Abstract
(−)-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(−)-RO363] is a highly selective β1-adrenergic receptor (β1AR) agonist. To study the binding site of β1-selective agonist, chimeric β1/β2ARs and Ala-substituted β1ARs were constructed. Several key residues of β1AR [Leu110 and Thr117 in transmembrane domain (TMD) 2], and Phe359 in TMD 7] were found to be responsible for β1-selective binding of (−)-RO363, as determined by competitive binding. Based on these results, we built a three-dimensional model of the binding domain for (−)-RO363. The model indicated that TMD 2 and TMD 7 of β1AR form a binding pocket; the methoxyphenyl group ofN-substituent of (−)-RO363 seems to locate within the cavity surrounded by Leu110, Thr117, and Phe359. The amino acids Leu110 and Phe359 interact with the phenyl ring of (−)-RO363, whereas Thr117 forms hydrogen bond with the methoxy group of (−)-RO363. To examine the interaction of these residues with β1AR in an active state, each of the amino acids was changed to Ala in a constitutively active (CA)-β1AR mutant. The degree of decrease in the affinity of CA-β1AR for (−)-RO363 was essentially the same as that of wild-type β1AR when mutated at Leu110 and Thr117. However, the affinity was decreased in Ala-substituted mutant of Phe359 compared with that of wild-type β1AR. These results indicated that Leu110 and Thr117 are necessary for the initial binding of (−)-RO363 with β1-selectivity, and interaction of Phe359 with the N-substituent of (−)-RO363 in an active state is stronger than in the resting state.
β-Adrenergic receptors are members of the G protein-coupled receptor superfamily with seven transmembrane helices and are classified into three subtypes: β1, β2, and β3. β1-, β2-, and β3-adrenergic receptors are predominantly expressed in the heart, trachea, and adipose tissue, respectively. Therefore, several β1-selective agonists are used for the improvement of the function of failing heart, whereas β2-selective agonists are used for treatment of bronchial asthma. To design a better drug, it is important to determine the binding sites for these agonists. Endogenous catecholamine such as norepinephrine and epinephrine, and synthetic catecholamine such as isoproterenol are full but nonselective agonists of β-adrenergic receptors. Ligand binding domains of β-adrenergic receptor for these agonists were shown to be located within transmembrane domains (Dixon et al., 1987; Dohlman et al., 1988; Wong et al., 1988; Hockerman et al., 1996). Site-directed mutagenesis studies of β2-adrenergic receptor have identified several key residues for isoproterenol binding. The amino group of catecholamine was revealed to interact with Asp113 in transmembrane domain 3 of β2-adrenergic receptor (Strader et al., 1987,1988, 1989a). The two catechol hydroxyl groups of agonists were shown to form hydrogen bond with Ser203 (Sato et al., 1999) as well as Ser204 and Ser207 (Strader et al., 1989b) in transmembrane domain 5. Furthermore, the catechol phenyl group and β-hydroxyl group of catecholamine were reported to interact with Phe290 (Strader et al., 1994) and Asn293 (Wieland et al., 1996), respectively, in transmembrane domain 6. Because these residues in β2-adrenergic receptor are conserved among three subtypes, these residues are not responsible for the subtype-selective binding.
Several groups have studied the binding sites for subtype-selective ligands, by using the chimeric receptors of β1- and β2-adrenergic receptor. Frielle et al. (1988) suggested that transmembrane domain 6 and transmembrane domain 7 play an important role in binding of β1-selective antagonist betaxolol and β2-selective antagonist ICI118,551. The binding sites of the β1- and β2-selective antagonists were also analyzed byMarullo et al. (1990). They reported that the subtype-selective binding of antagonists cannot be determined by single transmembrane domain. It has been reported that transmembrane domain 4 is responsible for the β1-selective binding of an endogenous agonist norepinephrine (Frielle et al., 1988; Dixon et al., 1989). However, key amino acids or subdomains of each β-adrenergic receptor for subtype-selective agonists have not been investigated in detail.
We have studied the binding domains for β1- and β2-selective agonists and have reported that transmembrane domains 2 and 7 of β1- and β2-adrenergic receptors form a binding pocket, and that Leu110, Thr117, and Val120 in transmembrane domain 2 of β1-adrenergic receptor or Tyr308 in transmembrane domain 7 of β2-adrenergic receptor are major determinants for β1- or β2-selective agonists, respectively (Isogaya et al., 1998, 1999; Kikkawa et al., 1998). However, β1-selectivity of T-0509 and denopamine [Ki(β2)/Ki(β1)] used in the previous study was at most 10-fold. Therefore, we could not determine the interaction of each amino acid in transmembrane domain 2 with β1-adrenergic receptor in an active state.
