Clathrin box in G protein-coupled receptor kinase 2.

beta(1)-Adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. However, beta(1)AR can internalize as G protein-coupled receptor kinase 2 (GRK2) is fused to its carboxyl terminus. Internalization of the beta(1)AR and GRK2 fusion protein (beta(1)AR/GRK2) is dependent on dynamin but independent of beta-arrestin and phosphorylation. The beta(1)AR/GRK2 fusion protein internalizes via clathrin-coated pits and is found to co-localize with the endosome that contains transferrin. The fusion proteins consisting of beta(1)AR and various portions of GRK2 reveal that the residues 498-502 in the carboxyl-terminal domain of GRK2 are critical to promote internalization of the fusion proteins. This domain contains a consensus sequence of a clathrin-binding motif defined as a clathrin box. In vitro binding assays show that the residues 498-502 of GRK2 bind the amino-terminal domain of clathrin heavy chain to almost the same extent as beta-arrestin1. The mutation of the clathrin box in the carboxyl-terminal domain of GRK2 results in the loss of the ability to promote internalization of the fusion protein. GRK2 activity increases and then decreases as the concentration of clathrin heavy chain increases. Taken together, these results imply that GRK2 contains a functional clathrin box and directly interacts with clathrin to modulate its function.

Low affinity of beta1-adrenergic receptor for beta-arrestins explains the resistance to agonist-induced internalization.

It has been reported that beta-arrestin is essential for the internalization of many G protein-coupled receptors. Since beta1-adrenergic receptor (beta1AR) shows the resistance to agonist-induced internalization, we examine the interaction of beta-arrestin with beta1AR with three different approaches: translocation of beta-arrestin to the plasma membrane, direct binding of in vitro translated beta-arrestin to intracellular domains of beta1- and beta2ARs, inhibition of beta1- and beta2AR-stimulated adenylyl cyclase activities by beta-arrestin. The enhanced green fluorescent protein (EGFP)-tagged beta-arrestin 2 (beta-arrestin 2-GFP) translocates to and stays at the plasma membrane by beta2AR stimulation. Beta-arrestin 2-GFP also translocates to the plasma membrane upon beta1AR stimulation. However, it returns to the cytoplasm 10 - 30 min after agonist stimulation. The amount of beta-arrestin bound to the third intracellular loop and the carboxyl tail of beta1AR is lower than that of beta2AR. The fusion protein of beta-arrestin 1 with glutathione-S-transferase inhibits the beta1- and beta2AR-stimulated adenylyl cyclase activities. However, inhibition of the beta1AR-stimulated activity requires a higher amount of the fusion protein than that of the beta2AR-stimulated activity. These results suggest that affinity of beta1AR for beta-arrestins is lower than that of beta2AR, and explains the resistance to agonist-induced internalization. This conclusion is further supported by the finding that beta-arrestin can induce internalization of beta1AR when beta-arrestin 1 fused to the carboxyl tail of beta1AR.

G alpha(i) and G alpha(o) are target proteins of reactive oxygen species.

Reactive oxygen species (ROS) have been identified as central mediators in certain signalling events. In the heart, ROS have important functions in ischaemia/reperfusion-induced cardiac injury and in cytokine-stimulated hypertrophy. Extracellular signal-regulated kinase (ERK) is one of the ROS-responsive serine/threonine kinases. Previous studies showed that tyrosine kinases and small G proteins are involved in the activation of ERK by ROS; however, the initial target protein of ROS that leads to ERK activation remains unknown. Here we show that inhibition of the betagamma-subunit of G protein (G betagamma) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. The G betagamma-responsive ERK activation induced by H2O2 is independent of ligands binding to Gi-coupled receptors, but requires phosphatidylinositol-3-kinase and Src activation. In in vitro studies, however, treatment with H2O2 increases [35S]GTP-gammaS binding to cardiac membranes and directly activates purified heterotrimeric Gi and Go but not Gs. Analysis using heterotrimeric Go and its individual subunits indicates that H2O2 modifies G alpha(o) but not G betagamma, which leads to subunit dissociation. We conclude that G alpha(i) and G alpha(o) are critical targets of oxidative stress for activation of ERK.

