©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Critical Determinants of -Adrenergic Receptor Subtype Selective Agonist Binding (*)

(Received for publication, April 26, 1995; and in revised form, July 25, 1995)

John Hwa (1) (2) Robert M. Graham (2) (3) Dianne M. Perez (1)(§)

From the  (1)Department of Molecular Cardiology, The Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, 44195, the (2)Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, 44106, and the (3)Victor Chang Cardiac Research Institute, St. Vincents Medical Centre, Darlinghurst, New South Wales 2010, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

alpha(1)-Adrenergic receptor (AR) subtypes mediate many effects of the sympathetic nervous system. The three cloned subtypes (alpha-AR, alpha-AR, alpha-AR), although structurally similar, bind a series of ligands with different relative potencies. This is particularly true for the alpha-AR, which recognizes a number of agonists and antagonists with 5-50-fold higher affinity than the alpha- or alpha- subtypes. Since ligands bind to receptor-residues that are located in the transmembrane spanning domains, we hypothesize that subtype differences in ligand recognition are due to differences in the binding properties of nonconserved transmembrane residues. Using site-directed mutagenesis, selected putative ligand-binding residues in the alpha-AR were converted, either individually or in combination, to the corresponding residues in the alpha-AR. Mutation of two such residues (of approximately 172 amino acids in the transmembrane domains) converted the agonist binding profile entirely to that of the alpha-AR. Over 80% of this conversion was due to an Ala Val substitution; the remainder was due to the additional substitution of Leu Met. To confirm that Ala and Leu are indeed critical for agonist subtype-selectivity, the equivalent residues in the alpha-AR (Val and Met) were reversed of that of the alpha-AR. Correspondingly, the agonist-binding profile of this double alpha-AR mutant reverted to that of the alpha-AR. From these data, in conjunction with macromolecular modeling of the ligand-binding pocket, a model has been developed, which indicates that the determinants of these two residues for agonist subtype-selectivity are due not only to interactions between their side chains and specific ligand moieties but also to a critical interaction between these two amino acids.


INTRODUCTION

alpha(1)-Adrenergic receptors (alpha(1)-ARs) are members of the seven transmembrane G-protein-coupled receptor family that are activated by norepinephrine released from sympathetic nerve endings and by epinephrine released from the adrenal medulla. They are part of a larger subset of related but distinct adrenergic receptors, which include the beta- (beta(1), beta(2), beta(3)) and the alpha(2)- (alpha, alpha, alpha) adrenergic receptors. alpha(1)-ARs are expressed in a wide variety of tissues including the brain, liver, myocardium, and vascular smooth muscle(2, 3) . Thus far, three alpha(1)-AR subtypes have been cloned and pharmacologically characterized, the alpha-AR(4) , the alpha-AR(2, 5) , and the alpha-AR(6, 7) . The alpha-subtype has a 5-50-fold higher affinity for the agonists oxymetazoline and methoxamine and for the antagonists 5-methylurapidil, (+)-niguldipine, and phentolamine when compared with the alpha- or the alpha-subtype(2, 7) . However, there are currently no good subtype-selective drugs readily available that can discriminate between the subtypes, based on 100-1000-fold differences in affinity.

Recent studies have indicated that alpha(1)-subtypes can mediate different and sometimes opposing physiological effects. In particular the alpha-AR appears to promote automaticity and arrhythmias during myocardial ischemia, whereas the alpha-AR subtype can activate a Na/K pump leading to cell hyperpolarization, thus decreasing the propensity for abnormal heart rhythms(8) . In addition, it is now known that the alpha-subtype regulates the predominant dynamic component involved in the development of benign prostatic hypertrophy(9) . Hence, development of better alpha(1)-subtype-selective drugs to either block or enhance function may help in combating subtype-related disorders.

