Phe310 in Transmembrane VI of the alpha 1B-Adrenergic Receptor Is a Key Switch Residue Involved in Activation and Catecholamine Ring Aromatic Bonding*

Songhai ChenDagger , Ming XuDagger , Fang LinDagger , Darren LeeDagger , Peter RiekDagger , and Robert M. GrahamDagger §

From the Dagger  Molecular Cardiology Unit, Victor Chang Cardiac Research Institute, St. Vincent's Hospital, Sydney 2010, Australia and the § School of Biochemistry and Molecular Genetics, University of New South Wales, Kensington, New South Wales 2033, Australia

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Pharmacophore mapping of adrenergic receptors indicates that the phenyl ring of catecholamine agonists is involved in receptor binding and activation. Here we evaluated Phe310, Phe311, and Phe303 in transmembrane VI (TMVI), as well as Tyr348 in TMVII of the alpha 1B-adrenergic receptor (alpha 1B-AR), which have been implicated in a catechol-ring interaction. Neither catecholamine docking studies nor mutagenesis studies of Phe311, Phe303, or Tyr348 supported a role for these residues in catechol-ring binding. By contrast, docking studies indicated that the Phe310 side chain is well positioned to interact with the catechol-ring, and substituted cysteine accessibility method studies revealed that the side chain of the 310, but not 311 residue, is both solvent accessible and directed into the agonist-binding pocket. Also, saturation mutagenesis of both Phe310 and Phe311 revealed for the former, but not for the latter, a direct relationship between side chain volume and agonist affinity, and that aromaticity is essential for wild-type agonist binding, and for both wild-type agonist potency and efficacy. Moreover, studies of Phe310 mutants combined with a previously described constitutively active alpha 1B-AR mutant, A293E, indicated that although not required for spontaneous receptor isomerization from the basal state, R, to a partially activated conformation R', interaction of Phe310 with catecholamine agonists is essential for isomerization from R' to the fully activated state, R*.

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INTRODUCTION
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alpha 1-Adrenergic receptors (alpha 1-AR)1 are members of the heptahelical superfamily that share a common structural motif of seven putative alpha -helical transmembrane spanning regions linked by three extra- and three intracellular loops, an extracellular N terminus and intracellular C-terminal tail. Transmembrane signaling by all alpha 1-AR subtypes (alpha 1A, alpha 1B, and alpha 1D) in response to the natural catecholamine agonists, norepinephrine and epinephrine, is mediated by G-proteins of the Gq/11 family or in some instances, the Gh family of tissue transglutaminases (1, 2). Based on certain key structural features, alpha 1-ARs are more closely related to rhodopsin or the type A subfamily of GPCRs that includes beta -ARs, than to the calcitonin (type B) or metabotropic (type C) subfamilies.

Binding of catecholamines by both alpha 1- and beta -ARs involves the formation of a salt bridge between the basic aliphatic nitrogen atom common to all sympathomimetic amines and an aspartate (Asp125 in the hamster alpha 1B-AR; Asp113 in the hamster beta 2-AR) in the third transmembrane segment (TMIII) (3, 4). With rhodopsin, light induced isomerization of the retinal chromophore leads to deprotonation of a Schiff base linking the chromophore to Lys296 in TMVII (5). In the ground state the protonated Schiff base is ionically bonded to a TMIII acidic residue (Glu113) that is equivalent to Asp125 and Asp113 in the alpha 1B and beta 2-ARs, respectively (5). With the alpha 1B-AR there is evidence that receptor activation also is due to disruption of an ionic interaction between the TMIII aspartate and a TMVII lysine (Lys331), due to competition between the catechol protonated amine and the TMVII lysine, for binding to the TMIII aspartate (3). The TMIII aspartate thus serves as a counterion and most likely is an important residue for agonist binding and activation of all adrenergic receptors.

For the beta 2-AR, two serine residues, Ser204 and Ser207, which are conserved in most adrenergic receptors, have been proposed to hydrogen bond with the meta- and para-hydroxyls, respectively, of the catechol ring (6). Mutation of either serine to an alanine results in a 30-fold decrease in affinity for catecholamine agonists, and each serine contributes about 50% to receptor activation. Thus, binding of both catechol hydroxyls is required for full agonist activity. By contrast, agonist binding to the alpha 1A-AR involves an interaction between the meta-hydroxyl and Ser188 (equivalent to Ser203, not Ser204 in the beta 2-AR) that plays a major role in receptor activation, being responsible for 70-90% of the wild-type response. An interaction between the para-hydroxyl and Ser192 (equivalent to Ser207 in the beta 2-AR), on the other hand, contributes minimally to receptor activation (7). Moreover, since the interacting serines in the alpha 1A-AR are separated by four residues, whereas those in the beta 2-AR are separated by only three residues, docking of the catecholamine ring is in a more planar orientation in the alpha 1A-AR, and is rotated by about 120° to that in the beta 2-AR.

This altered catechol ring orientation may also contribute to other agonist docking differences between alpha 1- and beta -ARs. For example, stereoselectivity of binding and activation has been attributed, in part, to a hydrogen bond interaction between the chiral benzylic hydroxyl group attached to the beta -carbon atom of catecholamines, and Asn293 in TMVI of the beta 2-AR (8). Although stereoselectivity of catecholamine binding and activation is preserved with alpha 1-ARs, the determinants of stereoselectivity have not been defined, and the Asn293 equivalent is replaced by a residue (leucine or methionine) lacking hydrogen-bonding potential. This finding again provides evidence that some of the catecholamine-binding and activation residues in alpha 1-ARs are distinct from those in beta -ARs.

Previous studies of the hamster beta 2-AR suggested that a phenylalanine in TMVI (Phe290, equivalent to Phe311 in the alpha 1B-AR, see Fig. 1), which is conserved only in biogenic amine-binding GPCRs, is involved in forming an aromatic-aromatic interaction with the phenyl ring of catecholamines (9). This conclusion was based on the finding of a 10-fold decrease in agonist, but not antagonist, binding with mutation of Phe290 to methionine. However, no additional studies were performed to exclude a nonspecific global or local change in receptor structure with this Phe290 mutation, or to evaluate the role of the potential aromatic-aromatic interaction in receptor activation. In addition, substitution of an adjacent TMVI phenylalanine (Phe289; equivalent to Phe310 in the alpha 1B-AR) to alanine, resulted in a 1000-fold decrease in agonist affinity with no change in antagonist binding. Finally, Tyr326 in TMVII was also suggested to potentially be involved in a catechol ring interaction, since substitution of this residue with leucine decreased agonist, but not antagonist binding by 10-fold (9).


