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Originally published In Press as doi:10.1074/jbc.M403703200 on June 11, 2004

J. Biol. Chem., Vol. 279, Issue 34, 35326-35333, August 20, 2004
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Subtype-selective Noncompetitive or Competitive Inhibition of Human {alpha}1-Adrenergic Receptors by {rho}-TIA*

Zhongjian Chen{ddagger}, George Rogge{ddagger}, Chris Hague{ddagger}, Dianne Alewood§, Barbara Colless§, Richard J. Lewis§, and Kenneth P. Minneman{ddagger}

From the {ddagger}Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322 and §Xenome Ltd., 50 Meiers Road, Indooroopilly 4068, Queensland, Australia

Received for publication, April 2, 2004 , and in revised form, May 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The 19-amino acid conopeptide ({rho}-TIA) was shown previously to antagonize noncompetitively {alpha}1B-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human {alpha}1-AR subtypes ({alpha}1A, {alpha}1B, and {alpha}1D). Radioligand binding assays showed that {rho}-TIA was 10-fold selective for human {alpha}1B-over {alpha}1A- and {alpha}1D-ARs. As observed with hamster {alpha}1B-ARs, {rho}-TIA decreased the number of binding sites (Bmax) for human {alpha}1B-ARs without changing affinity (KD), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, {rho}-TIA had opposite effects at human {alpha}1A-ARs and {alpha}1D-ARs, decreasing KD without changing Bmax, suggesting it acts competitively at these subtypes. {rho}-TIA reduced maximal NE-stimulated [3H]inositol phosphate formation in HEK293 cells expressing human {alpha}1B-ARs but competitively inhibited responses in cells expressing {alpha}1A- or {alpha}1D-ARs. Truncation mutants showed that the amino-terminal domains of {alpha}1B- or {alpha}1D-ARs are not involved in interaction with {rho}-TIA. Alanine-scanning mutagenesis of {rho}-TIA showed F18A had an increased selectivity for {alpha}1B-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at {alpha}1B-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus {rho}-TIA noncompetitively inhibits {alpha}1B-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at {alpha}1B-ARs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}1-Adrenergic receptors (ARs)1 are heptahelical G protein-coupled receptors (GPCRs) that mediate important physiological responses to norepinephrine (NE) and epinephrine as diverse as smooth muscle contraction, glycogenolysis, and myocardial inotropy (1, 2). Molecular cloning and pharmacological studies have identified three subtypes of {alpha}1-ARs: {alpha}1A-, {alpha}1B-, and {alpha}1D-ARs (3–10). Although all three {alpha}1-AR subtypes signal through the same Gq/11 signaling pathway (11), {alpha}1-AR subtypes can differ in their tissue distributions (1), cellular localization (12, 13), activation of transcriptional responses (14), and association with intracellular proteins (15–17), suggesting that {alpha}1-AR subtypes perform specific physiological functions. Furthermore, recent data obtained from both {alpha}1-AR overexpressed (18–21) and knockout mice (22–25) indicate that {alpha}1-ARs play important physiological roles in the cardiovascular system and central nervous system. However, attempts to pharmacologically isolate specific responses to particular subtypes have proven difficult because of the lack of highly {alpha}1B-AR-selective antagonists.

Recently, a 19-amino acid conopeptide, {rho}-TIA, was isolated from the venom of the fish-hunting cone snail Conus tulipa (26, 27). This highly charged peptide (amino acid sequence FNWRCCLIPACRRNHKKFC) forms a distinct structure with two disulfide bonds between cysteines 5 and 11 and cysteines 6 and 19 (26). {rho}-TIA was found to be a selective {alpha}1-AR antagonist, as it inhibited {alpha}1-AR-mediated contraction of rat vas deferens without affecting ATP or {alpha}2-AR-mediated responses (26, 27). Studies on recombinant hamster {alpha}1B-ARs found that increasing concentrations of {rho}-TIA progressively reduced the density of radioligand binding sites without changing radioligand affinity, suggesting that {rho}-TIA is a noncompetitive, possibly allosteric, inhibitor of {alpha}1B-ARs. In addition, radioligand binding assays performed using recombinant rodent {alpha}1-AR subtypes revealed a 2–5-fold higher affinity for {rho}-TIA at {alpha}1B-ARs (27).

Allosteric modulators of GPCRs have received increasing attention over the last few years for their potential specificity and lack of undesirable effects (28). Unlike drugs that act on the conserved orthosteric binding sites of closely related receptor subtypes, actions of allosteric modulators have been found to be highly specific for particular receptor subtypes, because they often interact with less highly conserved sites (28). Although {rho}-TIA also inhibits rodent {alpha}1A- and {alpha}1D-ARs, its mode of inhibition has not yet been determined. In addition, its effects on human subtypes are still unknown.