In order to design selective drugs, an understanding of subtype differences in the ligand binding pockets would be invaluable. Although the residues forming the alpha(1)-AR binding-pocket have not been defined, key interactions are likely to be similar to those of the beta-adrenergic receptor, since both receptor families are activated by the catecholamines, norepinephrine, and epinephrine. Based on studies of the beta(2)-AR, key points of interaction are believed to occur between Asp in the third transmembrane helix (TMIII) with that of the protonated amine of the agonists, and between two serine residues (TMV; Ser and Ser) that hydrogen-bond with the meta- and parahydroxyl groups of the catechol ring. These critical binding contacts with the natural ligands are well conserved between the subtypes. However, there are 48 amino acid differences in the transmembrane domains between the alpha-AR and the alpha-AR. We hypothesize that only a few of these nonconserved residues are critical for the ligand binding specificity of each alpha(1)-AR subtype since only a subset of these nonconserved residues occur in the regions of the transmembrane domain thought to be involved in ligand binding.

Therefore, rather than sequentially mutating all amino acids that differ between the alpha(1)-subtypes, we have developed simple criteria to select those residues that may be critical for ligand binding and thus are involved in subtype differences in ligand binding profiles. Eight alpha-AR residues were mutated individually to the corresponding residue in the alpha-AR. Two of these changes, when combined in a double mutant, conferred on the alpha-AR the agonist binding properties of the alpha-AR. A single change, Ala Val (^3)in TMV, made the greatest contribution to the change in ligand binding. Reversal of these two residues in the alpha-AR to those in the alpha-AR reversed the agonist binding profile back to that of the alpha-AR.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis

The construct used was the hamster alpha-AR cDNA (1600 bp) (4) including an EcoRI restriction site at the 5` end and a region encoding an octapeptide tag (ID4), at the end of the coding region, which was used to evaluate membrane expression with the monoclonal antibody (anti-ID4). The attachment of this epitope after the coding region does not affect protein expression or the function of the receptor. The construct terminates with a stop codon and a NotI restriction site. The EcoRI/NotI sites permitted insertion into the modified eukaryotic expression vector pMT2`(7) . Site-directed mutagenesis was performed as described previously utilizing the oligonucleotide-mediated double primer M13 method(10) . The alpha-AR cDNA was divided into two 800-bp fragments by digestion with the restriction endonuclease BamHI. Each of the fragments were inserted individually into an M13 phagemid. The full-length 1600-bp receptor gene was not inserted into M13 since such a large insert would be more susceptible to spontaneous mutations. Mutagenesis utilized a 20-base mutagenic primer encoding the codon mismatches to achieve the desired point mutation(s) and a universal primer(-20) for extension on single-stranded M13 templates. After transformation of the single-stranded products into DH5alphaF` cells, plaques were screened for the mutation on nitrocellulose lifts and probed with the P-end-labeled mutagenic primer. The efficiency of mutagenic incorporation was 5% of total plaques. Positive plaques were purified, and the DNA was isolated and sequenced by the dideoxy chain termination method (Sequenase, Amersham Corp.). The mutated fragment (800 bp) was then ligated with its complementary wild-type fragment (800 bp) into pMT2` for transfection into COS-1 cells. Sequencing was utilized to confirm that the fragments were in the correct orientation and that the appropriate mutation was present. Combination mutations were generated using unique restriction sites located within the alpha-AR cDNA. Reverse mutations that incorporated equivalent point mutations into the rat alpha-AR were also performed using similar methods.

Transfection of COS-1 Cells

COS-1 cells (ATCC) were grown in Dulbecco's modified Eagle's medium containing penicillin, streptomycin, glutamine, and 5% fetal bovine serum. At 60-80% confluency, transient transfection was performed according to published procedures(7) . Plasmid DNA was purified by RNase treatment and column chromatography utilizing the Wizard Maxiprep kit (Fisher). In brief, transfection involved initial washing of the cells with Hanks' balanced salt solution followed by the addition of the DNA mixture (10 µg/3 times 10^6 cells) in DEAE-dextran. After a 5-h incubation and a further wash, 10% dimethyl sulfoxide was used to shock the cells, and 0.1 mM chloroquine was added for 2 h. Cells were harvested 72 h posttransfection.

