The Role of the Seventh Transmembrane Region in High Affinity Binding of a β2-Selective Agonist TA-2005
- Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan
Abstract
To determine the structural basis for binding subtype selective agonists in the β-adrenergic receptor (βAR), we examined the interaction of the mutant β2AR and chimeric β1/β2AR with a selective β2AR agonist, TA-2005 (8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxyphenyl)-1-methylethyl]amino]ethyl] carbostyril hydrochloride). The β2AR mutant with Ala substituted for Ser204 (S204A) significantly decreased the affinities for TA-2005, des-8-hydroxy-TA-2005 derivative (compound I), and isoproterenol. In contrast, a S207A mutation slightly decreased the affinities for TA-2005 and compound I, although the affinity for isoproterenol was decreased dramatically. The EC50 values of TA-2005 to activate adenylyl cyclase were not changed in either the S204A- or S207A-β2AR. In contrast with TA-2005, the EC50 values of compound I were reduced in the S204A-β2AR but not in the S207A-β2AR. These results suggest that Ser204 is important for high affinity binding but not necessary to activate adenylyl cyclase. Although TA-2005 was highly selective at the β2AR, the compounds lackingp-methoxyphenyl-ethyl (compound II) orp-methoxyphenyl-methylethyl groups (compound III) on the amine portion of TA-2005 lost β2AR subtype selectivity. When the second and seventh transmembrane (TM) region but not the TM1 region of the β2AR were replaced with the corresponding regions of the β1AR, the affinities of the chimeras for TA-2005 decreased compared with those of the wild type β2AR. Furthermore, substitution of the TM7 region of the β1AR with the corresponding region of the β2AR significantly increased the affinities for TA-2005. The affinities for isoproterenol and compounds II and III were not affected in the chimeras. These data suggest that the TM7 region of the β2AR plays an important role in β2-selective agonist binding. To determine the specific amino acid which confers this high affinity binding of TA-2005 to the β2AR, an alanine-scanning mutagenesis approach was employed. All amino acids that were different from those of the β1AR were individually changed to alanine. One mutant receptor (Y308A-β2AR) out of 10 point-mutated β2ARs showed a dramatically reduced affinity for TA-2005. These results indicate that Tyr308 is an essential amino acid for high affinity binding of the β2-selective agonist TA-2005.
ARs are members of the G protein-coupled receptor superfamily and are classified into three groups (i.e., α1, α2, and β) (Bylund et al., 1994;Hieble et al., 1995). βARs consist of three subtypes, β1, β2, and β3. Endogenous agonists, norepinephrine, epinephrine, and the synthetic full agonist isoproterenol bind to the β2AR and induced conformational changes to activate the G protein. Mutagenesis experiments have revealed that the binding sites of isoproterenol on the β2AR seem to be located in the TM regions (Dixon et al., 1987; Dohlmanet al., 1988). It is assumed that aspartic acid at position 113 in the TM3 region of the β2AR 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 βAR subtypes (70% for β1 versus β2, 70% for β1 versus β3, 63% for β2 versus β3), the β1AR and the β2AR 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 β1/β2ARs to analyze the binding domains of β1- and β2-selective ligands. The gradual replacement of the TM regions of the β2AR with those of the β1AR result in receptors that show a gradual loss of β2AR selectivity and a gain in β1AR selectivity. The β1ARs with homologous replacement show a gradual loss of β1AR selectivity and a gain in β2AR selectivity. Frielle et al.(1988) have concluded that the TM4 region is a major determinant of the β1 and β2AR selectivity of agonist norepinephrine and that the TM6 or TM7 regions play a major role in determining β2AR selectivity for the β2AR antagonist ICI 118551 or a β1AR selectivity for β1AR antagonist betaxolol. Another group has employed the approach to randomly exchange the TM regions of the βARs 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 β1 and β2ARs 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 βAR confer the subtype selectivity, especially for agonists, has so far been only tentative.
TA-2005 is a non-catechol β2AR agonist with ap-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 β1and β2ARs, TA-2005 has a high selectivity as well as a high affinity for the β2AR 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). Vosset al. (1992) have also reported that TA-2005 shows a high potency for the β2AR and a long duration of action after removal of the drug using both guinea pig tracheal muscle relaxation and bovine trapezium muscle binding experiments.
The chemical structures of TA-2005, compounds I–III derivatives of TA-2005, and isoproterenol.