In this study, we compared the effects of {rho}-TIA at all three human {alpha}1-AR subtypes stably expressed in human embryonic kidney (HEK293) cells. We found that {rho}-TIA acts noncompetitively at human {alpha}1B-ARs but competitively at {alpha}1A- and {alpha}1D-ARs and that the isoleucine at position 8 in {rho}-TIA is required for noncompetitive inhibition. In addition, {rho}-TIA is the first highly selective antagonist for human {alpha}1B-ARs, which will be useful in functional characterization of this receptor.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Materials were obtained from the following sources: HEK293 cells, American Type Culture Collection (Manassas, VA); penicillin, streptomycin, phosphate-buffered saline (PBS), (–)-norepinephrine (NE) bitartrate, phentolamine mesylate, Sigma; carrier-free Na[125I], Amersham Biosciences; myo-[3H]inositol (1 mCi/ml), American Radiolabeled Chemicals (St. Louis, MO); Dulbecco's modified Eagle's medium with 4.5 g/liter glucose and L-glutamine, Mediatech (Herndon, VA); {[2-{beta}-(4-hydroxyphenyl)ethylaminomethyl]tetralone} from Dr. Giuseppe Romeo (University of Catania, Italy); [3H]prazosin, PerkinElmer Life Sciences; Fmoc-Rink amide resin (Polymer labs, 0.73 mmol/g, Scientex Australia); Fmoc amino acids (Novabiochem).

Peptide Synthesis—{rho}-TIA and its analogs were assembled using Fmoc chemistry. Chain assembly of the peptides was performed on a manual shaker system using HBTU activation protocols (29) or on an ACT396 synthesizer using HBTU in situ activation protocols. The Fmoc protecting group was removed using 50% piperidine in dimethylformamide, and dimethylformamide was used as both the coupling solvent and for flow washes throughout the cycle. Where possible, the progress of the assembly was monitored by quantitative ninhydrin monitoring (30). Peptide was deprotected and cleaved from the resin by stirring at room temperature in trifluoroacetic acid:H2O:triisopropyl silane: ethanedithiol (90:5:2.5:2.5) for 2–3 h. Cold diethyl ether was then added to the filtered mixture, and the precipitated peptide was collected by centrifugation. The final product was dissolved in 50% aqueous acetonitrile and lyophilized to yield a white solid. The crude, reduced peptide was examined by reverse phase high pressure liquid chromatography for purity, and the correct molecular weight was confirmed by electrospray mass spectrometry. Pure, reduced peptide was oxidized using 30% isopropyl alcohol, 0.1 M ammonium bicarbonate, pH 7.7, for 24 h, and then the major peak was purified to >95% purity and characterized by reverse phase high pressure liquid chromatography/mass spectrometry prior to further use.

Cell Culture and Transfection—HEK293 cells stably expressing human {alpha}1A- (31), {alpha}1B- (7), or {alpha}1D- (9) ARs have been previously described (32). Cells were propagated in Dulbecco's modified Eagle's medium containing 10% calf serum, 4.5g/liter glucose, 100 mg/liter streptomycin, and 105 units/liter penicillin at 37 °C in a humidified atmosphere with 5% CO2. Confluent 150-mm plates were subcultured at a ratio of 1:4 or 1:6 for transfection and cultured for 24–48 h. For cells expressing N-truncated constructs (see below), HEK293 cells were transfected with 10 µg of DNA of each construct for 3 h using Superfect® transfection reagent and were used for experimentation 48–72 h after transfection.

Membrane Preparation—Confluent 150-mm plates were washed with PBS (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, pH 7.4) and harvested by scraping. Cells were collected by centrifugation at 30,000 x g for 10 min, resuspended in PBS, and homogenized with a Polytron. This process was then repeated, and final membranes were collected in 0.5 ml of PBS.

Radioligand Binding—Receptor density was determined by saturation binding assays with the {alpha}1-AR-specific antagonist 125 I-BE (20–800 pM) (33) or [3H]prazosin (200–5000 pM) (34). Membranes were incubated with the indicated concentrations of either 125I-BE or [3H]prazosin at 37 °C in a water bath for 20 min. After incubation, samples were filtered through a wet Whatman GF/B paper under vacuum. Filter papers were washed twice with cold wash buffer (10 mM Tris-HCl, pH 7.4), and radioactivity was measured by gamma counting. Nonspecific binding was determined as binding in the presence of 10 µM phentolamine. For the washout experiments, membranes were aliquoted into two sets and incubated with or without 30 nM {rho}-TIA. For each wash, each sample was diluted 2.5-fold with PBS, homogenized, and resuspended in the appropriate volume of PBS. 125I-BE was added with or without phentolamine; samples were incubated at 37 °C and filtered, and radioactivity was measured by gamma counting. The amount of specific binding inhibited by 30 nM {rho}-TIA was calculated after each wash. Nonspecific binding was defined as binding remaining in the presence of 10 µM phentolamine.