Assessment of affinities of propranolol and bopindolol to membranes from COS-7 cell transiently transfected with beta-1- and beta-2-adrenoceptors using a radioligand-binding assay method.

This study was performed to assess the affinities of propranolol, bopindolol, its two metabolites (18-502, 20-785), pindolol, metoprolol, and atenolol to beta(1)- and beta(2)-adrenoceptor (beta(1)- and beta(2)-AR) subtypes using the membranes of COS-7 cells transiently expressing beta(1)- and beta(2)-AR subtypes. Radioligand-binding assays were performed and the results were compared with those (pKi or pA(2) values) obtained from the membrane-enriched fractions from the rat heart, cerebral cortex, bovine heart, tracheal smooth muscle or guinea-pig heart muscle. The pKi values of propranolol, bopindolol, its two metabolites, atenolol, pindolol and metoprolol to beta(1)-AR subtypes obtained from COS-7 cell membranes were 9.02 +/- 0.04, 7.44 +/- 0.12, 9.38 +/- 0.31, 6. 65 +/- 0.16, 5.55 +/- 0.14, 8.17 +/- 0.15 and 5.99 +/- 0.13, respectively. The rank order of pKi values for these agents to beta-(2)-ARs in COS-7 cell membranes was the same as that of beta(1)-ARs. In addition, good correlations were observed between pKi values of homogenates from various tissues and those of transfected COS-7 cell membranes to beta(1)- and beta(2)-ARs. Although good correlations were also observed between pA(2) values obtained from tracheal smooth muscle (beta(2)-ARs) and pKi values obtained from transfected COS-7 cell membranes to beta(2)-ARs, low correlation coefficient values to beta(1)-ARs were observed, however. In conclusion, these results suggested that binding characteristics of (3)H-CGP-12177 to beta-AR subtypes in these membranes from transfected COS-7 cells are similar to those from membrane fractions of various tissues.

Interaction with beta-arrestin determines the difference in internalization behavor between beta1- and beta2-adrenergic receptors.

The beta(1)-adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. As beta-arrestin is important for internalization, we examine the interaction of beta-arrestin with beta(1)AR with three different methods: intracellular trafficking of beta-arrestin, binding of in vitro translated beta-arrestin to intracellular domains of beta(1)- and beta(2)ARs, and inhibition of betaAR-stimulated adenylyl cyclase activities by beta-arrestin. The green fluorescent protein-tagged beta-arrestin 2 translocates to and stays at the plasma membrane by beta(2)AR stimulation. Although green fluorescent protein-tagged beta-arrestin 2 also translocates to the plasma membrane, it returns to the cytoplasm 10-30 min after beta(1)AR stimulation. The binding of in vitro translated beta-arrestin 1 and beta-arrestin 2 to the third intracellular loop and the carboxyl tail of beta(1)AR is lower than that of beta(2)AR. The fusion protein of beta-arrestin 1 with glutathione S-transferase inhibits the beta(1)- and beta(2)AR-stimulated adenylyl cyclase activities, although inhibition of the beta(1)AR-stimulated activity requires a higher concentration of the fusion protein than that of the beta(2)AR-stimulated activity. These results suggest that weak interaction of beta(1)AR with beta-arrestins explains the resistance to agonist-induced internalization. This is further supported by the finding that beta-arrestin can induce internalization of beta(1)AR when beta-arrestin 1 does not dissociate from beta(1)AR by fusing to the carboxyl tail of beta(1)AR.

[Simplified method for gene transfer and expression by recombinant adenoviruses].