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Fig. 1.   Sequence alignment of TM VI residues of hamster alpha 1B and other G protein-coupled receptors. Sequences were aligned to maximize homology within this region using the GCG program "Pileup." The conserved phenylalanines corresponding to Phe310 and Phe311 in the hamster alpha 1B-AR are shaded and in bold type, while Phe303, which is highly conserved among all G protein-coupled receptors is boxed. The dashed line at the top delineates the transmembrane residues of helix VI.

Here we show, based on macromolecular modeling studies, in which the planar orientation of the phenyl ring in alpha 1-ARs was taken into consideration when docking catecholamines, that interaction with the phenyl ring involves Phe310 in TMVI and not Phe311 in TMVI, or Tyr348 in TMVII (equivalent to Tyr326 in the beta 2-AR). Furthermore, based on mutagenesis studies coupled with the evaluation of group-specific catecholamine analogs, as well as SCAM (substituted cysteine accessibility method) studies, we provide evidence that Phe310 is critically involved both in forming an aromatic-aromatic interaction with the catecholamine phenyl ring and in receptor activation.

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Materials-- (-)-Epinephrine, (-)-norepinephrine, (±)-synephrine, (±)-halostachine, dopamine, phenethylamine, prazosin, phentolamine hydrochloride, lithium chloride, and dl-propranolol were purchased from Sigma. 5-Methylurapidil was from Research Biochemicals International. AG 1-X8 (100-200 mesh, formate form) was from Bio-Rad, and fetal calf serum and culture media from Trace Biosciences, Australia. myo-[3H]Inositol (80 Ci/mmol) was from Amersham. [125I]HEAT (2200 Ci/mmol) was from Du Pont. Methanethiosulfonate ethylammonium (MTSEA) was from Toronto Research Chemicals Inc. Other chemicals were of the highest grade available commercially.

Site-directed Mutagenesis-- The construct used was the hamster alpha 1B-AR cDNA with an octapeptide tag (1D4) at the end of coding region in the modified eukaryotic expressing vector, pMT2' (10). The presence of the 1D4 epitope at the C-terminal of alpha 1B-AR does not affect its expression and function (data not shown). Site-directed mutagenesis was performed as described previously (2, 11). Briefly, two primers, one carrying the nucleotide change(s) to produce the desired amino acid mutation in the alpha 1B-AR sequence, the other carrying the nucleotide changes to convert a unique restriction site (ClaI) to another restriction site (NarI) in a non-essential region of the vector, pMT2', were simultaneously annealed to the denatured template and a new second strand DNA containing both primers was synthesized by treatment with T4 polymerase. The DNA was then digested with ClaI to linearize re-annealed parental plasmid and the reaction mixture used to transform Escherichia coli cells (BMH 71-18 mutS cells). Transformants were grown en mass in liquid media and used to isolate plasmid DNA. The resulting DNA was digested with ClaI again to linearize remaining parental plasmid, and transformed into DH5alpha -cells. Transformants were plated and plasmid DNA prepared and sequenced to confirm the presence of the desired mutation.

Cell Culture and Transfection-- COS-1 cells (American Type Culture Collection, Manassas, VA) were cultured and transiently transfected with the indicated constructs using the DEAE-dextran method, as described previously (2, 12, 13). The transfection efficiency was 30-40%, as determined by in situ staining of cells transfected with pSVLacZ, a plasmid encoding the reporter, beta -galactosidase, and treatment of the cells with 0.2% 5-bromo-4-chloro-3-indoyl-beta -D-galactoside. Cells were harvested 72 h post-transfection.

Membrane Preparation-- Membranes were prepared from transfected COS-1 cells, as described previously (2, 10). The membranes were resuspended in HEM buffer (20 mM HEPES, pH 7.5, 1.5 mM EGTA, 12.5 mM MgCl2) containing 10% (v/v) glycerol, and stored at -70 °C. Protein concentration was determined by the Bradford method (14).

Western Blotting-- Membranes (50 µg of protein) were dissolved in 1% CHAPS and SDS sample buffer overnight at 4 °C, and then subjected to SDS-polyacrylamide gel electrophoresis, as described previously (2, 12). The resolved proteins were electroblotted onto Immobilon-P membranes and then immunostained for detection using the ECL chemiluminescence system (Amersham), as described previously (2). alpha 1B-AR was detected using a monoclonal antibody against the 1D4 epitope (12).

Ligand Binding-- The ligand binding characteristics of the membrane expressed receptors were determined in a series of radioligand binding studies performed exactly as described previously (2, 10), using [125I]HEAT, an alpha 1-specific antagonist, as the radioligand. For saturation binding studies, the concentrations of [125I]HEAT used ranged from 10-fold below to 10-fold above the Kd value, whereas for competition studies, a concentration near the Kd of the radioligand value was used. Ki values were determined using the Cheng-Prusoff equation (15). The membrane concentration used in these studies was selected to allow binding of less than 10% of the total radioligand added. To avoid interassay differences replicate studies with the wild-type alpha 1B-AR were performed with the analysis of each mutant. Binding data were analyzed using the iterative, nonlinear, curve-fitting program, Prism. For comparison of the fit to a one-site or two-site model, the F test was used, p < 0.05 was considered to be statistically significant.

Reaction with MTSEA and Binding Assays in Intact Cells-- Transfected COS-1 cells were harvested by trypsinization. After washing with phosphate-buffered saline, cells were resuspended in 1.4 ml of HEPES buffer (140 mM NaCl, 5.4 mM KCl, 1 mM EDTA, 0.006% bovine serum albumin, 25 mM HEPES, pH 7.4) as described previously (16). Aliquots (80 µl) of the cell suspension were incubated with 20 µl of freshly prepared MTSEA at the stated concentrations at room temperature for 2 min. Cell suspensions were then diluted 20-fold, and 100-µl aliquots were used to assay for [125I]HEAT (600 pM) binding in the presence or absence of 0.1 mM phentolamine in a total volume of 250 µl in triplicate. The result was analyzed as described above.