[3H]InsP Formation—Accumulation of [3H]inositol phosphates (InsPs) was determined in confluent 96-well plates as described previously (35). Cells were loaded with [3H]inositol (1 µCi/well) at the time of seeding and grown for 1–2 days until confluent. On the day of the experiments, wells were washed carefully with Krebs-Ringer bicarbonate (KRB) buffer (120 mM NaCl, 5.5 mM KCl, 2.5 mM CaCl2, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 20 mM NaHCO3, 11 mM glucose, 0.029 mM Na2EDTA) containing 10 mM LiCl, and then incubated with 0.1 ml of Li-KRB containing drugs at the indicated concentrations. Conopeptides were prepared in 50 µl of Li-KRB and added into the wells before adding an equal volume of Li-KRB containing appropriate NE concentrations. After incubating at 37 °C (in 95%O2/5%CO2) for 1 h, reactions were stopped by adding 10 mM formic acid. Samples were sonicated for 10 s, and [3H]InsPs were isolated by anion exchange chromatography as described (35). Data were normalized to % maximal stimulation caused by 100 µM NE alone. NE caused no stimulation of [3H]InsP formation in nontransfected HEK293 cells at concentrations up to 300 µM. Schild plot was generated as described previously (36).

Generation of Amino-terminal Truncated Receptors—Amino-terminally truncated human {alpha}1D-ARs ({Delta}1–79) were constructed in pRSVICAT vectors as described previously (37). Amino-terminally truncated human {alpha}1B-ARs ({Delta}1–38) were generated by PCR using specific primers on human {alpha}1B-AR cDNA in pDT, subcloned, and sequenced.

Data Analysis—Data are expressed as mean ± S.E. of results obtained from the indicated number of observations. For radioligand binding, calculations of KD and Bmax for saturation analysis, and IC50 values for inhibition of specific binding by peptides were fit by nonlinear regression using Prism (GraphPad). Global fitting was used to define the mode of inhibition of {rho}-TIA from saturation analysis (either shared KD or shared Bmax). Concentration-response curves for stimulation of [3H]InsP formation were also analyzed by nonlinear regression. One-way analysis of variance with post hoc t test performed by the Tukey method was used where indicated. Values of p < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
{rho}-TIA Inhibition of Specific 125I-BE Binding in HEK293 Cell Membranes Expressing Human {alpha}1A-, {alpha}1B-, or {alpha}1D-ARs—Because {rho}-TIA showed noncompetitive inhibition at hamster {alpha}1B-ARs (26), we investigated the inhibitory effects of {rho}-TIA at human {alpha}1A-, {alpha}1B-, and {alpha}1D-ARs. Increasing concentrations of {rho}-TIA inhibited specific 125I-BE binding to membranes prepared from HEK293 cells stably expressing individual {alpha}1-AR subtypes (Fig. 1). However, unlike the limited selectivity observed previously with the rodent clones (27), {rho}-TIA was more potent and showed a 10-fold {alpha}1B selectivity. IC50 values for {rho}-TIA were 18 nM (logIC50 –7.4 ± 0.08) at {alpha}1A-ARs; 2 nM (logIC50 –8.4 ± 0.10) at {alpha}1B-ARs; and 25 nM (logIC50 –7.3 ± 0.13) at {alpha}1D-ARs. The difference in potency of {rho}-TIA was significant between {alpha}1B-ARs and {alpha}1A- and {alpha}1D-ARs (p < 0.001) but not between {alpha}1A-ARs and {alpha}1D-ARs.



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  		FIG. 1.
Concentration-dependent inhibition of specific 125I-BE binding by {rho}-TIA in membranes prepared from HEK293 cells stably transfected with individual {alpha}1-AR subtypes. Symbols represent the mean ± S.E. of four determinations.

 
Mode of Inhibition—To determine whether {rho}-TIA acts noncompetitively at each human {alpha}1-AR subtype, saturation binding assays with the selective antagonist 125I-BE were performed on membranes prepared from HEK293 cells expressing individual {alpha}1-AR subtypes in the presence or absence of 100 nM {rho}-TIA (Fig. 2). Nonlinear regression analysis was used to determine whether inhibition by {rho}-TIA was competitive (shared Bmax) or noncompetitive (shared KD). It was found that inhibition of specific 125I-BE binding to human {alpha}1B-ARs was clearly noncompetitive (p < 0.005), whereas inhibition of binding to human {alpha}1A- and {alpha}1D-ARs was competitive (p < 0.01 for {alpha}1A- and p < 0.05 for {alpha}1D-ARs). To confirm that these findings could be generalized to another {alpha}1-AR radioligand, the same experiments were repeated using [3H]prazosin in place of 125I-BE. In agreement with our previous experiments, {rho}-TIA noncompetitively inhibited {alpha}1B-AR binding (p < 0.005) but was competitive at the {alpha}1A-AR (p < 0.05) and {alpha}1D-AR (p < 0.01) subtypes (data not shown). Therefore, these observations suggest {rho}-TIA acts differently at the {alpha}1-AR subtypes as a noncompetitive inhibitor at human {alpha}1B-ARs but as a competitive inhibitor at {alpha}1A- and {alpha}1D-ARs.