(−)-RO363 is one of the β1-selective agonists with structural similarity to T-0509 and denopamine (Fig.1). The affinity of (−)-RO363 for β1-adrenergic receptor is about 100- and 3000-fold higher than for β2- and β3-adrenergic receptors, respectively (McPherson et al., 1984; Molenaar et al., 1997). Therefore, we used (−)-RO363 as a tool to examine the contribution of key amino acids responsible for the β1-selective binding to the binding in the resting and an active states. We constructed chimeric β1/β2-adrenergic receptors and Ala-substituted β1-adrenergic receptors, and found that several key amino acids play an essential role in β1-selective binding of (−)-RO363, by competitive binding assay. To analyze a role of these amino acids in an active state, we constructed double mutants, in which Leu323 located at intracellular third loop was changed to Lys to transform the receptor into a constitutively active form, and key amino acids responsible for β1-selective binding were changed to Ala. Based on these results, we built a three-dimensional model of the binding domain for (−)-RO363.
Experimental Procedures
Materials.
The plasmids pBC-β1 and -β2 encoding human β1- and β2-adrenergic receptors were kindly provided by Dr. R. J. Lefkowitz (Duke University, Durham, NC). The plasmid encoding a constitutively active β1-adrenergic receptor mutant (L323K) was described by Lattion et al. (1999). (±)-Propranolol, (−)-isoproterenol, DEAE-dextran, and GTP were purchased from Sigma-Aldrich (St. Louis, MO).125I-Cyanopindolol was obtained from Amersham Biosciences, Inc. (Piscataway, NJ). Dulbecco's modified Eagle's medium was obtained from Invitrogen (Carlsbad, CA). Fetal bovine serum was from JRH Biosciences (Lenexa, KS).Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA). (−)-RO363 was provided by Roche Applied Science (Palo Alto, CA).
Construction of Chimeric Receptors and Mutant Receptors.
Chimeric β1/β2-adrenergic receptors were constructed by PCR, as described by Kikkawa et al. (1998). The structures of these chimeras are shown in Fig.2. The positions and amino acids of the junctions for the individual chimeras are as follows: chimera 1 (CH1), β1Met1-Ala84/β2Lys60-Leu413; chimera 2 (CH2), β2Met1-Phe71/β1Ile97-Cys131/β2Glu107-Leu413; chimera 3 (CH3), β2Met1-Val295/β1Lys347-Pro381/β2Asp331-Leu413; chimera 4 (CH4), β2Met1-Phe71/β1Ile97-Cys131/β2Glu107-Val295/β1Lys347-Pro381/β2Asp331-Leu413; chimera 5 (CH5), β2Met1-Ala59/β1Lys85-Val477; chimera 6 (CH6), β1Met1-Phe96/β2Ile72-Cys106/β1Glu132-Val477; chimera 7 (CH7), β1Met1-Val346/β2His296-Pro330/β1Asp382-Val477; and chimera 8 (CH8), β1Met1-Phe96/β2Ile72-Cys106/β1Glu132-Met1-Val346/β2His296-Pro330/β1Asp382-Val477. Ala-substituted mutants of β1-adrenergic receptor were constructed by PCR with QuickChange site-directed mutagenesis kit (Stratagene). After confirming the mutation, the PCR products were ligated with the rest of β1-adrenergic receptor sequences to obtain full-length mutated β1-adrenergic receptors. Ala-substituted mutants of constitutively active β1-adrenergic receptor were constructed as follows. L323K-mutant of β1-adrenergic receptor shows constitutive activity (Lattion et al., 1999). The fragment encoding L323K-mutation was digested from this constitutively active mutant and ligated with the rest of β1-adrenergic receptor sequence, which encodes Ala-substituted sequences, to obtain full-length constitutively active β1-adrenergic receptor mutants. These cDNAs were cloned into mammalian expression vector pCMV5 or pEF/myc/cyto.
Transient Expression of Chimeric and Mutant Receptors.
COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 10 μg/ml gentamicin. For radioligand binding assay, COS-7 cells were seeded at 1.0 to 1.5 × 106 cells/100-mm dish. The next day, the plasmid constructs were transfected by DEAE-dextran method as described (Isogaya et al., 1998, 1999; Kikkawa et al., 1998). Two days after the transfection, the cells were harvested for preparation of crude membrane fraction.
Radioligand Binding Assay.
Two days after the transfection, COS-7 cells were rinsed three times with 10 ml of ice-cold phosphate-buffered saline and mechanically detached in 1 ml of ice-cold lysis buffer containing 10 mM Tris, pH 7.4, 5 mM EDTA, 5 mM EGTA, 1 μg/ml benzamidine, 10 μg/ml soybean trypsin inhibitor (type II-S), and 5 μg/ml leupeptin. The cell lysate was centrifuged at 45,000g for 10 min at 4°C. The pellet containing crude membrane fraction was resuspended in 1 ml of ice-cold lysis buffer with Potter type homogenizer, frozen, and stored at −80°C until use. The concentration of membrane protein was determined by the method of Lowry et al. (1951). Membrane protein (0.1–5 μg) was used for the binding studies. The membrane was incubated with 50 pM125I-cyanopindolol in 75 mM Tris, pH 7.4, 12.5 mM MgCl2, 2 mM EDTA, and various concentrations of (−)-RO363 in the presence of 0.1 mM GTP for 1 h at 37°C. Nonspecific binding was determined in the presence of 5 μM (±)-propranolol. The reaction mixture was filtered over Whatman GF/C filters (Whatman, Maidstone, UK). The filters were washed with ice-cold buffer containing 25 mM Tris, pH 7.4, and 1 mM MgCl2. The bound125I-cyanopindolol on the filters was measured with a gamma counter.
Data Analysis.
The results are expressed as mean ± standard error of n determinations. Equilibrium dissociation constants were determined from saturation isotherms. The competition curves for determination of IC50 andKi values were analyzed by Prism software (GraphPad Software, San Diego, CA). Statistical significance was evaluated by one-way analysis of variance for multiple comparisons. Analysis of variance post hoc comparisons were assessed with Dunnett's test.
Molecular Modeling.
The model of the α-carbon template based on the structure of rhodopsin was presented by Baldwin et al. (1997) and was used as described previously (Isogaya et al., 1999). (−)-RO363-β1-adrenergic receptor complex was built by the following assumption: Asp138(corresponding to Asp113 of β2-adrenergic receptor) with the protonated amine, Ser229 (Ser204 of β2-adrenergic receptor) and Ser232 (Ser207 of β2-adrenergic receptor) with the catechol hydroxyl groups, and Phe341(Phe290 of β2-adrenergic receptor) with the catechol phenyl ring. The procedures of docking and energy minimization were described previously (Tanimura et al., 1994).
Results
Affinities of (−)-RO363 for β1/β2Chimeric Receptors.
The receptor binding analysis with human recombinant β1- and β2-adrenergic receptors showed that (−)-RO363 has 40-fold higher affinity for β1-adrenergic receptor than β2-adrenergic receptor (Table1). To determine the domain responsible for this selectivity, we constructed eight chimeric receptors of β1- and β2-adrenergic receptors (Fig. 2). The binding characteristics of these chimeric receptors for (−)-RO363 are summarized in Table 1. No significant differences were found when transmembrane domain 1 of β1- or β2-adrenergic receptors was replaced with the corresponding regions of β2- or β1-adrenergic receptors (CH5 or CH1). This result indicates that transmembrane domain 1 of β1-adrenergic receptor does not participate in the subtype-selective binding of (−)-RO363. The replacement of transmembrane domain 2 or transmembrane domain 7 of β1-adrenergic receptor (CH6 or CH7) decreased the affinity of (−)-RO363 for the resulting chimeras (33- or 5.5-fold), whereas the replacement of transmembrane domain 2 or transmembrane domain 7 of β2-adrenergic receptor (CH2 or CH3) increased the affinity for these chimeras (21- or 3.6-fold). Moreover, the replacement of transmembrane domain 2 together with transmembrane domain 7 of β1- or β2-adrenergic receptors with the corresponding regions of β2- or β1-adrenergic receptors (CH8 or CH4) resulted in the chimera that showed essentially the same affinities as those of wild type β2- or β1-adrenergic receptors. The affinity of (−)-RO363 for CH4 (β2-adrenergic receptor with transmembrane domains 2 and 7 of β1-adrenergic receptor) was 58-fold higher than that for wild type β2-adrenergic receptor and was essentially the same as that of wild-type β1-adrenergic receptor. In contrast, the affinity of (−)-RO363 for CH8 (β1-adrenergic receptor with transmembrane domains 2 and 7 of β2-adrenergic receptor) was 55-fold lower than that for wild-type β1-adrenergic receptor, and was almost equivalent to that of wild-type β2-adrenergic receptor. These data strongly suggest that transmembrane domains 2 and 7 of β1-adrenergic receptor are responsible for the β1-selective binding of (−)-RO363.