Construction of an alpha 1B-AR Molecular Model and Catecholamine Docking-- The coordinates of the alpha -carbon positions were determined by overlay of putative alpha 1-AR transmembrane residues with the transmembrane coordinates of bacteriorhodopsin (17), with data files generated using the Insight II molecular modeling software from Biosym Technologies. The boundaries of the putative transmembrane domains were determined using an algorithm based on the weighted pairwise comparisons of adjacent residues (18). The positioning of each helix with respect to the adjacent helices was based where possible on data from alpha 1B-AR mutagenesis studies (10, 18, 19). The projections of the helices proposed by Baldwin (21) were used to determine the tilt of each helix. The model was minimized and conflicts adjusted to remove steric clashes based on dynamic runs, as described previously (22).

Phosphatidylinositol Hydrolysis in Intact Cells-- Phosphatidylinositol (PI) hydrolysis in intact, transfected COS-1 cells was determined exactly as described previously (2), except that for determining basal activation of PI hydrolysis, the cells were seeded onto 6-well plates 1 day after transfection. Results are expressed as the mean ± S.E. (error bars). An analysis of variance and the Student's t test were used to determine significant differences (p < 0.05).

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A model of the alpha 1B-AR developed previously (22) was refined to accommodate the findings of recent mutagenesis studies and used to dock the agonist, (-)-epinephrine, into the binding pocket. In particular the refined model takes into account an interhelical stacking interaction we have identified between Ala204 in TMV and Leu314 in TMVI (10, 20), as well as the planar orientation of the catechol ring. As shown in Fig. 2A, from this model it is evident that the TMVI Phe310 side chain is well positioned to interact with the catechol ring in a parallel stacked and displaced conformation, which is an energetically favored structure for benzene dimers (23). The TMVI Phe311 side chain, on the other hand, is directed toward TMV, or is projecting into the lipid bilayer, and Tyr348 is located toward the intracellular end of TMVII, well below the plane of the catecholamine ligand.


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Fig. 2.   Model and secondary structure of the hamster alpha 1B-AR. A, a model of the alpha 1B-AR showing epinephrine docked into the ligand-binding pocket. The receptor is modeled as it would appear looking down onto the membrane from the extracellular side. White circles with Roman numerals indicate the respective transmembrane helices. The projection of the helices from the extra- to intracellular surface of the membrane is indicated by the yellow cylinders. Epinephrine is shown interacting via its protonated amine with Asp125 in TMIII; via the catechol ring meta-hydroxyl with Ser207 in TMV, and via the catechol ring with Phe310 in TMVI. Previously identified interactions between Asp125 in TMIII and Lys331 in TMVII (3), and Ala204 in TMV and Leu314 in TMVI (10, 19), as well as the orientation of Phe311 are shown. B, secondary structure of the hamster alpha 1B-AR indicating the location of the native cysteine residues, including the putative, solvent inaccessible disulfide-linked extracellular pair, Cys119 and Cys195 (38), and the residues (Phe303, Phe310, Phe311, and Tyr348) evaluated in this study.

To more directly evaluate the involvement of Phe310 in catechol ring binding, it was mutated to alanine or leucine, and the resulting mutants (F310A and F310L), as well as the wild-type alpha 1B-AR, were then evaluated in terms of membrane expression, ligand binding, and stimulation of PI hydrolysis. As controls for the potential Phe310-catechol ring interaction, alanine and leucine mutants of two other TMVI phenylalanines, Phe311 and Phe303, and a leucine mutant of Tyr348 in TMVII, were also constructed and similarly evaluated (Fig. 2B). Saturation binding and immunoblotting studies indicated that the F303A, F303L, and Y348L mutants were expressed in the plasma membrane and processed (glycosylation) at levels almost equal to the wild-type alpha 1B-AR (Table I and Fig. 3). All three mutants showed only small decreases (1.7-2.2-fold) in affinity for the antagonist radioligand, [125I]HEAT (Table I) and no change (Y348L) or an increase (5-20-fold, F303A and F303L) in affinity for the agonists, norepinephrine, epinephrine, and phenylephrine (Fig. 4). As will be reported in a subsequent paper,2 the increased agonist affinities observed with the Phe303 mutants are due to the fact that these substitutions result in constitutive receptor activation. Taken together, therefore, these findings do not support involvement of Phe303 or Tyr348 in an interaction with the catechol ring.

                              
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Table I
Binding of [125I]HEAT by wild-type and mutant alpha 1B-ARs
[125I]HEAT saturation binding studies were carried out with membranes prepared from transfected COS-1 cells as described under "Experimental Procedures." Bmax values indicate the maximum number of binding sites/mg of membrane protein. Data are presented as mean ± S.E. of three to five independent experiments, each performed in duplicate.


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Fig. 3.   Western blot analysis of receptor proteins expressed in membranes from COS-1 cells transfected with wild-type or mutant hamster alpha 1B-AR cDNA. Cells were transfected with 2 µg of cDNA for each construct, except that 25 and 20 µg of the F311A and F311L constructs were used, respectively. Membranes (50 µg) prepared from the transfected cells were solubilized in 1% CHAPS, and then subjected to immunoblot analysis after 12% SDS-polyacrylamide gel electrophoresis resolution, using a monoclonal antiserum to the 1D4 epitope at the C-terminal tail of the receptors, as described under "Experimental Procedures." The position of molecular markers (kDa) is shown on the left, while that of the receptor is indicated by the arrows. The higher Mr species (approximately 80 kDa) is the fully glycosylated receptor, whereas the 55-kDa species corresponds to the unglycosylated protein. This was confirmed by treating membranes with N-glycanase to deglycosylate the receptor-protein, which resulted in loss of the 80-kDa species and visualization only of the 55-kDa species (data not shown).


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Fig. 4.   Binding of agonists to Phe right-arrow Ala (A) or Phe right-arrow Tyr to Leu (B) alpha 1B-AR mutants. The binding affinity of agonists was determined by competition binding studies using transfected COS-1 cell membrane, as described under "Experimental Procedures." The fold change in KiM) values for the various mutants, as compared with those determined for the wild-type alpha 1B-AR, is shown. The chemical structures of the agonists evaluated are shown below "B"; Epi, (-)-epinephrine; NE, (-)-norepinephrine; Pheny, (-)-phenylephrine; Syn, (±)-synephrine; Halo, (±)-halostachine; Phene, phenethylamine; Dopa, dopamine.

