Introduction

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ARs are members of the G protein-coupled receptor superfamily and are classified into three groups (i.e., ₁, ₂, and ) (Bylund et al., 1994; Hieble et al., 1995). ARs consist of three subtypes, ₁, ₂, and ₃. Endogenous agonists, norepinephrine, epinephrine, and the synthetic full agonist isoproterenol bind to the ₂AR and induced conformational changes to activate the G protein. Mutagenesis experiments have revealed that the binding sites of isoproterenol on the ₂AR seem to be located in the TM regions (Dixon et al., 1987; Dohlman et al., 1988). It is assumed that aspartic acid at position 113 in the TM3 region of the ₂AR form an ionic bond with the amino group of isoproterenol (Strader et al., 1987, 1988, 1989a). It is also assumed that the catechol hydroxyl groups of the agonist isoproterenol interact with the side chains of Ser204 and Ser207 in the TM5 region of the receptor (Strader et al., 1989b). Although the TM regions are well conserved among the three AR subtypes (70% for ₁ versus ₂, 70% for ₁ versus ₃, 63% for ₂ versus ₃), the ₁AR and the ₂AR show different affinities for the various synthetic agonists and antagonists including the endogenous agonist norepinephrine (Emorine et al., 1989; Frielle et al., 1987; Kobilka et al., 1987; Stiles and Lefkowitz, 1984). Frielle et al. (1988) have constructed a series of chimeric ₁/₂ARs to analyze the binding domains of ₁- and ₂-selective ligands. The gradual replacement of the TM regions of the ₂AR with those of the ₁AR result in receptors that show a gradual loss of ₂AR selectivity and a gain in ₁AR selectivity. The ₁ARs with homologous replacement show a gradual loss of ₁AR selectivity and a gain in ₂AR selectivity. Frielle et al. (1988) have concluded that the TM4 region is a major determinant of the ₁ and ₂AR selectivity of agonist norepinephrine and that the TM6 or TM7 regions play a major role in determining ₂AR selectivity for the ₂AR antagonist ICI 118551 or a ₁AR selectivity for ₁AR antagonist betaxolol. Another group has employed the approach to randomly exchange the TM regions of the ARs and determine the binding characteristics of subtype selective antagonists in these chimeric receptors (Marullo et al., 1990). They have shown that the TM region's contribution to the subtype selective binding of the ₁ and ₂ARs differed between ligands. However, the random exchange of the TM regions may underestimate or overestimate the contribution of some TM regions to binding of a subtype selective ligand, because more than one TM region mutates at once and it is possible that some TM region are not involved in the subtype selective binding. Thus, determining which regions of the AR confer the subtype selectivity, especially for agonists, has so far been only tentative.

TA-2005 is a non-catechol ₂AR agonist with a p-methoxyphenyl group on the amine side chain and a 8-hydroxyl group on the carbostyril aromatic ring (see Fig. 1). We have previously shown that, compared with other ₁ and ₂ARs, TA-2005 has a high selectivity as well as a high affinity for the ₂AR in pharmacological and radioligand-binding studies using isolated guinea pig tissues (Kikkawa et al., 1991). Based on in vivo studies, we have reported that TA-2005 has long lasting bronchodilating effects (Kikkawa et al., 1994). Voss et al. (1992) have also reported that TA-2005 shows a high potency for the ₂AR and a long duration of action after removal of the drug using both guinea pig tracheal muscle relaxation and bovine trapezium muscle binding experiments.

In the present study we putatively assigned the binding sites of the ₂AR for carbostyril moiety of TA-2005 and determined the specific amino acid to be responsible for ₂-selective binding. We made several site-directed mutant ₂ARs and eight chimeric ₁/₂ARs, which were expressed in COS-7 cells, then analyzed the binding characteristics of TA-2005 and derivatives for these receptors, and compared these characteristics with those of isoproterenol.