Recombinant adenoviruses are attracting a great deal of attention as a highly efficient gene delivery technology and used for in vitro and in vivo gene expression in science. However, among traditional methods, there have been many difficulties and no simple procedure to generate recombinant adenoviruses. Since almost of all these methods involve the process of homologous recombination in a mammalian packaging cell line, the problems are low efficiency of homologous recombination, the need for complicated techniques and the demand for a long time to generate recombinant viruses. These problems have prevented widespread use of adenovirus technology as effective gene transfer tools. In the last few years, there have been several significant and innovative advances in adenoviral technologies, which include a new generation of vectors, the ease of vector manipulation and improvement of the viral production system with homologous recombination in E. coli. Here, we describe the easier and more efficient viral production systems and provide a practical manual for generating recombinant adenoviruses based on one of such systems.

Analysis of domain responsible for desensitization of beta1-adrenergic receptor.

When the wild type beta1-adrenergic receptor (WT-beta1AR) was expressed in Sf9 cells, the beta1AR-stimulated adenylyl cyclase activities were desensitized by prior treatment with isoproterenol. The extent of beta1AR desensitization was not modified, and the onset was not promoted by the overexpression of G protein-coupled receptor kinase 2 (GRK2), GRK5 or GRK6. However, overexpression of the dominant negative mutant of GRK2 appeared to inhibit desensitization of the beta1AR. The change of the potential protein kinase A phosphorylation site located at the intracellular third loop did not affect beta1AR desensitization. Desensitization of the truncated mutant, in which nearly all of the serine and threonine residues from the carboxyl terminus were eliminated, was the same as that of the WT-beta1AR. A deletion mutant that lacked serine and threonine residues of the intracellular third loop was also desensitized by isoproterenol stimulation. Furthermore, the deletion of serine and threonine residues from both the intracellular third loop and carboxyl terminus did not affect desensitization of the beta1AR. These results suggested that phosphorylation by endogenous GRKs in Sf9 cells contributed to desensitization of the beta1AR and that the regions other than third intracellular loop and carboxyl terminus may be responsible for beta1AR desensitization.

Ser203 as well as Ser204 and Ser207 in fifth transmembrane domain of the human beta2-adrenoceptor contributes to agonist binding and receptor activation.

We examined the contribution of Ser203 of the human beta2-adrenoceptor (beta2-AR) to the interaction with isoprenaline. The affinity of (-)-isoprenaline was reduced by substitution of an alanine for Ser203, as well as for Ser204 and Ser207. An (-)-isoprenaline derivative with only one hydroxyl group, at the meta-position, showed reduced affinity for wild-type beta2-AR and S207A-beta2-AR and even lower affinities for S203A-beta2-AR and S204A-beta2-AR. By contrast, an (-)-isoprenaline derivative with only a para-hydroxyl group showed reduced affinity for wild-type beta2-AR but the serine to alanine mutations did not cause further decreases. The EC50 value for cyclic AMP generation in response to (-)-isoprenaline was increased, by about 120 fold, for each alanine-substituted beta2-AR mutant. These results suggest that Ser203 of the human beta2-AR is important for both ligand binding and receptor activation.

Binding pockets of the beta(1)- and beta(2)-adrenergic receptors for subtype-selective agonists.

We examined the subtype-selective binding site of the beta-adrenergic receptors (betaARs). The beta(1)/beta(2)-chimeric receptors showed the importance of the second and seventh transmembrane domains (TM2 and TM7) of the beta(2)AR for the binding of the beta(2)-selective agonists such as formoterol and procaterol. Alanine-substituted mutants of TM7 of the beta(2)AR showed that Tyr(308,) located at the top of TM7, mainly contributed to beta(2) selectivity. However, Tyr(308) interacted with formoterol and procaterol in two different ways. The results of Ala- and Phe-substituted mutants indicated that the phenyl group of Tyr(308) interacted with the phenyl group in the N-substituent of formoterol (hydrophobic interaction), and the hydroxyl group of Tyr(308) interacted with the protonated amine of procaterol (hydrophilic interaction). In contrast to beta(2)AR, TM2 is a major determinant that beta(1)-selective agonists such as denopamine and T-0509 bound the beta(1)AR with high affinity. Three amino acids (Leu(110), Thr(117), and Val(120)) in TM2 of the beta(1)AR were identified as major determinants for beta(1)-selective binding of these agonists. Three-dimensional models built on the basis of the predicted structure of rhodopsin showed that Tyr(308) of the beta(2)AR covered the binding pocket formed by TM2 and TM7 from the upper side, and Thr(117) of the beta(1)AR located in the middle of the binding pocket to provide a hydrogen bonding for the beta(1)-selective agonists. These data indicate that TM2 and TM7 of the betaAR formed the binding pocket that binds the betaAR subtype-selective agonists with high affinity.