Affinities of (−)-RO363 for Ala-Substituted β1-Adrenergic Receptor Mutants.
To examine further the binding region of (−)-RO363, eight amino acids in transmembrane domain 2 of β1-adrenergic receptor that are different from those of β2-adrenergic receptor were individually changed to Ala (Fig.3). Among these eight mutants, L110A- and T117A-β1-adrenergic receptors showed the significant decrease in the binding affinities of (−)-RO363 (6.5- and 2.5-fold, respectively), whereas the affinities of125I-cyanopindolol to these two mutants were unaltered (Table 2; Fig.4). We also replaced 10 amino acids in transmembrane domain 7, which are different from β2-adrenergic receptor, individually with Ala (Fig. 3). In this region, only replacement of Phe359 significantly decreased the affinity of (−)-RO363 (4.1-fold; Table 3; Fig. 4). The substitution of amino acids other than Phe359with Ala did not significantly change the affinity of (−)-RO363. The substitution of Phe361 in transmembrane domain 7 with Ala resulted in low expression of the receptor, and we could not analyze its binding characteristics (data not shown). These data indicated that Leu110, Thr117, and Phe359contribute to the β1-selective binding of (−)-RO363.
Effects of Multiple Ala-Substitution of Key Amino Acids on Affinity of (−)-RO363 for β1-Adrenergic Receptor.
Although Leu110, Thr117, and Phe359 were suggested to participate in β1-selective binding of (−)-RO363, the decreases in the affinity of L110A-, T117A-, and F359A-β1-adrenergic receptors were relatively small compared with the difference ofKi values of (−)-RO363 for wild-type β1- and β2-adrenergic receptors. To investigate the possibility that the key amino acids (Leu110, Thr117, and Phe359) form a binding pocket in a concerted manner, we changed two or three of these amino acids to Ala. L110A/T117A-, L110A/F359A-, and T117A/F359A-β1-adrenergic receptors showed 3.5-, 26-, and 21-fold lower affinity for (−)-RO363 than wild-type β1-adrenergic receptor, respectively (Table4; Fig. 5). Furthermore, the affinity of (−)-RO363 for the triple mutant L110A/T117A/F359A-β1-adrenergic receptor was essentially the same as that for wild-type β2-adrenergic receptor (Table 4; Fig. 5). Except for L110A/T117A-β1-adrenergic receptor, the affinities for the double or triple mutants were almost multiplicatively decreased, indicating that these amino acids contribute to the free energy of binding in an additive manner. These results suggest that these three amino acids form a binding pocket in a cooperative manner, and that this binding pocket is a major determinant for β1-selective, high-affinity binding of (−)-RO363. To test this hypothesis, we introduced two of these key amino acids (Leu110 and Thr117) into the corresponding positions of β2-adrenergic receptor (A85L/A92T-β2-adrenergic receptor). However, the level of expression of this mutant was too low to analyze (data not shown).
Role of Key Amino Acids in Active State of Receptor.
As mentioned above, it was suggested that Leu110, Thr117, and Phe359 are key amino acids in β1-selective binding of (−)-RO363. To examine a role of these amino acids in active state of the receptor, we introduced the mutation into a constitutively active β1-adrenergic receptor (L323K-β1-adrenergic receptor). As reported byLattion et al. (1999), this mutation confers the constitutive activity on β1-adrenergic receptor. (−)-RO363 showed 3.6-fold higher affinity for L323K-β1-adrenergic receptor than for wild-type β1-adrenergic receptor (Table5), consistent with the previous observation that the increase in the affinity for agonists reflect the property of constitutively active receptor. The affinity of (−)-RO363 for constitutively active β1-adrenergic receptor was significantly decreased by mutation of Leu110, Thr117, and Phe359 (4.8-, 5.8-, and 12.6-fold, respectively). Among three mutants, only F359A constitutively active β1-adrenergic receptor showed a larger decrease in the affinity of (−)-RO363 than F359A-β1-adrenergic receptor (Fig.6).