Membrane Preparation

Membranes were prepared as described previously(7) . Briefly, the cells were washed in Hanks' balanced salt solution and removed from the flask. To isolate the membranes, the cells were suspended in 0.25 mM sucrose with protease inhibitors and subjected to nitrogen cavitation at 400 p.s.i. for 20 min. The cells were then homogenized in a ``B'' glass Dounce homogenizer for 10 strokes and spun at low speed (1260 times g for 5 min) to remove nuclei and microsomes, and then centrifuged at 30,000 times g for 15 min. After two further washes with HEM (20 mM HEPES, pH 7.5, 1.5 mM EGTA, and 12.5 mM MgCl(2)) and recentrifugation at 30,000 times g, the pellet was resuspended in HEM containing 10% glycerol and stored at -70 °C. A Bradford protein assay was performed initially on known concentrations of bovine serum albumin followed by membrane preparations of the wild-type and mutant alpha-ARs.

Ligand Binding

The ligand binding characteristics of the expressed receptors were determined in a series of radioligand binding studies using [I]HEAT, an alpha(1)-specific antagonist as the radioprobe(7) . In brief, for competition studies, the procedure involved duplicate tubes containing (total volume, 250 µl) 200 pM [I]HEAT, HEM buffer, COS-1 membranes, and increasing amounts of unlabeled competing ligand (at 10 or more different dilutions). Nonspecific binding was determined by the addition of phentolamine (10M). For saturation binding studies, 200-1600 pM [I]HEAT was used. After 1 h of incubation at room temperature, the reactions were stopped by the addition of ice-cold HEM buffer and were filtered onto Whatman GF/C glass filters with a Brandel cell harvester. The filters were washed 5 times with ice-cold HEM. They were then analyzed for bound radioactivity using a Packard Auto- 500 counter. Data were analyzed using the interactive program LIGAND. IC values were converted to K(i) using the Cheng-Prusoff equation and a K(D) for [I] HEAT of 90 pM. K(i) values were expressed as a mean ± S.E. An analysis of variance and Student's t test was used to determine significant differences (p < 0.05). To detect small but significant differences, sets of mutations along with the wild-types alpha-AR and alpha-AR were evaluated simultaneously (i.e. in the same assay).

Molecular Modeling

The coordinates of the alpha-carbon positions were determined by an overlay of the putative alpha(1)-AR transmembrane residues with the transmembrane coordinates of bacteriorhodopsin(11) . Data files were generated with the Insight II molecular modeling software from Biosym Technologies. The boundaries of the putative transmembrane domains were determined by an algorithm based upon the weighted pairwise comparisons of adjacent residues(12) . The model was then minimized, and conflicts were adjusted as described previously(13) . Assumptions of key interactions of specific amino acids with agonist are based upon previous mutagenesis work and proposed models with the beta-AR(14) . Results of our mutagenesis were then used to refine the structure of the alpha(1)-AR ligand binding pocket.

Materials

Drugs were obtained from the following manufacturers.(-)-Epinephrine,(-)-norepinephrine, oxymetazoline, methoxamine, phenylephrine, and phentolamine were from Sigma; Cirazoline was a gift from Pfizer. [I]HEAT, was from DuPont NEN. 5-Methylurapidil and WB4101 were from Research Biochemicals International (Natick, MA).


RESULTS AND DISCUSSION

The alpha-AR and the alpha-AR, although structurally similar in their transmembrane domains, have some significant differences in their ligand-binding affinities for a number of agonists and antagonists. Determining the critical amino acids responsible for these differences in binding properties may assist in the design of better subtype selective drugs. We hypothesized that there are only a few nonconserved transmembrane amino acids that distinguish the alpha-AR agonist-binding pocket from that of the alpha-AR.

Selection Criteria

Our choice of the alpha-AR and the alpha-AR as the initial targets for mutagenesis was 2-fold. First, as already described, there are some important differences in the responses mediated by these two subtypes, which may be of pathophysiological and therapeutic relevance. Second, the alpha has a 5-50-fold greater affinity for a number of agonists and antagonists than the alpha-AR. This allowed us to detect even small but significant changes in affinity (e.g. 2-5-fold) with our individual mutations in converting the alpha-AR to the alpha-AR.