Negative regulation of alpha2-adrenergic receptor-mediated Gi signalling by a novel pathway.

Chinese hamster ovary (CHO) cells stably expressing alpha(2) adrenergic receptor (alpha(2)AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The alpha(2A)AR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of alpha(2A)AR-mediated inhibition was not observed when alpha(2A)AR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to G(i)-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates G(i) signalling in a protein-kinase-C-dependent manner.

Adrenergic alpha2C-receptors reside in rat striatal GABAergic projection neurons: comparison of radioligand binding and immunohistochemistry.

We have investigated the distribution of alpha2c-adrenergic receptors in the rat striatum and characterized the striatal neuron types expressing these receptors. Sequential double-labelled immunocytochemistry was performed with a polyclonal antibody against rat alpha2c-adrenoceptors and antibodies against GABA, Calbindin-D28k, parvalbumin and calretinin. The subregional distribution of alpha2c-adrenoceptor binding sites in the striatum was also quantitatively investigated using selective radioligands. Almost all lightly stained striatal GABAergic neurons, with the morphological characteristics of medium-sized spiny projection neurons (94% of GABAergic cells counted), contained alpha2c-adrenoceptor-immunoreactive structures. Intensely labelled GABAergic inteneurons (6%) were devoid of alpha2c-adrenoceptor immunoreactivity. The co-localization of calbindin- and alpha2c-adrenoceptor immunoreactivity in the majority of the cells confirmed the presence of alpha2c-adrenoceptors in the population of medium-sized spiny neurons. Furthermore, the alpha2c-adrenoceptor/calbindin double-labelling disclosed the existence of three neuronal subsets in the matrix compartment of the striatum: a large proportion (83%) of double-labelled neurons, a population of neurons (8%) that exhibited only alpha2c-adrenoceptor immunoreactivity without calbindin immunoreactivity, and a population of neurons (9%) immunoreactive for calbindin, but lacking alpha2c-adrenoceptors. In addition, alpha2c-adrenoceptor immunolabelled neurons were observed in calbindin-free striatal patches. Parvalbumin- and calretinin-positive neurons never displayed alpha2c-adrenoceptor immunoreactivity, confirming that striatal GABAergic interneurons are devoid of alpha2c-adrenoceptors. The present findings indicate that alpha2c-adrenoceptors are localized in GABAergic medium-sized spiny projection neurons but not in interneurons of the rat striatum, and that they may modulate both the direct and indirect pathways of the basal ganglia, as well as participate in the regulation of mesencephalic dopaminergic neurons.

Activation of Rac1 increases c-Jun NH(2)-terminal kinase activity and DNA fragmentation in a calcium-dependent manner in rat myoblast cell line H9c2.

We examined the role of intracellular Ca(2+) in c-Jun NH(2)-terminal kinase (JNK) activation and DNA fragmentation in the rat myoblast cell line H9c2 using small GTP-binding protein Rac1. A constitutively active mutant of Rac1 (V12-Rac1) increased JNK-responsive gene expression 6-fold, although this increase was attenuated by the intracellular Ca(2+) chelator BAPTA-AM. V12-Rac1 also increased the number of DNA fragmentated cells. However, V12-Rac1-mediated JNK activation was not affected by BAPTA-AM as determined by direct measurement of active forms, and V12-Rac1 did not affect intracellular Ca(2+) concentration. These results suggest that Rac1 can activate JNK and induces cell injury, but [Ca(2+)](i) is necessary for V12-Rac1 to induce DNA fragmentation downstream of JNK activation.

