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• W2051530695 abstract "The 19-amino acid conopeptide (ρ-TIA) was shown previously to antagonize noncompetitively α1B-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human α1-AR subtypes (α1A, α1B, and α1D). Radioligand binding assays showed that ρ-TIA was 10-fold selective for human α1B-over α1A- and α1D-ARs. As observed with hamster α1B-ARs, ρ-TIA decreased the number of binding sites (Bmax) for human α1B-ARs without changing affinity (KD), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, ρ-TIA had opposite effects at human α1A-ARs and α1D-ARs, decreasing KD without changing Bmax, suggesting it acts competitively at these subtypes. ρ-TIA reduced maximal NE-stimulated [3H]inositol phosphate formation in HEK293 cells expressing human α1B-ARs but competitively inhibited responses in cells expressing α1A- or α1D-ARs. Truncation mutants showed that the amino-terminal domains of α1B- or α1D-ARs are not involved in interaction with ρ-TIA. Alanine-scanning mutagenesis of ρ-TIA showed F18A had an increased selectivity for α1B-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at α1B-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus ρ-TIA noncompetitively inhibits α1B-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 α1B-ARs. The 19-amino acid conopeptide (ρ-TIA) was shown previously to antagonize noncompetitively α1B-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human α1-AR subtypes (α1A, α1B, and α1D). Radioligand binding assays showed that ρ-TIA was 10-fold selective for human α1B-over α1A- and α1D-ARs. As observed with hamster α1B-ARs, ρ-TIA decreased the number of binding sites (Bmax) for human α1B-ARs without changing affinity (KD), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, ρ-TIA had opposite effects at human α1A-ARs and α1D-ARs, decreasing KD without changing Bmax, suggesting it acts competitively at these subtypes. ρ-TIA reduced maximal NE-stimulated [3H]inositol phosphate formation in HEK293 cells expressing human α1B-ARs but competitively inhibited responses in cells expressing α1A- or α1D-ARs. Truncation mutants showed that the amino-terminal domains of α1B- or α1D-ARs are not involved in interaction with ρ-TIA. Alanine-scanning mutagenesis of ρ-TIA showed F18A had an increased selectivity for α1B-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at α1B-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus ρ-TIA noncompetitively inhibits α1B-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 α1B-ARs. α1-Adrenergic receptors (ARs) 1The 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. 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 (1Zhong H. Minneman K.P. Eur. J. Pharmacol. 1999; 375: 261-276Crossref PubMed Scopus (335) Google Scholar, 2Piascik M.T. Perez D.M. J. Pharmacol. Exp. Ther. 2001; 298: 403-410PubMed Google Scholar). Molecular cloning and pharmacological studies have identified three subtypes of α1-ARs: α1A-, α1B-, and α1D-ARs (3Morrow A.L. Battaglia G. Norman A.B. Creese I. Eur. J. Pharmacol. 1985; 109: 285-287Crossref PubMed Scopus (44) Google Scholar, 4Minneman K.P. Han C. Abel P.W. Mol. Pharmacol. 1988; 33: 509-514PubMed Google Scholar, 5Lomasney J.W. Cotecchia S. Lorenz W. Leung W.Y. Schwinn D.A. Yang-Feng T.L. Brownstein M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1991; 266: 6365-6369Abstract Full Text PDF PubMed Google Scholar, 6Cotecchia S. Schwinn D.A. Randall R.R. Lefkowitz R.J. Caron M.G. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7159-7163Crossref PubMed Scopus (484) Google Scholar, 7Ramarao C.S. Denker J.M. Perez D.M. Gaivin R.J. Riek R.P. Graham R.M. J. Biol. Chem. 1992; 267: 21936-21945Abstract Full Text PDF PubMed Google Scholar, 8Schwinn D.A. Lomasney J.W. Lorenz W. Szklut P.J. Fremeau Jr., R.T. Yang-Feng T.L. Caron M.G. Lefkowitz R.J. Cotecchia S. J. Biol. Chem. 1990; 265: 8183-8189Abstract Full Text PDF PubMed Google Scholar, 9Esbenshade T.A. Hirasawa A. Tsujimoto G. Tanaka T. Yano J. Minneman K.P. Murphy T.J. Mol. Pharmacol. 