Candidate residues potentially involved in subtype selectivity were selected for mutation from the 48 nonconserved transmembrane amino acids, based on the following criteria: 1) location in the upper half of the transmembrane domain, where previous studies indicate that small ligands are bound; 2) exclusion of the first transmembrane domain since, based on models of bacteriorhodopsin and rhodopsin, this appears not to be directly involved in forming the ligand binding pocket(11) ; 3) location on the transmembrane alpha-helices such that their side-chains are oriented toward the putative binding pocket or adjacent transmembrane domains rather than toward the surrounding phospholipid bilayer (orientation of the side-chains was determined based on consideration of models of the alpha-AR developed in our laboratory(13) , and on the model of rhodopsin developed by Baldwin (15) ); 4) lack of conservation not due merely to interspecies differences, since there is conservation of alpha(1)-subtype ligand-binding profiles across species.

Seven amino acids (Fig. 1) of the alpha-AR were identified that fulfilled the criteria, and these residues were mutated to the corresponding residues on the alpha-AR. An eighth residue Leu Phe was also mutated. Although this residue was predicted to be facing the hydrocarbon environment, it was selected because the corresponding alpha-AR residue represented a significant change in size and functional group. Moreover, if our prediction of its orientation was correct, mutation of this residue should not alter ligand binding and, thus, this mutant would serve as a negative control.


Figure 1: Schematic representation of the alpha-AR highlighting the eight amino acid residues selected for mutagenesis and their transmembrane location. TMI, -II, -III, -IV, -V, -VI, and -VII represent the seven transmembrane domains. The mutations were as follows: TMII, Ser Thr and Phe Ser; TMIV, Thr Leu and Leu Phe; TMV, Ala Val and Ser Ala; Ala Val and Leu Met.



Analysis of Single Mutations in Converting the alpha-AR to the alpha-AR

The eight point mutations were initially analyzed by their ability to bind a panel of agonists. K(i) values for six alpha(1)-AR agonists are shown in Table 1and Fig. 2. The Ala Val mutation had a 5-10-fold increased binding affinity over the wild-type alpha-AR (WTb) for oxymetazoline (p < 0.001), cirazoline (p < 0.001), and methoxamine (p < 0.001), a ligand binding profile that is more consistent with that of the alpha-AR (Fig. 2A). Paradoxically, this mutation also had increased affinity (3-5-fold) for the natural ligands epinephrine (p < 0.01) and norepinephrine (p < 0.001) as well as phenylephrine (p < 0.001), which all show no selectivity between these two alpha(1)-AR subtypes (Fig. 2B). The Leu Met mutant also demonstrated a significant increase in its affinity for methoxamine (p < 0.001), cirazoline (p < 0.001), and oxymetazoline (p < 0.05) (Fig. 2B) but not for epinephrine, norepinephrine, or phenylephrine (see Fig. 5). The other six point mutations including the Leu Phe mutation showed no significant change from an alpha-AR pharmacology (Table 1). Each mutation had its own characteristic effect on binding. However, only the Ala Val and Leu Met mutations significantly altered their ligand-binding profiles from that of an alpha-AR to that of an alpha-AR pharmacology.




Figure 2: A, the effects of the alpha-AR point mutations on the affinities for agonists that have a hydrophobic ortho group on their phenyl ring (y axis); B, binding affinities for agonists without a hydrophobic ortho group (y axis). For both panels, the x axis represents the alpha-AR (WTb) and the alpha-AR (WTa) along with the mutations that were statistically different (by a Student's t test) from the WTb. The z axis represents the difference in K(-log) from the WTb.




Figure 5: A, the reverse mutations of Leu Met and Ala Val (alpha-AR) was performed in the alpha-AR (Met Leu and Val Ala, respectively). Removal of the inhibitory effect of methionine (Met Leu) resulted in an increased affinity for agonists that lacked an ortho hydrophobic group (epinephrine, norepinephrine, and phenylephrine). This is equivalent to the Ala Val in the alpha-AR. B, the combination of Met Leu and Val Ala in the alpha-AR revealed an alpha-AR agonist pharmacology. Shown for comparison is the alpha-AR double mutant Ala Val/Leu Met.