[beta gamma subunit of heterotrimeric G protein as a new target molecule for drug development].

Although ischemia-reperfusion produces reactive oxygen species and induces injury of the heart, the mechanism leading to injury is largely unknown. Hydrogen peroxide (H2O2) is widely used for a reagent to mimic the action of reactive oxygen species produced by ischemia-reperfusion. Treatment of the rat neonatal myocytes with H2O2 resulted in activation of mitogen-activated protein kinases (MAPKs) such as extracellular signal regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38. To study the involvement of beta gamma subunit of heterotrimeric G protein in H2O2-induced activation of MAPKs, we expressed the carboxyl terminus of G protein-coupled receptor kinase 2 (GRK2-ct) which can bind beta gamma subunit and inhibit the interaction with various effector proteins. Expression of GRK2-ct inhibited the H2O2-induced activation of ERK by 70% and also inhibited the activation of Akt by 30%. In contrast with H2O2-induced activation of ERK, the activation of ERK induced by phorbol ester PMA and the activation of JNK and p38 induced by H2O2 were not affected by expression of GRK2-ct, indicating that the activation of ERK but not JNK and p38 is dependent on beta gamma subunit. Among several inhibitors for analyzing intracellular signaling pathways, wortmannin inhibited the activation of ERK by H2O2 treatment. These data suggest that treatment of the rat neonatal myocytes with H2O2 releases beta gamma subunit from heterotrimeric G protein, and leads to activation of ERK in part by phosphatidylinositol-3 kinase dependent pathway. Thus beta gamma subunit may be a novel target molecule to selectively modulate the intracellular signaling cascade.

[Production of various antibodies and single chain Fv molecule against signal-transducing proteins].

In the present review, we described the procedures of production of polyclonal and monoclonal antibodies and single chain Fv molecule (scFv). Among several animal species, the rabbit is the best animal for polyclonal antibody production and the mouse is the best animal for monoclonal antibody production. In this review, we discussed problems that might be encountered when trying to produce antibodies. Polyclonal antibodies are easily produced in rabbits by immunizing them with glutathione-S-transferase fusion proteins. However, it is difficult to eliminate nonspecifically reacting antibodies, even after the antibodies were purified from sera by an antigen column. Monoclonal antibody production is a time-consuming process, but it successful, will produce a very useful reagent due to no limitation of supply and constant quality. We described monoclonal antibody production by means of glutathione-S-transferase fusion protein. scFv is a portion of the antibody and is constructed by PCR of VH and VL regions of the antibody. We recommend that scFv should be constructed from a hybridoma that secretes monoclonal antibody, although some researchers have claimed that scFv can be constructed from the spleen of immunized mice. The expression of scFv is a promising approach to analyze the function of one of the subtypes, when the original monoclonal antibody can block the function of the protein.

Identification of a key amino acid of the beta2-adrenergic receptor for high affinity binding of salmeterol.

Transmembrane domains (TMDs) I, II, and VII of the beta2-adrenergic receptor (beta2AR) were replaced, individually or in combination, with the corresponding regions of the beta1AR, and vice versa. The beta2-selective binding of salmeterol was not affected by the exchange of TMD I between the beta1- and beta2ARs. The affinity of salmeterol was slightly decreased (32-fold) by replacement of TMD II of the beta2AR with the homologous region of the beta1AR; the affinity was strongly decreased (1870-fold) for the beta2AR with TMD VII of the beta1AR. The affinity of salmeterol was partially restored by the introduction of TMD VII, but not TMD II, of the beta2AR into the beta1AR. By analyzing alanine-substituted mutants, we found that Tyr308 in TMD VII was mainly responsible for the high affinity binding of salmeterol. Two salmeterol derivatives with the ether oxygen at different positions in the side chain showed 33- and 64-fold decreased affinities for the wild-type beta2AR, and a derivative with no ether oxygen showed 147-fold decreased affinity for the wild-type beta2AR. These results indicate that Tyr308 in TMD VII is the major amino acid conferring the beta2-selective binding of salmeterol to the beta2AR and that the position of the ether oxygen in the side chain is also important for beta2-selective binding. A three-dimensional model of the salmeterol-beta2AR complex shows that the phenyl group of Tyr308 interacts with methylene groups near the protonated amine of salmeterol and the ether oxygen interacts with Tyr316.