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  		FIG. 2.
Saturation binding of 125I-BE to HEK293 membranes expressing {alpha}1-AR subtypes in the presence or absence of 100 nM {rho}-TIA. Symbols represent the mean ± S.E. of three experiments performed in duplicate.

 
{rho}-TIA Binds to the Human {alpha}1B-AR in a Reversible Manner— Although previous experiments suggested that {rho}-TIA binds to human {alpha}1B-ARs noncompetitively, it is unclear if this binding is also reversible. To examine this, HEK293 cells stably expressing human {alpha}1B-ARs were preincubated with increasing concentrations of {rho}-TIA for 2 h. As shown in Fig. 3A, preincubation had no effect on {rho}-TIA potency for inhibiting 125I-BE binding, demonstrating that {rho}-TIA acts in a time-independent manner. In addition, repeated washes slowly reversed {rho}-TIA inhibition of 125I-BE binding, supporting the reversibility of this interaction (Fig. 3B). Thus, these data indicate {rho}-TIA binds in a noncompetitive, reversible manner to the human {alpha}1B-AR, which suggests that {rho}-TIA is likely to be an allosteric inhibitor.



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  		FIG. 3.
Reversibility of {rho}-TIA binding to {alpha}1B-ARs. A, membranes from HEK293 cells expressing {alpha}1B-ARs were preincubated with ({blacksquare}) or without ({circ}) {rho}-TIA for 2 h prior to examination of inhibition of specific 125I-BE binding. B, HEK293 cell membranes expressing {alpha}1B-ARs were incubated with or without 30 nM {rho}-TIA and subjected to repeated washing. Data are expressed as % of control specific binding ({blacksquare}).

 
{rho}-TIA Inhibition of NE-stimulated [3H]InsP Formation—Radioligand binding studies suggested that {rho}-TIA binds differentially to the human {alpha}1-AR subtypes, and we wanted to determine whether similar results could be obtained with functional responses. To address this question, the ability of {rho}-TIA in inhibiting NE-stimulated [3H]InsP formation in HEK293 cells stably expressing individual {alpha}1-AR subtypes was examined. Cells were preincubated with [3H]inositol and stimulated with increasing concentrations of NE for 1 h in the absence or presence of various concentrations of {rho}-TIA. As shown in Fig. 4, increasing concentrations of {rho}-TIA caused parallel rightward shifts in the NE concentration-response curve for {alpha}1A- and {alpha}1D-ARs, without causing significant decreases in the NE maximal responses. From the concentration-response curves, Schild plots were constructed, and {rho}-TIA pA2 values were calculated as –7.0 for {alpha}1A-ARs and –6.8 for {alpha}1D-ARs, which were similar to the KD values generated from radioligand binding studies. Slope values were not significantly different from unity at either subtype, suggesting that {rho}-TIA binds to a single population of binding sites. In direct contrast, increasing concentrations of {rho}-TIA caused sequential decreases in the NE maximal response to {alpha}1B-AR activation, indicative of noncompetitive inhibition. These findings further support the hypothesis that {rho}-TIA acts as a noncompetitive inhibitor at human {alpha}1B-ARs but competitively inhibits human {alpha}1A- and {alpha}1D-ARs.



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  		FIG. 4.
{rho}-TIA inhibition of NE-stimulated [3H]InsP formation in HEK293 cells stably expressing {alpha}1B-AR subtypes. Left, NE concentration-response curves for [3H]InsP formation in HEK293 cells stably expressing each {alpha}1-AR subtype were measured in the absence or presence of the indicated concentrations of {rho}-TIA. Symbols represent the mean ± S.E. of 6–9 determinations. Right, Schild plots for competitive inhibition by {rho}-TIA in {alpha}1A- (upper, {diamond}) or {alpha}1D- (lower, {square})-AR expressing cells.

 
Role of the {alpha}1-AR Amino Terminus in {rho}-TIA Binding—Previous studies (38) have reported that the amino-terminal regions of GPCRs are involved in the formation of the binding pocket for peptide ligands. The amino-terminal regions of {alpha}1-AR subtypes differ in size and display very little sequence homology (37, 39). To determine whether these domains are important for {rho}-TIA binding, we compared the potency of {rho}-TIA in inhibiting specific 125I-BE binding to both amino-terminal truncated and full-length {alpha}1B- and {alpha}1D-ARs. As shown in Fig. 5, {rho}-TIA inhibited specific 125I-BE binding to full-length and amino-terminally truncated {alpha}1B- and {alpha}1D-ARs with similar potency, suggesting that the amino-terminal domains of these {alpha}1-AR subtypes are not important for {rho}-TIA binding. We did not perform experiments using truncated {alpha}1A-ARs, as the {alpha}1A-AR has a very short amino terminus (25 amino acids) relative to the {alpha}1B-(45 amino acids) and {alpha}1D-AR (95 amino acids) subtypes and is unlikely to be involved in {rho}-TIA binding given that the amino-terminal regions of {alpha}1B- and {alpha}1D-ARs were not involved.