In the present study we putatively assigned the binding sites of the β2AR for carbostyril moiety of TA-2005 and determined the specific amino acid to be responsible for β2-selective binding. We made several site-directed mutant β2ARs and eight chimeric β1/β2ARs, 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
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-β1 and -β2 encoding for the human β1 and β2ARs 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.
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 β1 and β2ARs 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 β1 and β2ARs to the ligands (data not shown). Chimeric receptors and site-directed mutants of the β1 and β2ARs were constructed by the polymerase chain reaction method (Higuchi, 1989). The positions of the junctions for individual chimeric β1 and β2ARs are as follows (numbers refer to amino acid positions in the human β1 and β2AR sequences): CH1, β1 1–84/β260–413; CH2, β21–71/β1 97–131/β2107–413; CH3, β21–295/β1 347–381/β2331–413; CH4, β21–71/β1 97–131/β2107–295/β1 347–381/β2331–413; CH5, β21–59/β1 85–477; CH6, β1 1–96/β272–106/β1 132–477; CH7, β1 1–346/β2296–330/β1 382–477; CH8, β1 1–96/β272–106/β1 132–346/β2296–330/β1 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 orEcoRI and SalI sites of the mammalian expression vector pCMV5. The alanine-scanning point mutants of the β2AR were made by the Quick change method according to manufacture’s instructions (Stratagene, La Jolla, CA). The two oligonucleotides (33–36 base pairs) and theBglII/EcoRV fragment of the β2AR 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 β2ARs. 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 βAR 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-β2ARs 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 mmMgCl2 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 pm125I-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 theKi andKd 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 andKi 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
Interaction of TA-2005 with Ser204 and Ser207 of β2AR.
To investigate whether TA-2005 interacts with the same serine residues of the β2AR as isoproterenol, we assessed the affinities of compound I, the des-8-hydroxy derivative of TA-2005, for the S204A- and S207A-β2ARs. TheKd values of125I-CYP for the WT-, S204A-, and S207A-β2ARs, respectively, were 56, 34, and 32 pm. TA-2005 had much higher affinity for the WT-β2AR than for isoproterenol and the affinity of TA-2005 for the S204A-β2AR was decreased 56-fold but only slightly for the S207A-β2ARs (4-fold), as compared with the WT-β2AR (Fig.2) [see also Kikkawa et al.(1997)]. Isoproterenol bound to the S204A- and S207A-β2ARs with 27- and 13-fold lower affinities, respectively, than to the WT-β2AR. In the present study, although the affinity of compound I for the S204A-β2AR was decreased 22-fold, the affinity for the S207A-β2AR was essentially the same as that of the WT-β2AR (Fig. 2).
Competition binding of TA-2005 (A), isoproterenol (B), and compound I (C) to the wild-type and mutant β2ARs. Competition binding was performed with membranes prepared from COS-7 cells transfected with the WT-β2AR (•), S204A-β2AR (○), or S207A-β2AR (▵) expression constructs. The data are the mean ± standard error of three experiments done in duplicate.
The contribution of Ser204 and Ser207 to the binding of TA-2005 was also determined by a functional assay. We used JEG-3 cells for the cAMP accumulation assay with two reasons. First, the JEG-3 cells had low basal adenylyl cyclase activity. In addition there was no significant cAMP accumulation by isoproterenol stimulation, even with high concentration (100 μm) of isoproterenol. These two characteristics allowed us to detect small changes in the cAMP contents by agonist stimulation in transiently transfected cells. The EC50 values of isoproterenol in the S204A- and S207A-β2ARs were increased by 12- and 4.3-fold, respectively (Fig. 3), consistent with the previous report (Strader et al., 1989b). The EC50 values of TA-2005 and compound I were slightly increased in the S204A-β2ARs (2.0- and 7.6-fold, respectively) but not in the S207A-β2ARs. In the WT-, S204A-, and S207A-β2ARs, both compound I and TA-2005 activated the adenylyl cyclase to the same extent as isoproterenol.
Cyclic AMP production by TA-2005 (•), isoproterenol (○), and compound I (▵) through activation of the WT-β2 (A), S204A-β2 (B), or S207A-β2 (C) ARs. The cAMP accumulation assay was performed with JEG-3 cells transfected with one of the βAR constructs. The data are the mean ± standard error of three experiments done in duplicate.
Selectivity for the WT-β2AR.