1995; 47: 977-985PubMed Google Scholar, 10Perez D.M. Piascik M.T. Graham R.M. Mol. Pharmacol. 1991; 40: 876-883PubMed Google Scholar). Although all three α1-AR subtypes signal through the same Gq/11 signaling pathway (11Hague C. Chen Z. Uberti M. Minneman K.P. Life Sci. 2003; 74: 411-418Crossref PubMed Scopus (37) Google Scholar), α1-AR subtypes can differ in their tissue distributions (1Zhong H. Minneman K.P. Eur. J. Pharmacol. 1999; 375: 261-276Crossref PubMed Scopus (335) Google Scholar), cellular localization (12Chalothorn D. McCune D.F. Edelmann S.E. Garcia-Cazarin M.L. Tsujimoto G. Piascik M.T. Mol. Pharmacol. 2002; 61: 1008-1016Crossref PubMed Scopus (120) Google Scholar, 13Hague C. Uberti M.A. Chen Z. Hall R.A. Minneman K.P. J. Biol. Chem. 2004; 279: 15541-15549Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), activation of transcriptional responses (14Gonzalez-Cabrera P.J. Gaivin R.J. Yun J. Ross S.A. Papay R.S. McCune D.F. Rorabaugh B.R. Perez D.M. Mol. Pharmacol. 2003; 63: 1104-1116Crossref PubMed Scopus (32) Google Scholar), and association with intracellular proteins (15Xu Z. Hirasawa A. Shinoura H. Tsujimoto G. J. Biol. Chem. 1999; 274: 21149-21154Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 16Chen S. Lin F. Iismaa S. Lee K.N. Birckbichler P.J. Graham R.M. J. Biol. Chem. 1996; 271: 32385-32391Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 17Diviani D. Lattion A.L. Abuin L. Staub O. Cotecchia S. J. Biol. Chem. 2003; 278: 19331-19340Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), suggesting that α1-AR subtypes perform specific physiological functions. Furthermore, recent data obtained from both α1-AR overexpressed (18Lin F. Owens W.A. Chen S. Stevens M.E. Kesteven S. Arthur J.F. Woodcock E.A. Feneley M.P. Graham R.M. Circ. Res. 2001; 89: 343-350Crossref PubMed Scopus (120) Google Scholar, 19Milano C.A. Dolber P.C. Rockman H.A. Bond R.A. Venable M.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10109-10113Crossref PubMed Scopus (332) Google Scholar, 20Zuscik M.J. Sands S. Ross S.A. Waugh D.J. Gaivin R.J. Morilak D. Perez D.M. Nat. Med. 2000; 6: 1388-1394Crossref PubMed Scopus (116) Google Scholar, 21Zuscik M.J. Chalothorn D. Hellard D. Deighan C. McGee A. Daly C.J. Waugh D.J. Ross S.A. Gaivin R.J. Morehead A.J. Thomas J.D. Plow E.F. McGrath J.C. Piascik M.T. Perez D.M. J. Biol. Chem. 2001; 276: 13738-13743Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and knockout mice (22Rokosh D.G. Simpson P.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9474-9479Crossref PubMed Scopus (166) Google Scholar, 23Cavalli A. Lattion A.L. Hummler E. Nenniger M. Pedrazzini T. Aubert J.F. Michel M.C. Yang M. Lembo G. Vecchione C. Mostardini M. Schmidt A. Beermann F. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11589-11594Crossref PubMed Scopus (270) Google Scholar, 24Tanoue A. Nasa Y. Koshimizu T. Shinoura H. Oshikawa S. Kawai T. Sunada S. Takeo S. Tsujimoto G. J. Clin. Investig. 2002; 109: 765-775Crossref PubMed Scopus (188) Google Scholar, 25O'Connell T.D. Ishizaka S. Nakamura A. Swigart P.M. Rodrigo M.C. Simpson G.L. Cotecchia S. Rokosh D.G. Grossman W. Foster E. Simpson P.C. J. Clin. Investig. 2003; 111: 1783-1791Crossref PubMed Scopus (164) Google Scholar) indicate that α1-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 α1B-AR-selective antagonists. Recently, a 19-amino acid conopeptide, ρ-TIA, was isolated from the venom of the fish-hunting cone snail Conus tulipa (26Sharpe I.A. Gehrmann J. Loughnan M.L. Thomas L. Adams D.A. Atkins A. Palant E. Craik D.J. Adams D.J. Alewood P.F. Lewis R.J. Nat. Neurosci. 