Three-Dimensional Model of (−)-RO363-β1-Adrenergic Receptor Complex.
We constructed a three-dimensional model of (−)-RO363-β1-adrenergic receptor complex in the resting state to explain the results of binding studies (Fig.7). The structure of rhodopsin presented by Baldwin et al. (1997) was used as a model of the α-carbon template. The amino acids of β1-adrenergic receptor that interact with catecholamine (Asp138in transmembrane domain 3, Ser229 and Ser232 in transmembrane domain 5, and Phe341 in transmembrane domain 6) were assumed to interact with the catecholamine-like structure of (−)-RO363, except for Asn344. Because Asn344did not reach β-hydroxyl group of (−)-RO363, Asn344 may interact with ether oxygen instead of β-hydroxyl group of (−)-RO3632. As previously described by Isogaya et al. (1999), the half of amino acids in transmembrane domains 2 and 7 toward the extracellular space forms binding pocket of β2-selective agonists. As shown in Fig. 7, Phe359 and Leu110 are located at the top and the bottom of a binding pocket of (−)-RO363, respectively. Phe359 seems to participate in β1-selective, high-affinity binding of (−)-RO363 through hydrophobic interaction with the phenyl group ofN-substituent of (−)-RO363. On the other hand, Thr117 is located near the methoxy group ofN-substituent of (−)-RO363 and seems to interact through hydrogen bond.
Discussion
In this study, we analyzed the site on β1-adrenergic receptor conferring the β1-selective binding. Based on analysis of the binding characteristics of several chimeric β1/β2-adrenergic receptors, transmembrane domains 2 and 7 were found to be involved in the β1-selective binding of (−)-RO363. This result is consistent with our previous reports that transmembrane domains 2 and 7 of β1- and β2-adrenergic receptors form a binding pocket for the β1- and β2-selective agonists (Isogaya et al., 1998,1999; Kikkawa et al., 1998). In the previous report, we examined the contribution of each of amino acids in transmembrane domain 2 of β1-adrenergic receptor to subtype-selective binding, and found that Leu110, Thr117, and Val120 of β1-adrenergic receptor play an important role in the β1-selective binding of T-0509 and denopamine, which are β1-selective agonists with similar structure to (−)-RO363 (Isogaya et al., 1999; Fig. 1). Because the effect of the replacement of Val120with Ala was relatively small compared with those of Leu110 and Thr117, it suggested that Leu110 and Thr117 mainly determine β1-selective binding of T-0509 and denopamine. We also found in the present study that the replacement of Val120 did not significantly decrease the affinity of (−)-RO363 for β1-adrenergic receptor. These results indicate that Leu110 and Thr117 in transmembrane domain 2 play a major role in the subtype-selective binding of the β1-selective agonists containing dimethoxyphenyl group such as T-0509, denopamine, and (−)-RO363.
We have previously reported that transmembrane domain 7 of β2-adrenergic receptor played an essential role in β2-selective binding irrespective of structure of β2-selective agonists such as salmeterol, TA-2005, and so on. We demonstrated in the present study that transmembrane domain 7 of β1-adrenergic receptor is important for the subtype-selective binding. It is interesting to note that the particular amino acid in transmembrane domain 7 located at specific position (Tyr308 of β2-adrenergic receptor or Phe359 of β1-adrenergic receptor) plays an essential role in determining subtype-selective binding irrespective of receptor subtypes. We suggested in the previous reports that Tyr308 prevented theN-substituent of β2-selective agonists from freely moving into extracellular space like a cover. Phe359 of β1-adrenergic receptor locates at the position corresponding to Tyr308 of β2-adrenergic receptor. Therefore, Phe359 may work as a cover of the binding pocket formed by transmembrane domains 2 and 7 of β1-adrenergic receptor like Tyr308 of β2-adrenergic receptor.