Cellular and subcellular sites for noradrenergic action in the monkey dorsolateral prefrontal cortex as revealed by the immunocytochemical localization of noradrenergic receptors and axons.

A series of electron microscopic immunocytochemical studies was performed to analyze subcellular sites for noradrenergic modulation in monkey prefrontal cortex. One out of 12 noradrenergic varicosities, identified by dopamine beta-hydroxylase immunocytochemistry within single ultrathin sections, forms morphologically identifiable junctions with small dendrites and spines. Accordingly, alpha2-adrenergic receptors, almost all of which are of the A-subtype, that occur in spines are localized discretely over postsynaptic membranes. alpha2-Adrenergic receptors are also found at sites along axons, dendritic shafts and astrocytic processes lacking morphologically identifiable synaptic junctions, suggesting that these receptors are activated by volume transmission. In particular, axonal alpha2-adrenergic receptors occur mostly at pre-terminal regions, suggesting that axo-axonic interactions may mediate reduction of neurotransmitter release at sites other than axo-spinous junctions by closing voltage-dependent calcium channels. These results indicate that noradrenergic modulation of prefrontal cortex involves synaptic interactions at spines of pyramidal neurons and nonsynaptic volume transmission to glia, dendritic shafts and axons.

Domains of beta1 and beta2 adrenergic receptors to bind subtype selective agonists.

We studied the binding region of several beta1 and beta2 selective agonists by using chimeric beta1 and beta2ARs, and point-mutated beta2 adrenergic receptors (ARs). By replacing a single transmembrane domain (TMD) of beta1AR (or beta2AR) with the corresponding region of beta2AR (or beta1AR), we found that beta2 or beta1 selectivities were determined by TMD2 and TMD7 of beta2AR or by TMD2 of beta1AR, respectively. Alanine-substituted beta2AR mutants showed that tyrosine at position 308 in TMD7 played an important role in binding of beta2 selective agonists with high affinity. These data also suggested that the substituent on the amine portion was important for subtype selective agonist binding.

The role of the seventh transmembrane region in high affinity binding of a beta 2-selective agonist TA-2005.

To determine the structural basis for binding subtype selective agonists in the beta-adrenergic receptor (beta AR), we examined the interaction of the mutant beta 2AR and chimeric beta 1/beta 2AR with a selective beta 2AR agonist, TA-2005 (8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxyphenyl)-1-methyle thy l] amino]ethyl] carbostyril hydrochloride). The beta 2AR 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-beta 2AR. In contrast with TA-2005, the EC50 values of compound I were reduced in the S204A-beta 2AR but not in the S207A-beta 2AR. 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 beta 2AR, the compounds lacking p-methoxyphenyl-ethyl (compound II) or p-methoxyphenyl-methylethyl groups (compound III) on the amine portion of TA-2005 lost beta 2AR subtype selectivity. When the second and seventh transmembrane (TM) region but not the TM1 region of the beta 2AR were replaced with the corresponding regions of the beta 1AR, the affinities of the chimeras for TA-2005 decreased compared with those of the wild type beta 2AR. Furthermore, substitution of the TM7 region of the beta 1AR with the corresponding region of the beta 2AR 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 beta 2AR plays an important role in beta 2-selective agonist binding. To determine the specific amino acid which confers this high affinity binding of TA-2005 to the beta 2AR, an alanine-scanning mutagenesis approach was employed. All amino acids that were different from those of the beta 1AR were individually changed to alanine. One mutant receptor (Y308A-beta 2AR) out of 10 point-mutated beta 2ARs showed a dramatically reduced affinity for TA-2005. These results indicate that Tyr308 is an essential amino acid for high affinity binding of the beta 2-selective agonist TA-2005.