	Experimental Procedures

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Materials

[¹²⁵I]Iodocyanopindolol (2200 Ci/mmol) and [³H]adenine (24.0-27.0 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). [¹⁴C]cAMP (20.0-50.1 mCi/mmol) was obtained from DuPont-New England Nuclear Research (Boston, MA), American Radiolabeled Chemicals (St. Louis, MO), or Moravek Biochemicals (Brea, CA). The plasmid constructs pBC-₁ and -₂ encoding for the human ₁ and ₂ARs were kindly provided by Dr. R. J. Lefkowitz (Duke University, Durham, NC). The mammalian expression vector pEF-BOS was a gift of Dr. S. Nagata (Osaka University, Osaka, Japan). ()Isoproterenol, (±)propranolol, and DEAE-dextran were purchased from Sigma Chemical (St. Louis, MO). DMEM and gentamicin were from GIBCO/BRL (Grand Island, NY). Fetal bovine serum was obtained from JRH Biosciences (Lenexa, KS). Taq or Pfu DNA polymerases were from Takara (Siga, Japan) or Stratagene (La Jolla, CA), respectively. GTP was from Seikagaku (Tokyo, Japan). TA-2005 (8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxy-phenyl)-1-methylethyl] amino]ethyl] carbostyril, hydrochloride), compound I (5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxy-phenyl)-1-methylethyl]amino]ethyl]quinolin-2(1H)-one, hydrochloride), compound II (5-[(2-amino-1-hydroxy)ethyl]-8-hydroxycarbostyril, hydrochloride), and compound III (5-[(1-hydroxy-2-isopropylamino)ethyl]-8-hydroxycarbostyril, hydrochloride) were synthesized at the Lead Optimization Research Laboratory, Tanabe Seiyaku (Saitama, Japan). The structures of these compounds are shown in Fig. 1.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   The chemical structures of TA-2005, compounds I-III derivatives of TA-2005, and isoproterenol.

DNA constructions, cell transfection, and culture. The epitope sequence (YPYDVPDYA) recognized by monoclonal antibody 12CA5 (Wilson et al., 1984) was inserted at the amino terminus of human ₁ and ₂ARs to evaluate the expression of the receptors (Barak et al., 1994; Sato et al., 1996; Von Zastrow and Kobilka, 1992). The epitope did not change the binding characteristics of ₁ and ₂ARs to the ligands (data not shown). Chimeric receptors and site-directed mutants of the ₁ and ₂ARs were constructed by the polymerase chain reaction method (Higuchi, 1989). The positions of the junctions for individual chimeric ₁ and ₂ARs are as follows (numbers refer to amino acid positions in the human ₁ and ₂AR sequences): CH1, ₁ 1-84/₂ 60-413; CH2, ₂ 1-71/₁ 97-131/₂ 107-413; CH3, ₂ 1-295/₁ 347-381/₂ 331-413; CH4, ₂ 1-71/₁ 97-131/₂ 107-295/₁ 347-381/₂ 331-413; CH5, ₂ 1-59/₁ 85-477; CH6, ₁ 1-96/₂ 72-106/₁ 132-477; CH7, ₁ 1-346/₂ 296-330/₁ 382-477; CH8, ₁ 1-96/₂ 72-106/₁ 132-346/₂ 296-330/₁ 382-477. The sequences of the amplified regions were confirmed by the dideoxy chain termination method (Sanger et al., 1977). Chimeric and mutated cDNAs were inserted into the EcoRI and BamHI or EcoRI and SalI sites of the mammalian expression vector pCMV5. The alanine-scanning point mutants of the ₂AR were made by the Quick change method according to manufacture's instructions (Stratagene, La Jolla, CA). The two oligonucleotides (33-36 base pairs) and the BglII/EcoRV fragment of the ₂AR in pSL1190 (Pharmacia LKB, Uppsala, Sweden) were used as primers or as a template, respectively. After the sequences were confirmed by the dideoxy chain termination method, the rest of the coding regions were ligated to make point-mutated full-length ₂ARs. The resulting constructs were inserted into the XbaI site of pEF-BOS (Mizushima and Nagata, 1990). For the binding studies, these constructs were transfected into COS-7 cells by the DEAE-dextran method (Cullen, 1987). Before the day of transfection, the COS-7 cells were seeded at 1.5 × 10⁶ cells per 100-mm dish. The concentration of the chimeric or mutated AR cDNAs were 5 µg per 100-mm dish. All cells were maintained in DMEM containing 10% fetal bovine serum and gentamicin (10 µg/ml). Two to three days after the transfection, the cells were harvested for preparation of the crude membrane fraction. For the cAMP accumulation assay, the expression constructs of WT-, S204A-, and S207A-₂ARs were transfected into the JEG-3 cells as described above, except that the concentration of DEAE-dextran was reduced to 250 µg/ml and the JEG-3 cells were seeded at 1.5-2.0 × 10⁶ cells/100-mm dish. The JEG-3 cells were maintained in DMEM containing 10% fetal bovine serum and gentamicin (10 µg/ml).