To determine which portion of the TA-2005 molecule is important for β2AR selectivity, we synthesized two compounds that lack p-methoxyphenyl (compound II) orp-methoxyphenyl methylethyl groups (compound III), and examined the affinities of these compounds for the WT-β1 and β2ARs. TA-2005 showed a 53-fold higher selectivity for the β2AR than for the β1AR, whereas isoproterenol showed no selectivity for the β1 and β2ARs (Fig.4, A and B). In contrast to TA-2005, compounds II and III completely lost their βAR subtype selectivity (Fig. 4, C and D).
Competition binding of TA-2005 (A), isoproterenol (B), compound II (C), and compound III (D) to the WT-β1(○) and WT-β2 (•) ARs. Competition binding was performed with membranes prepared from COS-7 cells transfected with either of the βAR constructs. The data are the mean ± standard error of three experiments done in duplicate.
Affinity for β1/β2AR chimeras.
To determine the domain(s) of the β2-receptor that interact with the p-methoxyphenyl group on the amine portion of TA-2005, eight β1/β2chimeric 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 and2. TheKd values of the radioligand125I-CYP in the chimeric receptors were essentially the same as those of the WT-β1 and β2ARs except in CH4 with slightly low affinity of 125I-CYP.
Structures of chimeric β1/β2ARs. Thin lines, peptide sequences derived from the β1AR; thick lines, peptide sequences derived from the β2AR. The positions of the junctions are described in Experimental Procedures.
Effects of substitution of the TM regions with corresponding portion of the β1AR on the ligand-binding characteristics of the β2AR
Effects of substitution of the TM regions with corresponding portion of the β2AR on the ligand-binding characteristics of the β1AR
When the TM2 or TM7 regions of the β2AR were replaced with the corresponding regions of the β1AR (CH2 and CH3), the affinities of TA-2005 were significantly decreased by 7- and 8-fold, respectively (Table 1). In contrast, the affinities of isoproterenol and compound III did not change significantly in CH2 and CH3. The replacement of both the TM2 and TM7 regions of the β2AR with those of the β1AR (CH4) markedly decreased the affinity of TA-2005 (20-fold). The affinity of isoproterenol for CH4 increased, although the extent of increase was small (1.9-fold) (Table 1).
The affinities of TA-2005 were not increased by the replacement of the TM1 and TM2 regions of the β1AR with those of the β2AR (CH5 and CH6), although these affinities would be expected to increase if these regions were involved in the β2-selective binding (Table 2). When the TM7 region of the β1AR was replaced with homologous region of the β2AR (CH7), the affinity of TA-2005 increased 3-fold. Although the affinity of TA-2005 for CH8 was essentially the same as that for the WT-β2AR, the affinities for all of the ligands were also increased in the CH8 receptor (Table 2). These nonspecific increases in the affinity for all of the ligands obscured the contribution of the TM2 region to the β2selectivity.
Alanine-scanning mutants of β2ARs.
There are 10 positions in the TM7 region of the β2AR in which the amino acid residues are different from those of the β1AR. To identify the amino acid which is important in β2-selective agonist binding, each of the amino acids was changed to alanine. One mutant (Y308A-β2AR) out of 10 different alanine-substituted mutants, in which Tyr308 was changed to alanine, showed a dramatically decreased affinity for TA-2005 (Table3). Although the Y308A-β2AR also showed the decreased affinity for isoproterenol, the extent of the decrease in affinity was smaller than that of TA-2005. Furthermore, the Y308F-β2AR mutant, in which Tyr308 in the TM7 region of the β2AR was replaced with the corresponding amino acid residue (Phe) of the β1AR, showed decreased affinity for TA-2005 (Y308F-β2AR: 61 nm versus WT-β2AR: 12 nm, p< 0.05), although that of isoproterenol for Y308F-β2AR was essentially the same as that of the WT-β2AR (1800 and 900 nm, respectively). The replacement of Phe359 of the β1AR with Tyr (F359Y-β1AR), which is a complementary mutant of Y308F-β2AR, increased the affinity for TA-2005 by 2.5-fold. However, we have also observed that F359Y-β1AR decreased the affinity for125I-CYP by 19-fold, whereas it increased the affinity for isoproterenol by 7-fold (data not shown).