2001; 4: 902-907Crossref PubMed Scopus (221) Google Scholar, 27Sharpe I.A. Thomas L. Loughnan M. Motin L. Palant E. Croker D.E. Alewood D. Chen S. Graham R.M. Alewood P.F. Adams D.J. Lewis R.J. J. Biol. Chem. 2003; 278: 34451-34457Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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 (26Sharpe I.A. Gehrmann J. Loughnan M.L. Thomas L. Adams D.A. Atkins A. Palant E. Craik D.J. Adams D.J. Alewood P.F. Lewis R.J. Nat. Neurosci. 2001; 4: 902-907Crossref PubMed Scopus (221) Google Scholar). ρ-TIA was found to be a selective α1-AR antagonist, as it inhibited α1-AR-mediated contraction of rat vas deferens without affecting ATP or α2-AR-mediated responses (26Sharpe I.A. Gehrmann J. Loughnan M.L. Thomas L. Adams D.A. Atkins A. Palant E. Craik D.J. Adams D.J. Alewood P.F. Lewis R.J. Nat. Neurosci. 2001; 4: 902-907Crossref PubMed Scopus (221) Google Scholar, 27Sharpe I.A. Thomas L. Loughnan M. Motin L. Palant E. Croker D.E. Alewood D. Chen S. Graham R.M. Alewood P.F. Adams D.J. Lewis R.J. J. Biol. Chem. 2003; 278: 34451-34457Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Studies on recombinant hamster α1B-ARs found that increasing concentrations of ρ-TIA progressively reduced the density of radioligand binding sites without changing radioligand affinity, suggesting that ρ-TIA is a noncompetitive, possibly allosteric, inhibitor of α1B-ARs. In addition, radioligand binding assays performed using recombinant rodent α1-AR subtypes revealed a 2–5-fold higher affinity for ρ-TIA at α1B-ARs (27Sharpe I.A. Thomas L. Loughnan M. Motin L. Palant E. Croker D.E. Alewood D. Chen S. Graham R.M. Alewood P.F. Adams D.J. Lewis R.J. J. Biol. Chem. 2003; 278: 34451-34457Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Allosteric modulators of GPCRs have received increasing attention over the last few years for their potential specificity and lack of undesirable effects (28Christopoulos A. Kenakin T. Pharmacol. Rev. 2002; 54: 323-374Crossref PubMed Scopus (805) Google Scholar). 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 (28Christopoulos A. Kenakin T. Pharmacol. Rev. 2002; 54: 323-374Crossref PubMed Scopus (805) Google Scholar). Although ρ-TIA also inhibits rodent α1A- and α1D-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 ρ-TIA at all three human α1-AR subtypes stably expressed in human embryonic kidney (HEK293) cells. We found that ρ-TIA acts noncompetitively at human α1B-ARs but competitively at α1A- and α1D-ARs and that the isoleucine at position 8 in ρ-TIA is required for noncompetitive inhibition. In addition, ρ-TIA is the first highly selective antagonist for human α1B-ARs, which will be useful in functional characterization of this receptor. 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-β-(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—ρ-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 (29Schnolzer M. Alewood P. Jones A. Alewood D. Kent S.B. Int. J. Pept. Protein Res. 1992; 40: 180-193Crossref PubMed Scopus (938) Google Scholar) 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 (30Sarin V.K. Kent S.B. Tam J.P. Merrifield R.B. Anal. Biochem. 1981; 117: 147-157Crossref PubMed Scopus (991) Google Scholar). 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 α1A- (31Hirasawa A. Horie K. Tanaka T. Takagaki K. Murai M. Yano J. Tsujimoto G. Biochem. Biophys. Res. Commun. 1993; 195: 902-909Crossref PubMed Scopus (162) Google Scholar), α1B- (7Ramarao C.S. Denker J.M. Perez D.M. Gaivin R.J. Riek R.P. Graham R.M. J. Biol. Chem. 1992; 267: 21936-21945Abstract Full Text PDF PubMed Google Scholar), or α1D- (9Esbenshade T.A. Hirasawa A. Tsujimoto G. Tanaka T. Yano J. Minneman K.P. Murphy T.J. Mol. Pharmacol. 