Radioligand binding assay. The cells were rinsed twice with 10 ml of ice-cold phosphate-buffered saline and mechanically detached in 1 ml of an ice-cold buffer containing 5 mM Tris·HCl (pH 7.4) and 2 mM EDTA. The lysate was centrifuged at 45,000 × g for 10 min at 4°. The pellet containing membrane fraction was resuspended in 1 ml of buffer containing 75 mM Tris·HCl (pH 7.4), 12.5 mM MgCl₂ and 2 mM EDTA with Potter type homogenizer and stored at 80° until use. A competition binding assay was performed in duplicate using ~10 µg of membrane protein, 50 pM ¹²⁵I-CYP, and 0-100 µM unlabeled ligand in the presence of 100 µM GTP for 60 min at 37°. The binding reaction was terminated by rapid filtration over Whatman GF/C filters and washing with an ice-cold solution containing 25 mM Tris·HCl (pH 7.4) and 1 mM MgCl₂. Nonspecific binding was determined in the presence of 5 µM (±)propranolol. The protein concentration was determined by the method of Lowry et al. (1951).

cAMP accumulation assay. Two days after the transfection, JEG-3 cells were incubated overnight with [³H]adenine (2 µCi/ml). On day 4 the cAMP accumulation was measured in the absence of activator (basal activity) or in the presence of test compounds for 15 min at 37° with 1 mM 3-isobutyl-1-methylxanthine. The reaction was terminated by the addition of 1 ml of ice-cold stop solution containing 2.5% perchloric acid, 0.2 mM cAMP, and [¹⁴C]cAMP (about 10,000 cpm). After being neutralized with 4.2 M KOH, the precipitate was removed by centrifugation at 5,000 rpm for 5 min at 4° in a microcentrifuge. The supernatant was sequentially processed by Dowex and by aluminum oxide columns for isolation of [³H]cAMP.

Data analysis and statistics. All results are expressed as an arithmetic mean together with mean ± standard error of the mean for n determinations except the K_i and K_d values, which are expressed as geometric means with 95% confidence limits. Equilibrium dissociation constants were determined from the saturation isotherms. Radioligand binding data were analyzed by a nonlinear regression analysis to determine IC₅₀ and K_i values using PRISM software (GraphPAD Software, San Diego, CA). Statistical significance was assessed with the analysis of variance for multiple comparisons; a probability value of p < 0.05 was considered as a significant difference.