As shown in Table II, the Phe310 mutants showed binding affinities for the antagonists, phentolamine, prazosin, and 5-methylurapidil that were 1.4-44-fold less than observed with the wild-type receptor. However, their affinity for the radioligand [125I]HEAT was largely unchanged (Table I) and their plasma membrane expression was equivalent to that of wild-type receptor (Table I and Fig. 3).

                              
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Table II
Antagonist binding profiles of wild-type and mutant alpha 1B-ARs
Antagonist binding affinities (Ki values, nM) were determined in [125I]HEAT competition binding studies using membranes prepared from transfected COS-1 cells as described under "Experimental Procedures." Data are presented as mean ± S.E. of at least three independent experiments, each performed in duplicate. Values in parentheses are the ratio of Ki values of the mutant to the wild-type alpha 1B-ARs.

The Phe311 mutants also displayed decreased antagonist binding (5-94-fold, Table II). However, in contrast to the Phe310 mutants, plasma membrane expression was markedly impaired with both F311A and F311L, and could not be enhanced by increasing the amounts of plasmid transfected (Table I and Fig. 3). These findings suggest that whereas substitution of Phe310 with alanine or leucine results in the loss of receptor interactions involved in the binding of some but not all antagonists, mutation of Phe311 produces a global change in receptor conformation that impairs not only ligand binding, but also protein folding and membrane expression.

To further characterize the effects of alanine or leucine substitution at the Phe310 and Phe311 positions, the binding of group-specific agonists was evaluated. The agonists evaluated included the natural catecholamines, (-)-norepinephrine, and (-)-epinephrine, as well as (-)-phenylephrine, which is also a full agonist but lacks the para-hydroxyl at the 4-position of the catechol ring, and a group of partial agonists: (±)-synephrine, (±)-halostachine, phenethylamine, and dopamine, which lack a meta-hydroxyl at the 3-position of the catechol ring; both catechol ring hydroxyls; both catechol ring hydroxyls and the chiral hydroxyl on the beta -carbon; or only the chiral beta -hydroxyl, respectively. As shown in Fig. 4A, the F310A mutant showed affinity losses of up to 1000-fold for the full agonists, but smaller losses (10-50-fold) for the partial agonists. Consistent with the involvement of Phe310 in ligand binding, the F310L mutant in which the hydrophobicity but not the aromaticity of the wild-type phenylalanine residue is preserved, displayed lesser decreases in binding of both full agonists (30-40-fold) and partial agonists (3-10-fold), than the F310A mutant (Fig. 4B).

As shown in Table III, the free energy change (Delta Delta G) observed for the full agonists, with substitution of Phe310 with alanine, was close to 4 kcal/mol, but only about 2 kcal/mol for the partial agonists. For the leucine substitution the free energy change in agonist binding was approximately 2 kcal/mol for the full agonists, and <1 kcal/mol for the partial agonists. Given that the theoretical bond energy of an aromatic-aromatic interaction is approximately 2 kcal/mol (23), one interpretation of these findings is that the leucine substitution, because of the hydrophobic character of its side chain, but not the alanine substitution, which lacks both hydrophobicity and aromaticity, partially compensates for the wild-type phenylalanine. The greater free energy loss observed for full agonists with the alanine substitution suggests that the aromatic component of the Phe310 interaction with the catechol ring is not only essential for ligand binding, but for critical positioning of the catechol ring to allow optimal interaction of other ligand moieties with the receptor, such as the catecholamine meta-hydroxyl and Ser207. With partial agonists, which lack at least one of the critical moieties of full agonists and, thus, most likely have a less constrained binding geometry, positioning of the catechol ring by an aromatic interaction with the Phe310 side chain may be less critical. As a result, the free energy losses for partial agonists are less than those for full agonists, and almost exactly those anticipated (2 kcal/mol) for the loss of a single aromatic-aromatic interaction.

                              
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Table III
Free energy change of agonist binding to wild-type and mutant alpha 1B-ARs

Like the Phe310 mutants, the Phe311 mutants also displayed decreased affinity for both full and partial agonists (Fig. 4). However, unlike the Phe310 mutants, the loss of agonist affinity with leucine substitution of Phe311 was greater than with alanine for the full agonists (up to 1000-fold versus 50-100-fold), but the same or less for partial agonists (5-10-fold versus 8-25-fold). Thus, the loss of both aromaticity and hydrophobicity with alanine substitution of Phe311 could not be compensated by a residue with a hydrophobic side chain (leucine) and, in fact, such a residue caused a greater impairment of agonist binding. Together with the markedly decreased membrane expression observed for the Phe311 mutants, these findings indicate a strict requirement for the phenylalanine side chain to allow proper folding into the wild-type receptor conformation, perhaps due to a critical interhelical stacking interaction between Phe311 and residues in the TMV helix.

To further evaluate the postulate that Phe310 forms a critical aromatic-aromatic interaction with the catechol ring, whereas Phe311 is not directly involved in ligand binding but rather in global receptor structure, additional Phe310 and Phe311 mutants were constructed and their agonist-binding properties evaluated. As seen in Fig. 5, the Phe310 mutants produced graded decreases in agonist affinity. Whereas the affinity for epinephrine, phenylephrine, and halostachine was up to 1000-fold lower with the F310A mutant than with the wild-type receptor, substitution of Phe310 with a tryptophan, which although slightly larger than the native phenylalanine, preserves both its hydrophobicity and aromaticity, resulted in a receptor protein with near wild-type agonist affinities. Substitution with a tyrosine, which also has an aromatic side chain, however, resulted in decreases in agonist affinity (25-30-fold) that were comparable to those observed with the F310L mutation. This may be due to the fact that the tyrosine ring contains an hydroxyl moiety. As a result, its side chain, unlike those of phenylalanine or tryptophan, is unlikely to be planar, and also has H-bonding potential. Thus, a tyrosine at the 310 position may perturb optimal positioning of the catechol ring due either to a steric clash or to loss of a favorable planar stacking interaction. Other substitutions at the 310 position with smaller hydrophobic or beta -branched residues (valine and isoleucine) or polar residues (asparagine) resulted in significantly greater reductions in agonist affinity than did tyrosine, leucine, or phenylalanine. Not surprisingly, therefore, a positive and highly significant correlation was evident between the volume of the substituent side chain and agonist affinity (Fig. 5). This correlation indicates a van der Waals component to the bonding between the catechol ring and the side chain at the 310 position. This is not inconsistent with an aromatic-aromatic interaction, which involves both a dipole-dipole and a van der Waals component (23). In keeping with an aromatic-aromatic interaction is the finding that the wild-type receptor and the F310W mutant, which both have a planar aromatic side chain at the 310 position, bind with considerably higher affinity than the F310L mutant. In the case of this latter mutant the 310 side chain is of similar volume to phenylalanine, but rather than being aromatic and planar, is aliphatic and bulky.