The structures of β1-selective agonists [denopamine, T-0509, and (−)-RO363] used for the previous and present studies are similar to each other. They possess a dimethoxyphenyl group as N-substituent. One of β1-selective agonists structurally different from these agonists is xamoterol. However, the affinity of xamoterol for β1-adrenergic receptor is at most 10-fold higher than for β2-adrenergic receptor as determined by binding experiment with recombinant β1- and β2-adrenergic receptors. Therefore, it is necessary to develop a structurally different β1-selective agonist for demonstrating the importance of Leu110, Thr117, and Phe359 of transmembrane domains 2 and 7, and generalization of the present model. The present model of the binding pocket helps to design new β1-selective agonists.
One of the chimeras (CH7), in which transmembrane domain 7 of β1-adrenergic receptor was replaced with that of β2-adrenergic receptor, did not decrease the affinity of (−)-RO363. However, we considered the possibility that the hydrophobic nature of the substituted amino acid may compensate the interaction of the chimera with N-substituent of (−)-RO363 because transmembrane domain 7 of β2-adrenergic receptor plays an essential role in the β2-selective binding. Therefore, we changed to Ala each of amino acids in transmembrane domain 7 of β1-adrenergic receptor that is different from β2-adrenergic receptor. Among them, F359A-β1-adrenergic receptor mutant only showed the significantly decreased affinity of (−)-RO363 for β1-adrenergic receptor. The position of Phe359 of β1-adrenergic receptor is exactly the same as that of Tyr308 of β2-adrenergic receptor, which determines the subtype-selective binding of the β2-selective agonists (Isogaya et al., 1998, 1999; Kikkawa et al., 1998). The homology of amino acids between the upper halves of transmembrane domains 2 and 7 of β1- and β2-adrenergic receptors is 54% (Fig. 3). Therefore, it may be feasible to imagine that the region of β1-adrenergic receptor formed by the amino acids different from β2-adrenergic receptor such as Leu110, Thr117, and Phe359 determines β1-selective binding. Although the β2-selectivity is mainly determined by one amino acid in transmembrane domain 7 (Tyr308), β1-selectivity for (−)-RO363 does not seem to be determined by a single amino acid. To examine whether Leu110, Thr117, and Phe359 work for subtype-selective binding of (−)-RO363 in a concerted manner, these three residues were replaced with Ala individually or in combination. L110A/F359A-, T117A/F359A-, and L110A/T117A/F359A-β1-adrenergic receptors showed lower affinity than the mutants with a single substitution. This result supports our conclusion that Leu110, Thr117, and Phe359 form the binding site in a concerted manner (Fig. 5; Table 4). However, the affinity of (−)-RO363 for L110A/T117A-β1-adrenergic receptor was essentially the same as those of L110A- or T117A-β1-adrenergic receptor with unknown reason (Fig. 5; Table 4). Molecular modeling of the (−)-RO363-β1-adrenergic receptor complex supports the notion that β1-selective agonist binding site is formed by three amino acids Leu110, Thr117, and Phe359 (Fig. 7). The model shows thatN-substituent of (−)-RO363 can locate at the space surrounded by these three key amino acids. T-0509 and denopamine, which we used in the previous study, have the same structure (dimethoxyphenyl group) of N-substituent as (−)-RO363. However, these two agonists show lower β1-selectivity than (−)-RO363. The model suggests that N-substituent of T-0509 and denopamine interacts with these amino acids, but the interaction is not enough to be tight to produce strong binding. The difference of the affinities between (−)-RO363 and T-0509 and denopamine is explained by the fact that the (−)-RO363 molecule is longer than T-0509 and denopamine by two atoms. Thus, the pocket formed by Leu110, Thr117, and Phe359 can contribute to the β1-selective binding of (−)-RO363 in a more efficient way.