	Results

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Interaction of TA-2005 with Ser204 and Ser207 of ₂AR. To investigate whether TA-2005 interacts with the same serine residues of the ₂AR as isoproterenol, we assessed the affinities of compound I, the des-8-hydroxy derivative of TA-2005, for the S204A- and S207A-₂ARs. The K_d values of ¹²⁵I-CYP for the WT-, S204A-, and S207A-₂ARs, respectively, were 56, 34, and 32 pM. TA-2005 had much higher affinity for the WT-₂AR than for isoproterenol and the affinity of TA-2005 for the S204A-₂AR was decreased 56-fold but only slightly for the S207A-₂ARs (4-fold), as compared with the WT-₂AR (Fig. 2) [see also Kikkawa et al. (1997)]. Isoproterenol bound to the S204A- and S207A-₂ARs with 27- and 13-fold lower affinities, respectively, than to the WT-₂AR. In the present study, although the affinity of compound I for the S204A-₂AR was decreased 22-fold, the affinity for the S207A-₂AR was essentially the same as that of the WT-₂AR (Fig. 2).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Competition binding of TA-2005 (A), isoproterenol (B), and compound I (C) to the wild-type and mutant 2ARs. Competition binding was performed with membranes prepared from COS-7 cells transfected with the WT-2AR (), S204A-2AR (), or S207A-2AR () expression constructs. The data are the mean ± standard error of three experiments done in duplicate.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Cyclic AMP production by TA-2005 (), isoproterenol (), and compound I () through activation of the WT-2 (A), S204A-2 (B), or S207A-2 (C) ARs. The cAMP accumulation assay was performed with JEG-3 cells transfected with one of the AR constructs. The data are the mean ± standard error of three experiments done in duplicate.

Selectivity for the WT-₂AR. To determine which portion of the TA-2005 molecule is important for ₂AR selectivity, we synthesized two compounds that lack p-methoxyphenyl (compound II) or p-methoxyphenyl methylethyl groups (compound III), and examined the affinities of these compounds for the WT-₁ and ₂ARs. TA-2005 showed a 53-fold higher selectivity for the ₂AR than for the ₁AR, whereas isoproterenol showed no selectivity for the ₁ and ₂ARs (Fig. 4, A and B). In contrast to TA-2005, compounds II and III completely lost their AR subtype selectivity (Fig. 4, C and D).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Competition binding of TA-2005 (A), isoproterenol (B), compound II (C), and compound III (D) to the WT-1 () and WT-2 () ARs. Competition binding was performed with membranes prepared from COS-7 cells transfected with either of the AR constructs. The data are the mean ± standard error of three experiments done in duplicate.

Affinity for ₁/₂AR chimeras. To determine the domain(s) of the ₂-receptor that interact with the p-methoxyphenyl group on the amine portion of TA-2005, eight ₁/₂ chimeric receptors were constructed and expressed in COS-7 cells (see Fig. 5 for structures). The ligand-binding properties of the resultant chimeric receptors are summarized in Tables 1 and 2. The K_d values of the radioligand ¹²⁵I-CYP in the chimeric receptors were essentially the same as those of the WT-₁ and ₂ARs except in CH4 with slightly low affinity of ¹²⁵I-CYP.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Structures of chimeric 1/2ARs. Thin lines, peptide sequences derived from the 1AR; thick lines, peptide sequences derived from the 2AR. The positions of the junctions are described in Experimental Procedures.

View this table: [in this window] [in a new window]   TABLE 1 Effects of substitution of the TM regions with corresponding portion of the 1AR on the ligand-binding characteristics of the 2AR The binding of ligands to the WT- and CH-ARs was measured by competition with 50 pM 125I-CYP. The data were analyzed using the nonlinear least-squares regression computer program as described in Experimental Procedures. The results are shown as the means and 95% confidence limits of three to four separate experiments.

View this table: [in this window] [in a new window]   TABLE 2 Effects of substitution of the TM regions with corresponding portion of the 2AR on the ligand-binding characteristics of the 1AR The binding of ligands to the WT- and CH-ARs was measured by competition with 50 pM 125I-CYP. The data were analyzed using the nonlinear least-squares regression computer program as described under Experimental Procedures. The results are shown as the means and 95% confidence limits from three to four separate experiments.