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Fig. 5.   Correlation between the change in agonist binding affinity and the volume of the substituent amino acid side chain at Phe310 (A-C) and Phe311 (D-F). The affinity for (-)-epinephrine (A and D), (-)-phenylephrine (B and E) and (±)-halostachine (C and F) binding by the various Phe310 or Phe311 mutants was determined as detailed in the legend to Fig. 4. The relationship between the change in agonist binding affinity and the volume of the substituent amino acid side chain was analyzed by linear regression. The goodness of fit, indicated by the r2 and p values, and the slope of each regression line are shown.

In contrast to the Phe310 mutations, no correlation was observed between the volume of the substituent side chain at the 311 position and agonist affinity (Fig. 5). Thus, small polar residues, such as asparagine, produced lesser decreases in affinity than larger hydrophobic residues, such as leucine or isoleucine.

If Phe310 indeed interacts with the catechol ring, as suggested by the above findings, it should be solvent accessible with its side chain projecting into the agonist-binding pocket. By contrast, Phe311, which we speculate projects toward TMV, should be much less solvent accessible. To directly evaluate the solvent accessibility of Phe310 and Phe311, we constructed the cysteine mutants, F310C and F311C, and used the SCAM (16, 24) to test their sensitivity to derivatization with the polar cysteine-modifying reagent, MTSEA. This compound is 2500 times more soluble in water than in n-octanol, fits into a cylinder of about 0.6 nm in diameter by 1 nm in length, and specifically adds its -SCH2CH2NH3+ moiety to reduced sulfhydryls to form mixed disulfides (24). Given the size of the -SCH2CH2NH3+ moiety, derivitization of a residue projecting into the binding pocket should sterically hinder ligand binding, whereas derivitization of a residue projecting toward an adjacent helix or into the lipid bilayer will occur much more slowly, and will either not influence ligand binding or will produce a more global change in receptor structure.

To evaluate the accessibility of Phe310 and Phe311 to MTSEA derivitization, we initially evaluated the accessibility of the 16 native cysteines (Fig. 2B) in the wild-type alpha 1B-AR. As shown in Fig. 6, MTSEA modification of intact COS-1 cells expressing the wild-type alpha 1B-AR irreversibly inhibited binding of the radioligand [125I]HEAT. This inhibition could be rescued by treatment with the reducing agent, dithiothreitol, and could be prevented by pretreatment with the alpha 1-AR antagonist, phentolamine (data not shown). This indicates that MTSEA specifically modified cysteine residues rather than producing a nonspecific disruption of receptor structure. As will be detailed in a subsequent paper,2 mutagenesis of three native cysteines (Cys128, Cys129, and Cys137) to serines, significantly reduced both the sensitivity and reactivity of the alpha 1B-AR to MTSEA modification (Fig. 6, A and B). Importantly, the triple mutant (C128S/C129S/C137S) displayed similar binding affinities for various alpha 1-AR antagonists ([125I]HEAT and prazosin) and agonists (epinephrine and cirazoline), and similar activity in stimulating PI hydrolysis, as the wild-type receptor (Table IV). This triple mutant was thus used as a template for SCAM studies of F310C and F311C.


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Fig. 6.   Effect of MTSEA on [125I]HEAT binding to wild-type and mutant alpha 1B-ARs. COS-1 cells transfected with plasmids encoding the wild-type (black-square) or mutant (, C128S/C129S/C137S (3C); open circle , F310C/C128S/C129S/C137S (F310/3C); , F311C/C128S/C129S/C137S (F311/3C)) alpha 1B-ARs were incubated with the indicated concentrations of MTSEA for 2 min (A); or incubated with 1 mM MTSEA for the times indicated (B), or pretreated with (-)-epinephrine (0.1 mM for the wild-type alpha 1B-AR and C128S/C129S/C137S, 10 mM for F310C/C128S/C129S/C137S, and 50 mM for F311C/C128S/C129S/C137S) for 30 min at room temperature before incubation with 5 mM MTSEA for 2 min, and then washed three times with phosphate-buffered saline (C). Specific [125I]HEAT binding to intact cells was then determined as detailed under "Experimental Procedures." *** indicates significant difference (p < 0.001) versus [125I]HEAT binding in the absence of (-)-epinephrine pretreatment.

                              
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Table IV
Ligand binding and functional properties of wild-type and mutant alpha 1B-ARs
Ligand-binding affinity (Ki) was determined from [125I]HEAT competition binding studies except the affinity of [125I]HEAT (Kd), which was determined by saturation binding as described under "Experimental Procedures." Activation of PI hydrolysis by the wild-type alpha 1B-AR and the mutant C128S/C129S/C137S was determined in intact COS-1 cells stimulated with 10-4 M epinephrine for 30 min as detailed under "Experimental Procedures."