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  		FIG. 5.
Inhibition of specific 125I-BE binding by {rho}-TIA in membranes from HEK293 cells expressing full-length or amino-terminal truncated {Delta}1–38{alpha}1B-(left) or {Delta}1–79{alpha}1D-ARs (right). Symbols represent the mean ± S.E. of three to four determinations.

 
Effect of Alanine Scanning Mutagenesis on Selectivity of {rho}-TIA for {alpha}1-AR Subtypes—To examine the importance of individual amino acids in conferring {alpha}1-AR subtype selectivity, {rho}-TIA was subjected to alanine scanning mutagenesis of non-cysteine residues. {rho}-TIA and alanine-substituted analog potencies for inhibiting 125I-BE binding at {alpha}1-AR subtypes are displayed in Fig. 6. The majority of the peptide analogs examined retained their {alpha}1B-AR selectivity, although it was somewhat lower than that of the parent compound. In a few instances, there was a loss of {alpha}1B-AR selectivity (R4A and I8A). A number of analogs (F1A, L7A, and R12A) bound with lower affinity to {alpha}1B-ARs than {rho}-TIA but had a higher degree of selectivity between the {alpha}1B-ARs and {alpha}1A- and {alpha}1D-AR subtypes. Most interesting, the F18A mutation resulted in an increase in subtype selectivity without a loss in {alpha}1B-AR affinity (Figs. 6 and 7). Mutants with high {alpha}1B-AR selectivity (F1A, N14A, K16A, and F18A) (Fig. 7) maintained their noncompetitive inhibition at {alpha}1B-ARs by progressively decreasing Bmax without affecting KD (Fig. 8). These results suggest that modification of {rho}-TIA can increase subtype selectivity without changing its noncompetitive properties in inhibition of {alpha}1B-ARs. Among all the alanine-substituted analogs, R4A had the lowest affinity for all three {alpha}1-AR subtypes. The R4A mutation was shown previously to impart low affinity binding to hamster {alpha}1B-ARs (27), because of a disruption between the positively charged arginine on {rho}-TIA and a complementary negatively charged residue on the {alpha}1B-AR. In support of these findings, alanine substitution of arginine at positions 12 and 13 resulted in a decrease in {rho}-TIA inhibitory potency (Fig. 6). Therefore, these data suggest that the charged nature of the {rho}-TIA plays a role in imparting high affinity interactions with the {alpha}1-AR subtypes but does not contribute to its subtype selectivity.



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  		FIG. 6.
Effect of alanine substitution on the potency of {rho}-TIA in competing for specific 125I-BE binding in membranes from HEK293 cells expressing human {alpha}1-AR subtypes. Non-cysteine amino acids of {rho}-TIA were systematically replaced with alanine as described under "Experimental Procedures." Bars represent the mean IC50 values ± S.E. calculated by nonlinear regression.

 



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  		FIG. 7.
Effect of selected alanine substitution on {rho}-TIA inhibition potency. Competition curves for inhibition of 125I-BE binding by the alanine-substituted analogs F1A (upper left), N14A (upper right), K16A (lower left), and F18A (lower right) to HEK293 membranes expressing individual {alpha}1-AR subtypes are displayed. Symbols represent the mean ± S.E. of four determinations.

 



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  		FIG. 8.
Noncompetitive binding of alanine-substituted {rho}-TIA analogs to {alpha}1B-ARs. 125I-BE saturation binding was performed on HEK293 membranes expressing {alpha}1B-ARs in the presence or absence of increasing concentrations of F1A (upper left), N14A (upper right), K16A (lower left), or F18A (lower right). Symbols represent the mean ± S.E. of four determinations.

 
Effect of Amino Acid Substitution at Position 18 in {rho}-TIA—In the previous figure, the F18A-TIA peptide analog showed an increased subtype selectivity in comparison to {rho}-TIA without loss in affinity. To determine whether the {alpha}1-AR subtype selectivity of {rho}-TIA could be further enhanced, the phenylalanine at position 18 was modified by amino acid substitution. The position 18-modified peptide analogs and their potencies for inhibiting 125I-BE binding to {alpha}1-AR subtypes are listed in Table I. All position 18 analogs had decreased potencies for inhibiting 125I-BE binding to {alpha}1B-ARs in comparison to the parent peptide. In addition, amino acid substitution did not enhance the binding to the {alpha}1A- or {alpha}1D-AR subtypes nor were there any further increases in {alpha}1-AR subtype selectivity relative to the F18A analog. Of all the peptide analogs screened, the F18N analog displayed the highest level of subtype selectivity, with a 30-fold difference in potency between {alpha}1B-ARs over {alpha}1A- and {alpha}1D-AR subtypes. However, in comparison to the F18A analog, F18N bound to {alpha}1B-ARs with lower affinity. Therefore, substituting the phenylalanine at position 18 with alanine or asparagine increases the {alpha}1-AR subtype selectivity of {rho}-TIA but does not increase the {alpha}1B-AR binding affinity.