Our results indicate that with agonist binding, individual amino acids, particularly in the 5th and 6th transmembrane domains, are critical in defining the agonist binding pocket between the alpha-AR and the alpha-AR. Each agonist is similarly effected by changes in critical residues but not to the same degree. The Ala Val mutation appears to explain most of the higher affinity for agonists as seen with the alpha-AR. The increased affinity observed with the substitution of valine for alanine may be related to an increased hydrophobic interaction between the valine and the aromatic ring of the ligands. The increase in binding affinity with the Leu Met may be due to an increased interaction as a result of the extended chain length of the methionine residue with the ortho hydrophobic group found with many of the synthetic agonists. Supporting this is the progressive increase in affinity conferred by Leu Met with a progressive increase in size and hydrophobicity of the ortho side chain. The descending order of binding affinities (Fig. 2A) are cirazoline > methoxamine > oxymetazoline and their respective side chains are a cyclopropyl, a methoxy, and a methyl group.

Effects of Ser Ala (alpha) on the Agonist Binding Properties of alpha(1)-ARs

The serine involved in the Ser Ala (alpha) mutation corresponds to one of the proposed serines that binds to the catechol ring hydroxyls. Previous studies have shown that mutation of the equivalent serine residue in the beta(2)-adrenergic receptor (residue 204) to alanine, decreases binding affinity by 10-fold (14) and reduces the intrinsic activity of full agonists. Thus, it was proposed that for full agonist activity in the beta(2)-AR, both hydroxyls on the catechol ring hydrogen bonded to the two serines, Ser and Ser. Site-directed mutagenesis on the alpha-AR at the equivalent positions Ser and Ser suggested a role for Ser in hydrogen bonding, whereas Ser appeared not to be necessary in catecholamine binding(16) . In the alpha(1)-AR, the serine at position 208 (equivalent to Ser in the beta(2)-AR and Ser in the alpha-AR) appears not to be required for hydrogen bonding to the catechol ring of the natural ligands since the binding affinity of the Ser Ala (alpha) mutant was unaltered from that of the wild-type alpha-AR (Table 1). Consistent with this hypothesis, phenylephrine has only a single hydroxyl at the metaposition and is a full agonist for both the alpha-AR and the alpha-AR. Thus the metahydroxyl is likely to hydrogen bond to Ser (alpha), the serine equivalent to Ser on the beta(2)-AR and Ser on the alpha-AR. This indicates that determinants of agonist binding and intrinsic activity in the beta(2)-AR are likely not entirely conserved among the alpha-adrenergic receptors and that alpha(1)-ARs display a phenotype for hydroxyl binding similar to the alpha(2)-ARs.

Effects of the Point Mutations on Antagonist Affinity

Analysis of the eight alpha-AR mutants for alterations in antagonist binding revealed no significant changes particularly for Ala Val and Leu Met, which were shown to significantly alter agonist binding affinities (Table 2). This indicates that the determinants for agonist binding may be quite distinct from those for antagonist binding. Antagonists, as a result of their larger size, may extend to both the first and seventh transmembrane domain and perhaps bind below the upper half of the transmembrane domain. Our selection criteria for mutagenesis precluded the first transmembrane domain and the lower half of the binding pocket. Thus our selection criteria may have favored residues involved in agonist interactions.