Substitution of Phe310 or Phe311 with cysteine produced only a small decrease (3-15-fold) in [125I]HEAT affinity, which was not further impaired when either F310C or F311C was combined with the cysteine triple mutant to produce the quadruple mutants F310C/C128S/C129S/C137S and F311C/C128S/C129S/C137S (Table V). By contrast with the cysteine triple mutant (IC50 for MTSEA-induced inhibition of [125I]HEAT binding, >10 mM) additional substitution of Phe310 to cysteine in the mutant, F310C/C128S/C129S/C137S, restored sensitivity to MTSEA (IC50 0.98 ± 0.23 mM, p < 0.001 as compared with the triple mutant) toward that observed with the wild-type receptor (0.33 ± 0.06 mM) (Fig. 6A). However, sensitivity to MTSEA was not restored with additional substitution of Phe311 to cysteine in the mutant, F311C/C128S/C129S/C137S (IC50 > 10 mM) (Fig. 6A). In agreement with these findings, the reactivity of Phe310 to modification with MTSEA in the F310C/C128S/C129S/C137S mutant was rapid and identical to that observed with the wild-type receptor (t1/2 = 1-3 s; Fig. 6B). By contrast, the reaction kinetics for MTSEA modification of Phe311 in the F311C/C128S/C129S/C137S mutant were extremely slow and identical to those observed with the triple mutant, C128S/C1295/C137S (t1/2 > 600 s) (Fig. 6B). Nevertheless, given that MTSEA-induced receptor inactivation was evaluated with the antagonist, [125I]HEAT, and given that the residues involved in antagonist and agonist binding may not be identical, these findings do not exclude the possibility that Phe310 forms part of the antagonist, but not agonist-binding pocket. To address this issue, receptor-protection studies were performed to evaluate if agonist could protect against MTSEA inactivation. As shown in Fig. 6C, (-)-epinephrine fully protected both the wild-type receptor and the F310C/C128S/C129S/C137S mutant. In contrast, the small amount of inhibition of [125I]HEAT binding observed with MTSEA treatment of both the F311C/C128S/C129S/C137S and C128S/C129S/C137S mutants was unaltered by (-)-epinephrine pretreatment (Fig. 6C). Thus, the effect of MTSEA on [125I]HEAT binding to the F311C/C128S/C129S/137S mutant is not due to derivatization of Phe311. Moreover, in keeping with this contention is the finding that, in contrast to the effect of MTSEA on the F311C quadruple mutant, substituting Phe311 with a variety of other residues that have considerably smaller side chain volumes (<= 200 Å, Fig. 5, D-F) than that of the charged MTSEA moiety, -SCH2CH2NH3+ (volume = 283 Å3), markedly perturbs [125I]HEAT binding (Fig. 5, D-F). Taken together therefore, these data indicate that the side chain of Phe310, but not Phe311, is both solvent accessible and directed toward the agonist-binding pocket.

                              
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Table V
Binding of [125I]HEAT by wild-type and mutant alpha 1B-ARs
The binding affinity of [125I]HEAT (Kd, pM) was determined by saturation binding studies performed as described under "Experimental Procedures." Values in parentheses are the fold change in the dissociation constant compared to that of the wild-type alpha 1B-AR.

To determine the role of Phe310 and Phe311 in receptor activation, the ability of substitution mutants to mediate agonist-stimulated PI hydrolysis was compared with that of the wild-type alpha 1B-AR. With the F310L mutant, the maximal response was unaltered but the potency of the epinephrine-stimulated PI response was decreased by about 10-fold (EC50 = 0.57 µM versus 0.08 µM for the wild-type alpha 1B-AR), which is comparable to its decrease in epinephrine binding affinity (Fig. 7A). However, the F310A mutant showed a significant decrease not only in potency (EC50 = 120 µM versus 0.08 µM for the wild-type alpha 1B-AR), but also in efficacy of epinephrine-stimulated PI hydrolysis (Emax = 60% of the wild-type response) (Fig. 7A). This decrease in agonist efficacy was also observed with two other catecholamine analogues, halostachine and phenethylamine, which lack either the catechol para-hydroxyl, or both catechol hydroxyls and the chiral hydroxyl on the beta -carbon, respectively (Fig. 7, B and C). Like the Phe310 mutants, alanine and leucine substitutions at the 311 position also affected (-)-epinephrine-stimulated PI hydrolysis. However, in this case the leucine substituent produced a greater pertubation of receptor signaling (decreased potency, EC50 = 219 µM versus 0.08 µM for the wild-type alpha 1B-AR, and efficacy, Emax = 40% of the wild-type response) than the alanine substituent (decreased potency only, EC50 = 4.9 µM). Together with the alterations in agonist binding observed above with these Phe310 and Phe311 mutants, the data can be interpreted to indicate that at the 310 position, substitution of the native phenylalanine with a hydrophobic residue, such as leucine, can partially compensate for the loss of both aromaticity and hydrophobicity associated with an alanine substitution. At the 311 position, however, the greater perturbation of receptor signaling with leucine than with alanine may be due to the bulky, non-planar leucine side chain disrupting interhelical packing. In keeping with the requirement of an aromatic side chain at the 310 position for both agonist binding and receptor activation, is the finding that a mutant in which Phe310 was replaced with another aromatic residue, tryptophan, displayed not only near wild-type agonist affinities, but also wild-type PI hydrolysis (Fig. 7, A-C).


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Fig. 7.   (-)-Epinephrine (A), (±)-halostachine (B), and phenethylamine (C) stimulated PI hydrolysis. COS-1 cells expressing the wild-type () or mutant (black-square, F310W; open circle , F310L; , F310A; black-diamond , F311A; diamond , F311L) alpha 1B-ARs were stimulated with the indicated concentrations of each agonist for 30 min. Total inositol phosphate generation was determined as described under "Experimental Procedures" and expressed as the percentage of the (-)-epinephrine-stimulated maximal response with the wild-type alpha 1B-AR. The expression levels of the wild-type and F310W, F310L, F310A, F311A, and F311L mutants were 5.1 ± 0.4, 6.2 ± 0.6, 4.6 ± 0.8, 4.2 ± 0.6, 1.0 ± 0.1, and 2.1 ± 0.3 pmol/mg protein, respectively.

Finally, to confirm that the effects of the F310A and F310L mutations on PI signaling are not due to a global change in receptor structure, we combined them with a previously described constitutively active mutant, A293E (25, 26), to produce the double mutants, F310A/A293E and F310L/A293E. Since Ala293 is about four helical turns below Phe310, it is possible that a structural change caused by the alanine or leucine substitutions will be transmitted to Ala293, thus altering its structure and constitutive signaling activity. As shown in Fig. 8, signaling in the absence of agonist by the F310A or F310L mutants was not different from that observed with the wild-type alpha 1B-AR. Thus, F310A and F310L are not constitutively active. Moreover, increased basal signaling by the A293E mutant, which is dependent on its level of expression, was not altered with the double mutants, F310A/A293E or F310L/A293E (Fig. 8, inset). As seen in Fig. 9A, compared with the wild-type alpha 1B-AR, the A293E mutant showed all the hallmarks of a constitutively active receptor, viz, increased basal PI signaling, and a decreased EC50 and increased Emax for epinephrine-stimulated PI hydrolysis. Both double mutants showed similar increases in basal PI signaling to that observed with the A293E mutant, alone. However, like the single mutant, F310L, the F310L/A293E double mutant displayed a decreased potency for epinephrine-stimulated PI hydrolysis (EC50 = 113 nM versus 0.81 nM for A293E) without a change in efficacy (Emax). Similarly, like the F310A single mutant, the F310A/A293E double mutant displayed a greater decrease in agonist potency (EC50 = 8020 nM) than F310L/A293E, as well as a decrease in efficacy (Fig. 9A).