Alanine-scanning mutants of ₂ARs. There are 10 positions in the TM7 region of the ₂AR in which the amino acid residues are different from those of the ₁AR. To identify the amino acid which is important in ₂-selective agonist binding, each of the amino acids was changed to alanine. One mutant (Y308A-₂AR) out of 10 different alanine-substituted mutants, in which Tyr308 was changed to alanine, showed a dramatically decreased affinity for TA-2005 (Table 3). Although the Y308A-₂AR also showed the decreased affinity for isoproterenol, the extent of the decrease in affinity was smaller than that of TA-2005. Furthermore, the Y308F-₂AR mutant, in which Tyr308 in the TM7 region of the ₂AR was replaced with the corresponding amino acid residue (Phe) of the ₁AR, showed decreased affinity for TA-2005 (Y308F-₂AR: 61 nM versus WT-₂AR: 12 nM, p < 0.05), although that of isoproterenol for Y308F-₂AR was essentially the same as that of the WT-₂AR (1800 and 900 nM, respectively). The replacement of Phe359 of the ₁AR with Tyr (F359Y-₁AR), which is a complementary mutant of Y308F-₂AR, increased the affinity for TA-2005 by 2.5-fold. However, we have also observed that F359Y-₁AR decreased the affinity for ¹²⁵I-CYP by 19-fold, whereas it increased the affinity for isoproterenol by 7-fold (data not shown).

View this table: [in this window] [in a new window]   TABLE 3 Effects of substitution of single amino acid in the TM7 region of the 2AR with alanine on the ligand-binding characteristics The binding of ligands to the WT- and mutant-ARs was measured by competition with 50 pM 125I-CYP. The data were analyzed using the nonlinear least-squares regression computer program as described under Experimental Procedures. Results are shown as the means and 95% confidence limits from three separate experiments.

	Discussion

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Mutagenesis experiments have determined the binding sites of the nonselective agonists and selective antagonists for the ₂AR (Dixon et al., 1989; Frielle et al., 1988; Marullo et al., 1990). However, the binding sites of the selective ₂AR agonists are little known. We have studied the binding sites of TA-2005, one of the most selective ₂AR agonists with a hydroxy-carbostyril structure. TA-2005 has one hydroxyl group and one amido group at positions corresponding to the p- and m-hydroxyl groups of isoproterenol, respectively (Fig. 1).

At first, we compared the binding sites of the hydroxy-carbostyril moiety of TA-2005 to those of the catechol moiety of isoproterenol. For this purpose, we synthesized compound I and constructed two site-directed mutants in which Ser204 or Ser207 in the TM5 region were changed to alanine. It was proposed that these serines should interact with the m- and p-hydroxyl groups of isoproterenol (Strader et al., 1989b). Replacement of Ser204 or Ser207 with alanine decreased the affinities of isoproterenol and TA-2005, although the change in the affinity of TA-2005 for the S207A-₂AR was smaller than that of isoproterenol (Kikkawa et al., 1997). In contrast with binding experiments, the functional assay for TA-2005-stimulated cAMP accumulation showed no differences in the EC₅₀ values of TA-2005 in the WT-₂AR and the two mutants. These data suggest that Ser204 may be an important determinant for the high affinity binding of TA-2005 but not necessarily for the activation of adenylyl cyclase. It also suggests that one of the two serines in the TM5 region of the ₂AR may be enough to activate the adenylyl cyclase by TA-2005 with high affinity. Because the affinity of compound I for the S207A-₂AR was similar to that for the WT-₂AR, and the affinity of compound I decreased in the S204A-₂AR, it seems that the 8-hydroxyl group of TA-2005 may interact with the Ser207 hydroxyl group of the ₂AR. However, it is necessary to analyze the interaction of another derivatives of TA-2005 with more mutants to determine the precise interaction sites for the hydroxy-carbostyril moiety and the TM5 region of the ₂AR.

To determine the structural basis for the ₂AR selectivity, we assessed the affinities of TA-2005 and its derivatives for a series of chimeric ₁/₂ARs. Although TA-2005 had a 53 times higher affinity for the ₂AR than for the ₁AR, compound II and III which lacked the p-methoxyphenyl group of TA-2005 completely lost their ₂ selectivity. Kontoyianni et al. (1996) have suggested that, based on a computer-modeling technique, the large 2-phenylethyl N-substituent of TA-2005 can lie in a pocket formed by the TM2 and TM7 regions. Their binding model supports our finding that the replacement of either the TM2 or the TM7 regions of the ₂AR, or both with homologous regions of the ₁AR significantly decreases the affinities of TA-2005 but not of compound II and III. There have been several reports that the TM2 region of the gonadotropin-releasing hormone receptor should be in close the proximity to the TM7 region and that both regions participate in hormone binding (Arora et al., 1996; Awara et al., 1996; Davidoson et al., 1996; Zhou et al., 1994).