Analysis of Combined alpha-AR Mutations

Combination mutations were made to assess if the individual mutations were independent and/or additive in their affects on changing the binding-profiles to that of the alpha-AR. The initial combination (Ser Thr/Phe Ser, Leu Phe, and Ser Ala), involved mutations that by themselves had no major effect on ligand binding. The binding-profile of this combination was not different from that of the wild-type alpha-AR (Table 3, Fig. 3). When the two mutants that alone showed a significant change toward the alpha-AR (Leu Met/Ala Val) were combined, the ligand binding profile of this double mutant was now similar to that of the wild-type alpha-AR (Table 3, Fig. 3). Interestingly, this double mutant showed affinity changes that were additive for methoxamine, oxymetazoline, and cirazoline but inhibitory for epinephrine, norepinephrine, and phenylephrine. Thus for epinephrine, norepinephrine, and phenylephrine, the additional change of the Leu to methionine decreased the affinity observed for these ligands with the single Ala Val mutant. Fig. 4, a and b, represents a model that explains this phenomenon. All three agonists that showed an additive effect when Leu Met and Ala Val were combined have a hydrophobic group at the ortho position of the phenyl ring. Thus, the bulkier methionine residue may allow a hydrophobic interaction with this ortho substituent on the phenyl ring. As a result, the ortho group would no longer interact with the valine of the Ala Val mutation, and the valine side chain would be free to interact with the hydrophobic phenyl ring (Fig. 4A). Since both residues interact with different components on the agonist and assist each other, their interaction is additive and cooperative. In the absence of an ortho group on the ligand, the bulkier methionine may interact with the Val and inhibit this valine from interacting with the phenyl ring, due to the proximity of these two residues on the fifth and sixth transmembrane domains. Therefore, with the double mutant (Leu Met and Ala Val), the affinity of ligands lacking an ortho group is decreased (Fig. 4B). Thus, residues Leu and Ala together differentiate the alpha-AR agonist binding pocket from that of the alpha-AR.




Figure 3: Binding affinities for the combination alpha-AR mutations. Ser Thr/Phe Ser, Leu Phe, and Ser Ala represents the combination of four mutations that individually didn't significantly differ from the WTb. The Ala Val and Leu Met double mutant represents the combination of the two mutations that did significantly differ in their agonist binding affinity from that of the WTb toward that of the WTa.




Figure 4: A model of the Ala Val and Leu Met (alpha) mutations showing proposed interactions in the agonist binding pocket. The graph represents the individual mutations followed by the combination of Leu Met and Ala Val. The y axis shows K (-log) differences from the WTb. Below each graph is a model that describes the data. Oxymetazoline is used to represent an agonist with a hydrophobic ortho group and phenylephrine, an agonist without such a group. Only transmembranes 5 (TMIV) and 6 (TMVI) are shown. Only the phenyl ring and the ortho group of the ligand is represented. Arrows indicate the putative sites of interactions. A, for oxymetazoline, Leu Met interacts with the ortho methyl group, and Ala Val interacts with the phenyl ring. In combination the 314 position is pulled away by the ortho substituent from the 204 position allowing both residues to interact freely with the ligand, thus having an additive effect. B, for phenylephrine, the lack of an ortho group results in steric inhibition of the 204 position by the bulkier methionine, leading to an inhibitory effect when they are combined.



Reversing the Mutations in the alpha-AR

We performed the equivalent reverse mutations in the alpha-AR to confirm that the Leu Met and Ala Val mutations (alpha-AR residues) indeed define the critical residues differentiating the alpha-AR and alpha-AR agonist binding pocket (Table 4). The results showed that a Val Ala mutation (corresponding residue to the Ala Val mutation in the alpha-AR) of the alpha-AR reversed the binding affinity of the wild-type receptor toward that of the alpha-AR, particularly for oxymetazoline and methoxamine. Supporting our theory that a methionine (Leu Met in the alpha-AR) is involved in inhibiting the interaction of valine with the phenyl ring of the agonists, Met Leu (alpha-AR) relieved this inhibition, thus increasing the binding affinity for the agonists lacking ortho-substituents (epinephrine, norepinephrine, and phenylephrine) (Fig. 5A). In combination (Val Ala and Met Leu (alpha)) they had alpha-AR pharmacology, however there were still some residual differences with methoxamine and cirazoline (Fig. 5B). This indicates that there still may be other minor conformational differences in the agonist ligand binding pocket of the alpha-AR due to other nonconserved amino acids. However, the reversal of these mutations supports our hypothesis that the Leu and Ala in the alpha-AR and their corresponding alpha-AR residues Val and Met are the most critical determinants of the differences in agonist binding between these two subtypes.