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Fig. 8.   Basal PI hydrolysis. Total inositol phosphates generated in the absence of agonist were determined in intact COS-1 cells transfected with vector alone (mock), or plasmid encoding the wild-type (WT), or F310A or F310L mutant alpha 1B-ARs. Total inositol phosphates were quantitated as described under "Experimental Procedures" in cells incubated for 30 min in the presence of 10 mM LiCl. The expression levels of the wild-type, F310A, and F310L mutants were 6.2 ± 0.2, 5.7 ± 0.1, and 5.9 ± 0.3 pmol/mg protein, respectively. Inset, basal PI hydrolysis determined as described above in cells transfected with various amounts of plasmid encoding the wild-type (black-square) or mutant (, A293E; open circle , F310A/A293E; , F310L/A293E) alpha 1B-ARs, to produce the receptor expression levels (densities) shown. Receptor densities were determined from parallel saturation binding studies. Data points for each receptor construct did not differ from a linear relationship, as determined from test runs (p > 0.05). The slopes of the regression lines (0.68 ± 0.28, wild-type; 3.79 ± 1.65, A293E; 3.2 ± 1.31, F310A/A293E; 2.53 ± 0.86, F310L/A293E) provide an index of the amount of inositol phosphates generated per pmol of receptor.


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Fig. 9.   (-)-Epinephrine-stimulated PI hydrolysis and binding by wild-type and mutant alpha 1B-ARs. A, total inositol phosphates generated by COS-1 cells transfected with plasmid encoding the wild-type (black-square) or mutant (, A293E; open circle , F310L/A293E; , F310A/A293E) alpha 1B-ARs were determined in the presence of the indicated concentration of (-)-epinephrine, as described in the legend to Fig. 8. The concentration of (-)-epinephrine required to produce a half-maximal response (EC50) was 56.2 ± 1.2, 0.81 ± 0.13, 8020 ± 159, and 113 ± 23 nM, for the wild-type alpha 1B-AR and A293E, F310A/A293E, and F310L/A293E mutants, respectively. The respective receptor densities determined in parallel saturation binding studies, were 3.1 ± 0.2, 2.4 ± 0.4, 3.2 ± 0.9, and 2.9 ± 0.7 pmol/mg protein. B, membranes prepared from COS-1 cells transfected as detailed in A were used to determine the affinity for (-)-epinephrine in competition studies performed as described under "Experimental Procedures." Ki values of 0.75 ± 0.04, 0.017 ± 0.04, 64 ± 19.9, and 1.84 ± 0.82 µM were determined for the wild-type alpha 1B-AR and A293E, F310A/A293E, and F310L/A293E mutants, respectively.

Consistent with these changes in PI signaling, the increased epinephrine affinity observed with the A293E mutant (Ki = 0.017 µM) compared with the wild-type alpha 1B-AR (Ki = 0.75 µM), was perturbed by additional substitution of Phe310 with leucine in the F310L/A293E double mutant (Ki = 1.84 µM), and further decreased in the F310A/A293E double mutant (Ki = 64 µM) (Fig. 9B). Thus, the effects of alanine or leucine substitution of Phe310 on PI signaling do not suggest distortion of global receptor structure, but are entirely consistent with the involvement of Phe310 as a key switch residue involved in ligand binding and receptor activation.

Pharmacophore mapping of adrenergic agonists suggests that the catechol ring of ligands is important both for binding and receptor activity (27). In this study, we provide several lines of evidence to support the contention that Phe310 in TMVI of the alpha 1B-AR mediates such effects through the formation of an aromatic-aromatic interaction between its side chain and the catechol ring. First, computerized modeling of the alpha 1B-AR structure, and docking of catecholamines into the ligand-binding pocket, indicate that the Phe310 side chain is well positioned to interact with the catechol ring. Second, as required for a residue directly involved in ligand binding, SCAM studies reveal that the Phe310 side chain is not only solvent accessible but is directed into the agonist-binding pocket. Third, since substitution of Phe310 with a variety of other amino acids does not perturb membrane expression or post-translational processing (glycosylation) of the receptor, its effects on ligand binding and receptor activation are due to local interactions, and not to alterations in global receptor structure. In addition, even loss of all bonding potential with alanine substitution of Phe310 does not impair spontaneous isomerization of the receptor to a partially activated conformation, as is evident with the F310A/A293E double mutant. Fourth, and most importantly, the aromatic character of the Phe310 side chain is essential for wild-type agonist binding and, in terms of receptor signaling, for both wild-type agonist potency and efficacy. Thus, although substitution of Phe310 with a residue that has a hydrophobic side chain, such as leucine, partially compensates for the loss of phenylalanine at the 310 position, only an aromatic residue, e.g. tryptophan, can fully restore ligand-binding and -receptor activation.

Our results do not support the direct involvement of Phe311 in agonist binding as proposed by Strader et al. (9) for the equivalent residue (Phe290) in the beta 2-AR, or alternatively, indicate that the residues interacting with the catecholamine ring in the alpha 1B and beta 2-AR, differ. Thus, substitution of Phe311 with a variety of different residues markedly impaired receptor expression and post-translation processing, indicating that phenylalanine at the 311 position is essential for proper folding of the receptor protein. In addition, neither the volume nor the hydrophobicity of Phe311 substituent side chains could be directly related to agonist binding or receptor activation. Finally, and most importantly, SCAM studies indicated that the Phe311 side chain is neither solvent accessible nor directed into the ligand-binding pocket. Thus, effects of Phe311 substitutions on ligand binding and receptor activation are likely secondary to global changes in receptor structure, rather than to loss of an interaction directly involved in ligand binding.