To avoid the misleading conclusions that can arise from the use of loss-of-function mutants, in which chimeric receptor mutants lose their the ₂ selectivity, we made a series of gain-of-function mutants, in which the chimeric receptor mutants gained ₂ selectivity. The replacement of the TM7 but not the TM2 regions significantly increased the affinities of TA-2005. Although the ₁AR with both TM2 and TM7 regions of the ₂AR almost completely restored the ₂ selectivity to the level of the WT-₂AR, the resultant chimeric receptor (CH8) also increased the affinity of isoproterenol. This suggests that the TM7 region of the ₂AR contributes to ₂-selective agonist binding, but the contribution of the TM2 region to this binding is not definitive. It has recently been reported that the long lipophilic side chain of salmeterol, a ₂-selective agonist, interacts with residues 149-158 within the TM4 region of the ₂AR (Green et al., 1996). It has also been shown that the TM4 region is a domain necessary for the persistent binding of salmeterol to the ₂AR (i.e., exosite of the ₂AR). This region, however, does not seem to contribute to ₂-selective binding of salmeterol, because the ₂AR with the TM4 region of the ₁AR lost its ability to persistently bind salmeterol but still retained the ₂ selectivity. This suggests that the region that confers ₂ selectivity to the ₂AR is distinct from the exosite. These results also support our assumption that the TM7 region is important for the ₂-selective agonist TA-2005 in binding to the ₂AR with high affinity.

To identify the amino acid that is important for the high affinity binding of a ₂-selective agonist, each of the amino acids in TM7 region that are different from those of the ₁AR were changed to alanine. One mutant (Y308A-₂AR) out of 10 alanine-substituted mutants significantly decreased its affinity for TA-2005. Furthermore, the affinity of TA-2005 for the Y308F-₂AR was significantly decreased, although the affinity of isoproterenol was essentially the same as that of the WT-₂AR. These results suggest that Tyr308 is a major determinant for the binding of the ₂-selective agonist TA-2005 and Tyr308 may interact with the side chains of N-substituted TA-2005. Although the affinity of TA-2005 increased in the F359Y-₁AR, which is a complementary mutant of the Y308F-₂AR, the affinities for ¹²⁵I-CYP and isoproterenol were also changed dramatically. This indicates that the replacement of Phe of the ₁AR with Tyr may cause an overall structural change of the ₁AR and the substituted Tyr may provide an additional binding site for the ligands. Photoaffinity labeling experiments have shown the direct interaction between the TM7 region and the aryloxy portion of the AR antagonists such as pindolol, CGP-12177A, and CYP (Dohlman et al., 1988; Hockerman et al., 1996; Wong et al., 1988). Each of the three photoaffinity labels, [¹²⁵I]iodocyanopindolol-diaserine, [¹²⁵I]iodoasidobenzylpindolol, and ¹²⁵I-asidophenyl CGP12177A was incorporated at the TM6 an TM7 regions of the purified AR as well as other TM regions, depending on the photoaffinity label. These data support our assumption that N-substituent of the ligands can interact with Tyr308 in the TM7 region and contribute to subtype selective binding with high affinity.

From the results of chimeric and alanine-substituted mutants, we have concluded that Tyr308 in the TM7 region of the ₂AR is crucial for the high affinity binding of the ₂-selective agonist TA-2005. This is the first report to show that a specific residue in the TM7 region is involved in the binding of a ₂-selective agonist. It remains to be determined whether Tyr308 also plays an important role in the binding of other ₂-selective agonists such as salmeterol, formoterol and procaterol.