Modeling the Agonist Ligand Binding Pocket of the Two Subtypes

Previous work analyzing subtype specificity between the beta(1) and beta(2)-AR (17) indicated that residues in the middle portion of the beta-AR sequence, particularly around transmembrane regions 4 and 5, contribute predominantly to the subtype-specific binding of agonists. However, no single residue substitution appeared to be capable of altering the subtype specificity of the receptor. Analysis of an extensive number of chimeric beta(1) and beta(2) receptors functionally expressed in Escherichia coli(18) demonstrated that each of 11 selective ligands appeared to define its own ligand binding subsite. In contrast, our results indicate that at least for agonist binding, discrete amino acid interactions can account for subtype selectivity.

From our data we were able to model the subtype differences between their agonist binding pockets (Fig. 6, A and B). It appears that when an agonist is docked into the binding pocket, the presence of a hydrophobic ortho substituent on the phenyl ring will interact with the bulkier methionine residue at position 293 on the alpha-AR (Leu on the alpha-AR) thus increasing binding affinity significantly. Similarly the larger hydrophobic valine residue in the alpha-AR at position 185 (Ala in the alpha-AR) interacts directly with the phenyl ring. For agonists with an ortho hydrophobic substituent as with cirazoline, oxymetazoline, and methoxamine, the effect of these two residues is additive conferring upon the alpha-AR increased binding affinity. In the absence of the ortho group (phenylephrine, norepinephrine, and epinephrine) Met (alpha) sterically hinders Val (alpha) from interacting with the phenyl ring, thus there is no increase in affinity. These two residues in combination differentiate the agonist binding pocket of these two alpha(1)-AR subtypes. The metahydroxyl group appears to interact with the Ser as there was no difference in binding affinity between the wild-type alpha-AR and Ser (alpha) for phenylephrine, which contains only a metahydroxyl group. Thus each agonist docks into the ligand binding pocket in a tilted manner.


Figure 6: A, model of the alpha-AR showing the docking of oxymetazoline (oxy) in the mutated ligand binding pocket (Leu Met and Ala Val), which mimics that of the alpha-AR. The ortho methyl group interacts with Met, the phenyl ring with Val, and the metahydroxyl with Ser. B, with phenylephrine (PH) the lack of an ortho substituent allows steric interaction of Leu Met and Ala Val and therefore no difference is seen in agonist binding affinity. TMI, -II, -V, -VI, and -VII are shown; TMIII and -IV have been removed to allow visualization of the binding pocket.



From our data we have defined two important residues that are critical in differentiating the ligand binding pocket between two alpha(1)-AR receptor subtypes. We are able to model the interaction between these two residues and in so doing have provided a rational basis for the design of better subtype-selective agonists to allow optimal binding and receptor activation.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant NS19853 (to R. M. G. and D. M. P.), an American Heart Association Grant in aid (to D. M. P.), an educational grant from Glaxo Inc., and an Eccles Award (to R. M. G.) 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. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Cardiology, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-2058; Fax: 216-444-9263.

(^1)
The abbreviations used are: AR, adrenergic receptor; TM, transmembrane; bp, base pair(s); [I]HEAT, (±)-beta-([I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone; WT, wild type.

(^2)
In this paper we will use the standardized nomenclature system for alpha(1)-AR subtypes as recommended by the International Union of Pharmacology Committee on the Classification of Adrenergic Receptors. The cloned subtypes are designated lower case letters as alpha, alpha, and alpha. The pharmacologically described tissue subtypes are defined as the alpha, alpha and alpha, respectively(1) .

(^3)
Mutations indicate the position of the wild type and substituting residue. Thus, for example, in the Ala Val (alpha) mutation, the wild-type alanine residue at position 204 in the alpha-AR has been mutated to a valine.


ACKNOWLEDGEMENTS

We thank F. Brown of GLAXO, INC. for access to their alpha-AR model, R Peter Riek for his model of the alpha-AR and R. Gaivin for his technical assistance.


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