the A₁ adenosine and the D₂ dopamine receptor regardless of whether the inhibition of high affinity agonist binding or of agonist-stimulated binding of [³⁵S]GTPS was determined. Suramin was more potent than NF037 in uncoupling A₁ adenosine receptors, whereas the two compounds were equipotent in preventing the interaction of the D₂ dopamine receptor with G proteins. These observations complement and extend previous work that was carried out on A₁ adenosine receptors in human brain cortex and D₂ dopamine receptors in rat striatum. Here, we used both the rat and human A₁ adenosine receptor and therefore rule out species differences as a trivial explanation for the distinct activity profiles of the two compounds. Because the receptors were stably expressed in the same cell line, an effect of cellular heterogeneity also can be ruled out. In agreement with this conclusion, the activity profile of the compounds was indistinguishable from the findings obtained in HEK 293 cells when the receptors were transiently expressed in COS-7 cells to obtain a nonhuman tissue readout system (not shown). Finally, the receptors may have been targeted to different subcellular compartments that possibly differed in composition of G protein subunits; when heterologously expressed in a cell line derived from a polarized epithelium, ₂-adrenergic and A₁ adenosine receptors are localized in the basolateral and apical membrane, respectively (Saunders et al., 1996; Wozniak and Limbird, 1996). This potential source of error was eliminated by pretreating the cells with pertussis toxin and stably reconstituting high affinity agonist binding to the membranes with a defined G protein  subunit (rG_i-1). Thus, the higher affinity of NF037 for uncoupling the D₂ dopamine receptor (compared with its ability to uncouple A₁ adenosine receptors) is maintained even when the receptors are forced to interact with identical G protein  subunits. Uncoupling of the D₂ dopamine receptor/G protein complex by suramin analogues gave inhibition curves with varying slopes (Figs. 1C and 5C). On pertussis toxin treatment and reconstitution with G_i-1, the inhibition curves were shallower than in the control membranes. A steep slope (Hill coefficient ~ 2) suggests interference with a reaction different from the 1:1 mode of receptor/G protein coupling, such as through the formation of receptor dimers. Dimerization of G proteincoupled receptors might result in enhanced signaling efficacy as opposed to the monomeric form of receptor (Hebert et al., 1996). On the basis of evidence obtained with other types of G protein-coupled receptors (Hebert et al., 1996; Cvejic and Devi, 1997), it is attractive to speculate that the D₂ dopamine receptor in HEK 293 cells undergoes dimerization leading to steep inhibition curves with the suramin analogues. On pertussis toxin treatment of the membranes and reconstitution with G_i-1 shallow inhibition curves (Hill coefficient ~ 1) would suggest that the ability to dimerize is lost after manipulation of the membranes. Nevertheless, the slope of the inhibition curves but not the IC₅₀ values was independent of the fractional receptor occupancy generated in the inhibition experiments (see Fig. 7A). Thus, although we have no direct evidence to explain the changes in slopes, we believe that this discrepancy does not interfere with our conclusions. Finally, we stress that experiments with purified  subunit and [³H]suramin demonstrate a binding stoichiometry of 1:1 (Hill coefficient ~ 1.0; Hohenegger et al., 1998).

If the pertussis toxin-treated membranes were stably reconstituted with rG_i-1, large differences in reconstitution efficiencies were observed, and the rank order was human A₁ > rat A₁ > human D₂ receptor; the amount of G protein incorporated into the membrane was clearly not limiting because the  subunit was present in vast excess over the receptor level. We therefore hypothesized that the differences in reconstitution reflected the rank order of affinity of the individual receptors for rG_i-1. If correct, it was likewise sensible to assume that the affinities of individual receptors for the same G protein is the major determinant for the potency of suramin in differentially inhibiting receptor/G protein coupling. The difference in G protein affinity was confirmed by titrating the ability of exogenously added rG_i-1 to reconstitute high affinity binding to pertussis toxin-treated membranes. We previously determined the affinity of the human A₁ adenosine receptor expressed in E. coli in reconstitution experiments with individual forms of recombinant G protein  subunits (Jockers et al., 1994); the affinity currently observed for the interaction of the human A₁ adenosine receptor in pertussis toxin-treated HEK 293 membranes with rG_i-1. (~6 nM) was in reasonable agreement with that estimated in the earlier work (~15 nM). It was, on the other hand, somewhat surprising that the affinity of the D₂ dopamine receptor for rG_i-1. was so low.