The proximity between transmembrane domains 2 and 7 has been reported in several GPCRs such as gonadotropin-releasing hormone receptor (Zhou et al., 1994; Ballesteros et al., 1998), tachykinin neurokinin 2 receptor (Donnelly et al., 1999), serotonin 5-hydroxytryptamine2A receptor (Perlman et al., 1997), and thyrotropin-releasing hormone receptor (Sealfon et al., 1995). Two amino acids (Asp in the middle of transmembrane domain 2, and Asn in the middle of transmembrane domain 7) are highly conserved among GPCR superfamily (Gether and Kobilka, 1998). Interaction of these two amino acids seems to be a driving force to keep transmembrane domains 2 and 7 in a close position. Furthermore, Asn in the middle of transmembrane domain 1, and Asp-Arg-Tyr motif (DRY motif) in the cytoplasmic end of transmembrane domain 3 are also highly conserved (Ballesteros et al., 1998; Gether and Kobilka, 1998). The DRY motif plays an important role in activation of GPCRs, through the interaction with G protein and catalysis of GDP-GTP exchange of G protein α-subunit (Acharya and Karnik, 1996). Moreover, highly conserved Asn in transmembrane domain 7 of GPCRs forms NPXXY motif, which is involved in activation and internalization of GPCRs (Abdulaev and Ringe, 1998;Konvincka et al., 1998). Therefore, it is likely that these polar residues form a network responsible for activation of GPCRs induced by agonists. Actually, in α1B-adrenergic receptor (Scheer et al., 1996, 1997) and β2-adrenergic receptor (Rasmussen et al., 1999), mutations that disrupt the interactions between some of these highly conserved polar residues were found to evoke the agonist-independent activation of these receptors. Furthermore, in rhodopsin (Farrens et al., 1996; Sheikh et al., 1996) and β2-adrenergic receptor (Gether et al., 1997), it was reported that changes around transmembrane domains 3 and 6 occurred after receptor activation. These conformational changes seem to reflect the movement of transmembrane domains induced by agonist binding. However, it is remains to be determined how agonists induce these conformational changes at molecular level.N-substituent of (−)-RO363 seems to fit a binding pocket formed by transmembrane domains 2 and 7 of β1-adrenergic receptor. If this binding causes disruption of hydrophobic and hydrophilic interactions of these conserved polar amino acids in several transmembrane domains, it is possible that the β1-selective binding is dependent on the conformation induced by agonist. To examine this possibility, we replaced Leu110, Thr117, or Phe359, which is responsible for the β1-selectivity of (−)-RO363, of a constitutively active mutant of β1-adrenergic receptor with Ala. If a conformational change occurs around these amino acids, the degree of decrease in the affinity caused by Ala-substitution will be changed by alteration of distance between these amino acids and (−)-RO363. Although the replacement of Leu110 or Thr117 of constitutively active β1-adrenergic receptor decreased theKi value, the degree of reduction was essentially the same as that of wild-type β1-adrenergic receptor (Table 5; Fig. 7). Therefore, Leu110 and Thr117 seem to contribute to the initial stage of (−)-RO363 binding. However, Ala-substitution of Phe359 of constitutively active β1-adrenergic receptor caused a larger decrease in the affinity of (−)-RO363 than that of wild-type β1-adrenergic receptor (Table 5; Fig. 7). This result suggests that Phe359 in an active state locates in a closer position than in the resting state.
In conclusion, in β1AR, Leu110 and Thr117 in transmembrane domain 2, and Phe359 in transmembrane domain 7 are major determinants of β1-selectivity of (−)-RO363. By combining the present result with the previous observations, we propose that Leu110, Thr117, and Phe359 of β1-adrenergic receptor form in a concerted manner the subtype-selective binding pocket of β1-selective agonists possessing dimethoxyphenyl group as N-substituent.
Acknowledgments
We thank Dr. R. J. Lefkowitz for the pBC-β1 and pBC-β2plasmids. We also thank Roche Biosciences for (−)-RO363.
Footnotes
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↵1 Present address: Kyowa-Hakko Co., Simotogari 1179, Nagaizumi, Suntoh-gun, Shizuoka 411-0943, Japan.
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↵2 The present model indicates that β-hydroxyl group of (−)-RO363 interacts with Asp138, because the side chain containing β-hydroxyl group is flexible to rotate. However, we did not pursue this point, because the purpose of the present study is to identify the amino acid(s) for the β1-selective binding of (−)-RO363.
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This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan (to T.N. and H.K.) and the Organization for Pharmaceutical Safety and Research (to G.T.).
- Abbreviations:
- (−)-RO363
- (−)-1-(3,4-dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol
- PCR
- polymerase chain reaction
- CH
- chimera
- GPCR
- G protein-coupled receptor
- AR
- adrenergic receptor
- ICI118,551
- (±)-1-[2,3-(dihydro-7-methyl-1H-iden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol, T-0509, (−)-(R)-1-(3,4-dihydroxyphenl)-2-[(dimethoxyphenyl)amino]ethanol
- Received November 5, 2001.
- Accepted December 19, 2001.
- The American Society for Pharmacology and Experimental Therapeutics