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  		TABLE I
Comparison of the potencies of F18A and its various amino acid-substituted analogs for human {alpha}1-AR subtypes

The Phe-18 residue of {rho}-TIA was systematically replaced with different amino acids, and constructs were assayed for inhibiting specific 125I-BE binding to membranes prepared from HEK293 cells stably expressing individual receptor subtypes. Data are shown as mean log IC50 ± S.E. of 2–4 determinations.

 
In the previous alanine scanning study, a number of the analogs (F1A, L7A, P9A, N14A, K16A, and K17A) showed an equal or greater degree of {alpha}1-AR subtype selectivity in comparison to {rho}-TIA. Thus, {rho}-TIA analogs containing both the F18A and an additional alanine substitution were created to attempt to enhance the {alpha}1-AR subtype selectivity. Of the six analogs tested (F1A/F18A, L7A/F18A, P9A/F18A, N14A/F18A, K16A/F18A, and K17A/F18A), only the K17A/F18A (log IC50 {alpha}1A = –5.7 ± 0.06; {alpha}1B = –7.7 ± 0.06; {alpha}1D = –6.6 ± 0.19) and L7A-F18A (log IC50 {alpha}1A = –5.2 ± 0.10; {alpha}1B = –7.3 ± 0.11; {alpha}1D = –6.1 ± 0.03) double mutants maintained a level of {alpha}1-AR subtype selectivity equal to the F18A analog. All other double mutant analogs were not as selective as F18A and bound with lower affinity (data not shown). Furthermore, the L7A and K17A mutations were combined with the F18A mutation to attempt to enhance the selectivity of the peptide. Introducing all three alanine mutations (L7A/K17A/F18A) failed to increase the subtype selectivity and had significantly lower affinity (log IC50 {alpha}1A = –4.8 ± 0.27; {alpha}1B = –6.9 ± 0.13; {alpha}1D = –5.7 ± 0.08) for the {alpha}1-AR subtypes. Therefore, these data suggest that introducing a single alanine mutation at position 18 in {rho}-TIA yields the highest level of {alpha}1-AR subtype selectivity, while maintaining a high affinity interaction with the {alpha}1B-AR.

Importance of the Amino-terminal Portion of {rho}-TIA for Activity at {alpha}1-ARs—Previous studies (27) have shown that removal of the initial three amino acids from {rho}-TIA (TIA4–19) resulted in a decrease in the ability of {rho}-TIA to inhibit {alpha}1-AR-induced contraction. To examine the importance of the proximal three amino acids in maintaining {alpha}1-AR subtype selectivity of {rho}-TIA, we compared the potency of TIA4–19 for inhibiting 125I-BE binding to each of the three {alpha}1-AR subtypes. As shown in Fig. 6 (far right), TIA4–19 was ~10-fold more selective for the {alpha}1B-AR subtype over the {alpha}1A- and {alpha}1D-AR subtypes. However, the affinity of TIA4–19 was at least 1000-fold lower for all three subtypes relative to {rho}-TIA. These results suggest that removing the proximal three amino acids from {rho}-TIA does not abrogate the subtype selectivity of the peptide but is critical for maintaining high affinity binding to the {alpha}1-AR subtypes.

Isoleucine 8 Is Critical for {rho}-TIA Noncompetitive Inhibition at {alpha}1B-ARs—From the alanine scanning mutagenesis shown in Fig. 6, it is clear that the majority of {rho}-TIA analogs maintained a moderate degree of {alpha}1-AR subtype selectivity. However, substituting isoleucine at position 8 (I8A) resulted in an abolishment of {rho}-TIA selectivity (Fig. 6). From these results, we hypothesized that the mode of inhibition of {rho}-TIA at {alpha}1B-ARs may have been altered. To test this, we performed 125I-BE saturation binding experiments on HEK293 cells stably expressing {alpha}1B-ARs in the absence and presence of 300 nM I8A. As observed in Fig. 9A, I8A treatment had no significant effect on the {alpha}1B-AR Bmax but significantly changed the KD (Fig. 9A). These findings directly contrast with our previous results using {rho}-TIA, which significantly reduced the {alpha}1B-AR Bmax (Fig. 2). Thus, we examined the ability of I8A to inhibit NE-stimulated [3H]InsP formation in HEK293 cells stably expressing human {alpha}1B-ARs. Preincubation of cells with 3 µM I8A resulted in a 10-fold parallel shift to the right in the NE concentration-response curve without reducing the maximal response. Combined with the data from the saturation analysis, our findings suggest that isoleucine at position 8 is essential for the noncompetitive nature of {rho}-TIA at the {alpha}1B-AR subtype, and substitution of this amino acid results in a complete reversal of {rho}-TIA to a competitive antagonist at the {alpha}1B-AR.