The interaction between the D₂ dopamine receptor and rG_i-1. may have been impeded by the presence of octylglucoside. We therefore exploited the Cheng-Prusoff relation to independently estimate the affinity of the human D₂ dopamine receptor for its cognate G protein in HEK 293 membranes. This approach is valid if suramin competes with the agonist-liganded receptor for binding to the G protein  subunit. All available evidence supports this assumption: (1) suramin analogues bind directly to G protein subunits (Freissmuth et al., 1996), (2) they do not affect binding of antagonists or agonists to the receptor in the absence of receptor/G protein coupling (Beindl et al., 1996), (3) the inhibition of receptor/G protein coupling can be overcome by raising the concentration of active receptor in the membrane (see Fig. 7), and (4) if the site of action of suramin is on the G protein, the regression lines in the plot of IC₅₀ versus receptor occupancy are expected to yield similar y-axis intercepts; this was indeed observed. The affinity values estimated for the interaction between receptor and G protein were ~0.8 µM and ~10 nM for agonist-liganded D₂ dopamine receptor and the human A₁ adenosine receptor, respectively, and hence consistent with the findings in the reconstitution experiments. Based on our experimental observations, we conclude that the affinity of the individual receptors for rG_i-1 is inversely correlated to the potency of suramin in uncoupling the receptors. This conclusion predicts that a selective action of suramin on receptor/G protein coupling can also result from the difference in affinity of individual agonist-liganded receptors for the same G protein. This may also explain the earlier observation that in membranes from NG108-15 cells, suramin inhibited activation of pertussis toxin-sensitive G proteins by -opioid agonists but not by serum factors (Huang et al., 1990).

The structural basis for the different activity profile of suramin analogues in uncoupling A₁ adenosine (suramin > NF037) and D₂ dopamine receptors (NF037 = suramin) is not known. The contact points by which receptors interact with their cognate G proteins are formed by those segments of the receptor that are juxtaposed to the transmembrane spans. These discontiguous segments cooperatively support binding of the receptor to the G protein oligomer (Ernst et al., 1995; Gomeza et al., 1996) and determine the G protein specificity of the receptor (Wong et al., 1994; Liu and Wess, 1996; for review, see Gudermann et al., 1996b). The amino acids DRY (ERY in rhodopsin) at the beginning of the second intracellular loop are invariant and are required for G protein activation (Scheer et al., 1996). Apart from this triplet, only very few amino acids are conserved within the intracellular loops; hence, a clearcut consensus sequence that would allow to predict the G protein specificity of a given receptor cannot be deduced. In addition, the ability of a receptor to activate multiple G proteins is specified by distinct portions within the intracellular loops; the _2A-adrenergic receptor can couple to both G_s and G_i. These two coupling modes, however, require distinct amino acid stretches in the second and third intracellular loops (Eason and Ligett, 1996). It is even more striking that different amino acids in the third intracellular loop are required to support coupling of the _1B-adrenergic receptor to the closely related  subunits G_q, G_-14, and G_-16 (Wu et al., 1995). These findings predict that the surface the receptors cover on a given G protein  subunit varies in individual receptor/G protein tandems. This is indeed the case; if the five last amino acids in the carboxyl terminus are exchanged between G_s and G_q, some, but not all, receptors are capable of recruiting this mutated  subunit in a manner similar to their cognate G protein (Conklin et al., 1996). Hence, the contact sites that are formed in individual receptor/G protein tandems must be different to account for this observation. It is attractive to speculate that in the A₁ receptor/G_i-1 tandem, the receptor covers a larger area of the G protein  subunit than in the D₂ receptor/G_i-1 tandem. This hypothesis would explain both the higher affinity of the A₁ receptor for G_i-1 and the lower relative potency of NF037 in disrupting the A₁ receptor/G complex; in this model, suramin, which has two additional methyl groups, competes more efficiently than NF037 with the A₁ adenosine receptor for binding to the G protein, whereas the difference in surface covered by the two compounds does not affect the formation of the D₂ receptor/G protein complex. Taken together, our data show that two factors contribute to the selectivity of inhibitors of receptor/G protein tandem formation, namely (1) differences in affinity of individual receptors for the G protein (which determines the apparent IC₅₀ value of an inhibitor) and (2) differences in the contact site between individual receptors and the G protein (which gives rise to a distinct structure activity relation for inhibitors). Both aspects are relevant in the development of G protein inhibitors that may eventually be useful in vivo.