Based on SCAM studies of the dopamine D2 receptor, it has been suggested recently that all of the aromatic residues in TMVI of biogenic amine receptors are solvent accessible (28). Furthermore, it was postulated that these aromatic residues relayed information from the site of agonist-binding in the extracellular half of the TMVI helix to produce a conformational change at the intracellular end of the helix, resulting in receptor activation. While an attractive hypothesis, the SCAM conclusions were based entirely on antagonist-inactivation data, and were not confirmed by evaluating an effect on agonist binding.

Apart from defining a critical residue (Phe310) involved in ligand binding and signaling by the alpha 1B-AR, the findings of this study have potentially important ramifications for our understanding of the mechanisms of GPCR activation. For rhodopsin, activation involves initial disruption of an ionic bond between the protonated Schiff base formed by the interaction of 11-cis-retinal with Lys296 in TMVII and Glu113 in TMIII (5). Similarly, activation of the alpha 1B-AR involves disruption of an ionic bond linking Asp125 in TMIII and Lys331 in TMVII (3), and partial receptor activation can be induced by a moiety mimic (triethylamine) of the catecholamine protonated amine (29). For rhodopsin, activation has been shown to result in rigid body movement of the TMVI helix (30), and movement of this helix is also a feature of beta 2-AR (31) and alpha 1B-AR activation.2 Thus, it is likely that movement of TMIII, due to disruption of a constraining interaction with TMVII, and movement of TMVI, are common features of GPCR receptor activation. Movement of these helices, which are contiguous with the second and third intracellular loops, is also consistent with the known involvement of these loops in G-protein activation (12).

Given these considerations, and based on the findings of this study that implicate Phe310 in TMVI as a key switch residue in alpha 1B-AR activation, it seems reasonable to postulate that activation of adrenergic receptors involves initial disruption of the Asp125/Lys331 ionic interaction followed by Phe310/catechol ring-induced movement of TMVI. Indeed, the findings of our studies with the F310A or L/A293E double mutants support this model based on the following considerations: (i) central to the recently revised ternary complex model of GPCR activation is the finding that mutant receptors can exist in a constitutively active state that allows signaling in the absence of agonist; (ii) in support of this model is the finding that overexpression of wild-type receptors can also initiate biochemical responses in the absence of agonist; (iii) accordingly, it has been proposed that receptors spontaneously resonate between a basal state, R, and an active state, R*, with only the latter being able to productively interact with G-protein, and (iv) as a corollary, constitutively active mutants, which partially mimic the active state, represent an intermediate receptor conformation, R' (26). Based on these considerations and on the finding that agonists bind to constitutively active receptors with higher affinity than to wild-type receptors, it has been suggested that rather than inducing the active conformation upon binding, agonists merely select or "trap" the active conformation that results from spontaneous isomerization of R to R* (32). Nevertheless, recent structure-function studies of the angiotensin II (AT1) receptor provide compelling evidence for an R' conformation that is a distinct intermediate between R and R*, and that isomerization from the R' conformation to the active state involves an inductive step that requires agonist binding (33).

In the present study we demonstrate that little, if any, PI hydrolysis is observed with the wild-type alpha 1B-AR in the absence of agonist. Moreover, signaling in the absence of agonist was similar with the F310A and F310L mutants, and, importantly, was not reduced below that observed with the unliganded wild-type alpha 1B-AR, even though these mutants markedly impaired agonist-induced signaling. Thus the unliganded wild-type alpha 1B-AR receptor likely represents the true basal or R conformation, whereas the receptor maximally activated by high agonist concentrations represents the R* state. By contrast, and consistent with an R' conformation, basal signaling was readily apparent with the unliganded A293E mutant. Since the double mutants F310A/A293E and F310L/A293E also showed similar basal signaling in the absence of agonist, but impaired agonist-induced signaling, the Phe310-catechol ring interaction is likely not required for isomerization between R and R', but is critical for full receptor activation. Consistent with the above considerations, it seems reasonable to speculate that some other activation process, e.g. disruption of the Asp125/Lys331 ionic interaction, is required for transition from R to R', whereas the Phe310-catechol ring interaction mediates isomerization from R' to R*. As with the AT1 receptor (33), this latter requirement of a specific agonist-receptor interaction also supports the notion that isomerization from R' to R* is an agonist-dependent inductive process. Clearly additional studies, particularly aimed at directly evaluating receptor conformational states, based, for example, on electron spin resonance (30), NMR (34), fluorescence spectroscopy studies (35), Fourier transform infrared resonance spectroscopy (36), and surface plasmon resonance spectroscopy (37), will be required to more fully define the activation process at atomic resolution.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Siiri Iismaa and Professor Ahsan Husain for critical reading of the manuscript and Elaine Martin for expert secretarial assistance.

    Note Added in Proof

While the paper was in review, additional alpha 1B-AR mutants were constructed and evaluated in which Phe310 or Phe311 in both the wild-type and C128S/C129S/C137S receptors were substitute with lysine. Since the side chain of this residue mimics the MTSEA moiety that binds to cysteine, it should produce similar effects to MTSEA derivitization of F310C- or F311C-substituted wild-type or C128S/C129S/C137S receptors. All lysine-substituted mutants showed wild-type membrane expression (Western blotting and in situ immunofluorescence), and as observed with MTSEA derivitization of F310C-substituted receptors, the F310K mutants displayed loss of [125I]HEAT binding. However, unlike MTSEA derivitization of F311C-substituted mutants, which did not perturb [125I]HEAT binding (Fig. 6B), the F311K mutants displayed loss of radioligand binding. These findings further indicate the Phe311 is not directed into the ligand-binding pocket.

    FOOTNOTES

* This work was supported by a grant from the National Health and Medical Research Council, Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom requests for reprints should be addressed: Victor Chang Cardiac Research Institute, 384 Victoria St., Darlinghurst, NSW 2010, Australia. Fax: 612-9295-8501; E-mail: b.graham{at}victorchang.unsw.edu.au.

2 S. Chen, M. Xu, F. Lin, and R. M. Graham, unpublished data.

    ABBREVIATIONS

The abbreviations used are: alpha 1-AR, alpha 1-adrenergic receptor; SCAM, substituted cysteine accessability method; [125I]HEAT, 2-[beta -(4-hydroxyl-3-[125I]iodophenyl)ethylaminomethyl]tetralone; Emax, the maximal effector response; TM, transmembrane; MTSEA, methanethiosulfonate ethylammonium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PI, phosphatidylinositol; GPCR, G-protein coupled receptor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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