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  		FIG. 9.
Competitive binding of the {rho}-TIA analog I8A to {alpha}1B-ARs. A, 125I-BE saturation binding to HEK293 membranes stably expressing {alpha}1B-ARs in the absence ({circ}) or presence ({blacksquare}) of 300 nM I8A. Symbols represent the mean ± S.E. of four determinations. B, NE concentration-response curve for stimulation of [3H]InsP formation in HEK293 cells stably expressing {alpha}1B-ARs in the absence ({circ}) or presence ({blacksquare}) of 3 µM I8A. Symbols represent the mean ± S.E. of data from six determinations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown that the conopeptide {rho}-TIA is a highly selective {alpha}1B-AR antagonist. Through a combination of radioligand binding and functional assays, we have demonstrated that {rho}-TIA interacts differentially at the {alpha}1-AR subtypes as a noncompetitive antagonist at {alpha}1B-ARs and as a competitive antagonist at {alpha}1A- and {alpha}1D-ARs. The {alpha}1-AR amino-terminal regions do not appear to be important for binding to {rho}-TIA, as there were no changes observed in the {rho}-TIA binding between amino-terminal truncated and full-length receptors. Alanine scanning mutagenesis revealed that substituting phenylalanine at position 18 resulted in a substantial increase in subtype selectivity. In addition, substituting isoleucine at position 8 changed {rho}-TIA from being a noncompetitive antagonist at {alpha}1B-ARs to a competitive antagonist. Taken together, these findings indicate that {rho}-TIA is a new pharmacological tool that can be used to distinguish between {alpha}1-AR subtypes and may serve as a lead compound for the development of highly selective {alpha}1-AR agents.

In the past, development of therapeutic compounds that target {alpha}1-ARs has been driven by their important roles in the regulation of hypertension, benign prostatic hypertrophy, and nasal congestion (1, 11). However, few drugs have been produced that can adequately differentiate between {alpha}1-AR subtypes. The majority of {alpha}1-AR ligands developed has been shown to be {alpha}1A-AR-selective, including WB 4101 (40), 5-methylurapidil (41), tamsulosin (42), and (+)-niguldipine (43). A single highly selective {alpha}1D-AR antagonist exists, BMY 7378 (44). However, there has yet to be developed a highly selective {alpha}1B-AR antagonist (1). The most widely used {alpha}1B-AR ligand to date has been chloroethylclonidine (45), a site-directed alkylating agent that has been used for pharmacological characterization of {alpha}1-AR subtypes. However, the technical difficulties associated with using chloroethylclonidine (i.e. temperature and time dependence) have limited the usefulness of this compound. Spiperone and cyclazosin (46) are {alpha}1B-AR competitive antagonists that have been reported to be slightly selective for {alpha}1B-ARs over {alpha}1A- and {alpha}1D-ARs and therefore have limited use in functional applications. Therefore, the need for highly selective {alpha}1B-AR has remained unfulfilled. In this study, we have shown that {rho}-TIA is 10-fold selective for the {alpha}1B-AR subtype, and most important, acts as a noncompetitive antagonist at {alpha}1B-ARs and as a competitive antagonist at {alpha}1A- and {alpha}1D-AR subtypes. The dual nature of this ligand is novel for {alpha}1-AR pharmacological agents and will be highly useful for future studies involving characterization of {alpha}1-AR functional responses. In addition, we have shown that the subtype selectivity of this peptide can be further enhanced by modification of specific amino acid residues. Thus, {rho}-TIA can act as a template for the development of novel therapeutic compounds for treatment of diseases associated with {alpha}1-AR stimulation.

Recent studies using transgenic and knockout mice have implicated {alpha}1B-ARs in playing an important role in blood pressure regulation (23), cardiac remodeling (19, 21, 47), glucose homeostasis (48), and central nervous system control of locomotion (49). However, several studies (50) have suggested that {alpha}1A- and {alpha}1D-ARs can also contribute to the regulation of these physiological processes. Therefore, highly selective {alpha}1B-AR antagonists would be extremely valuable in clarifying the relative contributions of each subtype in regulating these important biological functions. Increasing concentrations of agonist can overcome {rho}-TIA inhibition of {alpha}1A- and {alpha}1D-ARs but are unable to surmount {rho}-TIA noncompetitive antagonism at {alpha}1B-ARs. Therefore, taking advantage of the differential modes of inhibition of {rho}-TIA will allow us to distinguish functional differences between {alpha}1-AR subtypes and may provide some clarity to current debates concerning {alpha}1-AR functional responses.