Effects of substitution of single amino acid in the TM7 region of the β2AR with alanine on the ligand-binding characteristics
Discussion
Mutagenesis experiments have determined the binding sites of the nonselective agonists and selective antagonists for the β2AR (Dixon et al., 1989; Frielleet al., 1988; Marullo et al., 1990). However, the binding sites of the selective β2AR agonists are little known. We have studied the binding sites of TA-2005, one of the most selective β2AR agonists with a hydroxy-carbostyril structure. TA-2005 has one hydroxyl group and one amido group at positions corresponding to the p- andm-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-β2AR 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 EC50values of TA-2005 in the WT-β2AR 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 β2AR may be enough to activate the adenylyl cyclase by TA-2005 with high affinity. Because the affinity of compound I for the S207A-β2AR was similar to that for the WT-β2AR, and the affinity of compound I decreased in the S204A-β2AR, it seems that the 8-hydroxyl group of TA-2005 may interact with the Ser207 hydroxyl group of the β2AR. 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 β2AR.
To determine the structural basis for the β2AR selectivity, we assessed the affinities of TA-2005 and its derivatives for a series of chimeric β1/β2ARs. Although TA-2005 had a 53 times higher affinity for the β2AR than for the β1AR, compound II and III which lacked the p-methoxyphenyl group of TA-2005 completely lost their β2selectivity. Kontoyianni et al. (1996) have suggested that, based on a computer-modeling technique, the large 2-phenylethylN-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 β2AR, or both with homologous regions of the β1AR 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; Zhouet 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 β2 selectivity, we made a series of gain-of-function mutants, in which the chimeric receptor mutants gained β2 selectivity. The replacement of the TM7 but not the TM2 regions significantly increased the affinities of TA-2005. Although the β1AR with both TM2 and TM7 regions of the β2AR almost completely restored the β2 selectivity to the level of the WT-β2AR, the resultant chimeric receptor (CH8) also increased the affinity of isoproterenol. This suggests that the TM7 region of the β2AR contributes to β2-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 β2-selective agonist, interacts with residues 149–158 within the TM4 region of the β2AR (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 β2AR (i.e., exosite of the β2AR). This region, however, does not seem to contribute to β2-selective binding of salmeterol, because the β2AR with the TM4 region of the β1AR lost its ability to persistently bind salmeterol but still retained the β2selectivity. This suggests that the region that confers β2 selectivity to the β2AR is distinct from the exosite. These results also support our assumption that the TM7 region is important for the β2-selective agonist TA-2005 in binding to the β2AR with high affinity.
To identify the amino acid that is important for the high affinity binding of a β2-selective agonist, each of the amino acids in TM7 region that are different from those of the β1AR were changed to alanine. One mutant (Y308A-β2AR) out of 10 alanine-substituted mutants significantly decreased its affinity for TA-2005. Furthermore, the affinity of TA-2005 for the Y308F-β2AR was significantly decreased, although the affinity of isoproterenol was essentially the same as that of the WT-β2AR. These results suggest that Tyr308 is a major determinant for the binding of the β2-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-β1AR, which is a complementary mutant of the Y308F-β2AR, the affinities for125I-CYP and isoproterenol were also changed dramatically. This indicates that the replacement of Phe of the β1AR with Tyr may cause an overall structural change of the β1AR 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 βAR 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, and125I-asidophenyl CGP12177A was incorporated at the TM6 an TM7 regions of the purified βAR 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 β2AR is crucial for the high affinity binding of the β2-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 β2-selective agonist. It remains to be determined whether Tyr308 also plays an important role in the binding of other β2-selective agonists such as salmeterol, formoterol and procaterol.
Acknowledgments
We are grateful to Drs. K. Naito and A. Saito (Tanabe Seiyaku) for their helpful suggestions, to Dr. P.W. Tsao for reviewing this manuscript, and to Dr. Inoue (Tanabe Seiyaku) who kindly synthesized the compounds for us. We also thank Dr. R. J. Lefkowitz for the pBC-β1 and β2 plasmids and Dr. S. Nagata for the pEF-BOS plasmid.
Footnotes
-
Send reprint requests to: Hideo Kikkawa, Ph.D., Lead Optimization Research Laboratory, Tanabe Seiyaku Co., Ltd., 2-2-50, Kawagishi, Toda-shi, Saitama 335, Japan. E-mail:hideo-k{at}tanabe.co.jp
- Abbreviations:
- AR
- adrenergic receptor
- TM
- transmembrane
- WT
- wild type
- CYP
- cyanopindolol
- DMEM
- Dulbecco’s modified Eagle’s medium
- CH
- chimera
-
- Received May 14, 1997.
- Accepted October 1, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