The most interesting aspect of this study was that {rho}-TIA binds to closely related receptor subtypes through different mechanisms. {rho}-TIA bound to {alpha}1B-ARs in a noncompetitive, reversible manner but displayed competitive antagonism at {alpha}1A- and {alpha}1D-ARs. These data suggest that {rho}-TIA may be a negative allosteric modulator at {alpha}1B-ARs, although further studies will be needed to substantiate this conclusion. This is similar to what has been observed in previous studies investigating benzodiazepines at all three {alpha}1-AR subtypes (51) or amiloride analogs (52) binding to {alpha}1A-ARs. Therefore, understanding the mechanism by which {rho}-TIA binds to each subtype may provide useful information for the development of novel {alpha}1-AR subtype-selective drugs. Theoretically, the highly charged and rigid structure of {rho}-TIA should limit accessibility to the binding site of the endogenous ligand in the hydrophilic pocket formed by the transmembrane domains (2). Therefore, we hypothesized that the amino-terminal domains may play a role in {rho}-TIA binding to {alpha}1-ARs. For example, the long amino terminus of the lutrophin/choriogonadotropin receptor confers high affinity peptide ligand binding (38). However, no substantial differences in {rho}-TIA binding were observed when comparing amino-terminal truncated receptors to full-length receptors, suggesting that the amino terminus does not contribute to either the affinity or competitive/noncompetitive nature of this ligand. Previous studies have found that peptide ligands including angiotensin (53), neuropeptide Y (54), and vasopression (55) bind to their respective GPCRs through a mechanism that involves the extracellular domains. The {alpha}1-AR extracellular loop domains are highly divergent among subtypes (25–40% homology) and thus may be responsible for the differential modes of inhibition of {rho}-TIA between the {alpha}1-AR subtypes. In fact, mutation of three adjacent amino acids in the second extracellular loop of {alpha}1B-ARs to corresponding amino acids found in the {alpha}1A-AR second extracellular loop was able to confer {alpha}1A-AR antagonist binding properties to the {alpha}1B-AR (56). Therefore, these findings suggest that {rho}-TIA may bind to a novel binding site on {alpha}1-ARs, which may be partially formed by the extracellular loops of the receptor.

Peptide ligands for GPCRs are routinely modified in order to enhance subtype selectivity and/or increase the potency of the ligand at the receptor (57). Normally, amino acid scan/substitution and partial truncation of a parent peptide ligand are performed in order to determine the key residues involved in the biological activity of the peptide. In previous studies, alanine scanning of neurotensin revealed that leucine at position 13 confers high affinity binding (58). In these studies, alanine scanning mutagenesis of {rho}-TIA revealed that alanine substitution of phenylalanine at position 18 enhances the subtype selectivity of {rho}-TIA, while maintaining the noncompetitive nature and high affinity interaction of {rho}-TIA at {alpha}1B-ARs. However, alternate amino acid substitutions at position 18 failed to create any increase in {alpha}1-AR subtype selectivity. Charged residues within the peptide ligand have been shown to be important in peptide-receptor interactions. For example, alanine substitution of arginine at positions 8 or 9 resulted in a 10–100-fold decrease in neurotensin binding affinity (58). Charged arginine residues at positions 4, 12, and 13 in {rho}-TIA appear to be important for conferring high affinity binding, as alanine substitution reduced the potency of {rho}-TIA at all three human {alpha}1-AR subtypes by 6–400-fold. In addition, the three amino-terminal amino acids of {rho}-TIA appear to be important, as amino-terminal truncation results in a decrease in {rho}-TIA binding affinity to all three subtypes. Combined with previous reports of the NMR structure of {rho}-TIA (26), these data suggest that the amino terminus of {rho}-TIA forms a flexible open end that serves to extend the backbone, therein facilitating the interaction of {rho}-TIA with the receptor. Taken together, our results may provide useful structural information for further analyses of peptide conformation using various conformational constraint approaches.

Another interesting finding of this study was that alanine substitution of isoleucine at position 8 changed {rho}-TIA from a noncompetitive antagonist at the {alpha}1B-AR to a competitive antagonist. In previous studies investigating calcitonin gene-related peptide (59) and opioid agonists (60), minor modifications in amino acid sequence have resulted in dramatic differences in ligand binding properties. Despite these findings, considerable efforts are still required to obtain a full understanding of the structure-activity relationship between {rho}-TIA and the {alpha}1-AR subtypes.

In summary, we found that {rho}-TIA is a noncompetitive inhibitor at human {alpha}1B-ARs, whereas at {alpha}1A- and {alpha}1D-AR subtypes it acts as a competitive inhibitor. Replacing isoleucine with alanine at position 8 converts the noncompetitive interaction of {rho}-TIA at {alpha}1B-ARs to a competitive interaction, supporting the conclusion that the binding site of {rho}-TIA is in close proximity to that of the endogenous ligand. By taking advantage of the selective nature and differential modes of inhibition of {rho}-TIA, this peptide should prove valuable in elucidating the relative contribution of {alpha}1-AR subtypes in mediating different functional responses.


   FOOTNOTES
 
* This work was supported by grants from the National Institutes of Health (to K. P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 404-727-5985; Fax: 404-727-0365; E-mail: kminneman{at}pharm.emory.edu.

1 The abbreviations used are: ARs, adrenergic receptors; GPCR, G protein-coupled receptor; NE, norepinephrine; HEK, human embryonic kidney; PBS, phosphate-buffered saline; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HBTU, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate. Back


   ACKNOWLEDGMENTS
 
Peptide assembly was carried out by Jason Hodonickzy and Brad Patterson at Xenome Ltd.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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