1995; 47: 977-985PubMed Google Scholar) ARs have been previously described (32Romeo G. Materia L. Manetti F. Cagnotto A. Mennini T. Nicoletti F. Botta M. Russo F. Minneman K.P. J. Med. Chem. 2003; 46: 2877-2894Crossref PubMed Scopus (42) Google Scholar). 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 × 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 α1-AR-specific antagonist 125 I-BE (20–800 pm) (33Minneman K.P. Fox A.W. Abel P.W. Mol. Pharmacol. 1983; 23: 359-368PubMed Google Scholar) or [3H]prazosin (200–5000 pm) (34Greengrass P. Bremner R. Eur. J. Pharmacol. 1979; 55: 323-326Crossref PubMed Scopus (433) Google Scholar). 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 ρ-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 ρ-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 (35Theroux T.L. Esbenshade T.A. Peavy R.D. Minneman K.P. Mol. Pharmacol. 1996; 50: 1376-1387PubMed Google Scholar). 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 (35Theroux T.L. Esbenshade T.A. Peavy R.D. Minneman K.P. Mol. Pharmacol. 1996; 50: 1376-1387PubMed Google Scholar). 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 (36Arunlakshana O. Schild H.O. Br. J. Pharmacol. 1959; 14: 48-58Crossref PubMed Scopus (4275) Google Scholar). Generation of Amino-terminal Truncated Receptors—Amino-terminally truncated human α1D-ARs (Δ1–79) were constructed in pRSVICAT vectors as described previously (37Hague C. Chen Z. Pupo A.S. Schulte N. Toews M.L. Minneman K.P. J. Pharm. Exp. Ther. 2004; 309: 388-397Crossref PubMed Scopus (63) Google Scholar). Amino-terminally truncated human α1B-ARs (Δ1–38) were generated by PCR using specific primers on human α1B-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 ρ-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. ρ-TIA Inhibition of Specific 125I-BE Binding in HEK293 Cell Membranes Expressing Human α1A-, α1B-, or α1D-ARs—Because ρ-TIA showed noncompetitive inhibition at hamster α1B-ARs (26Sharpe I.A. Gehrmann J. Loughnan M.L. Thomas L. Adams D.A. Atkins A. Palant E. Craik D.J. Adams D.J. Alewood P.F. Lewis R.J. Nat. Neurosci. 2001; 4: 902-907Crossref PubMed Scopus (221) Google Scholar), we investigated the inhibitory effects of ρ-TIA at human α1A-, α1B-, and α1D-ARs. Increasing concentrations of ρ-TIA inhibited specific 125I-BE binding to membranes prepared from HEK293 cells stably expressing individual α1-AR subtypes (Fig. 1). However, unlike the limited selectivity observed previously with the rodent clones (27Sharpe I.A. Thomas L. Loughnan M. Motin L. Palant E. Croker D.E. Alewood D. Chen S. Graham R.M. Alewood P.F. Adams D.J. Lewis R.J. J. Biol. Chem. 2003; 278: 34451-34457Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), ρ-TIA was more potent and showed a 10-fold α1B selectivity. IC50 values for ρ-TIA were 18 nm (logIC50 –7.4 ± 0.08) at α1A-ARs; 2 nm (logIC50 –8.4 ± 0.10) at α1B-ARs; and 25 nm (logIC50 –7.3 ± 0.13) at α1D-ARs. The difference in potency of ρ-TIA was significant between α1B-ARs and α1A- and α1D-ARs (p < 0.001) but not between α1A-ARs and α1D-ARs. Mode of Inhibition—To determine whether ρ-TIA acts noncompetitively at each human α1-AR subtype, saturation binding assays with the selective antagonist 125I-BE were performed on membranes prepared from HEK293 cells expressing individual α1-AR subtypes in the presence or absence of 100 nm ρ-TIA (Fig. 2). Nonlinear regression analysis was used to determine whether inhibition by ρ-TIA was competitive (shared Bmax) or noncompetitive (shared KD). It was found that inhibition of specific 125I-BE binding to human α1B-ARs was clearly noncompetitive (p < 0.005), whereas inhibition of binding to human α1A- and α1D-ARs was competitive (p < 0.01 for α1A- and p < 0.05 for α1D-ARs). To confirm that these findings could be generalized to another α1-AR radioligand, the same experiments were repeated using [3H]prazosin in place of 125I-BE. In agreement with our previous experiments, ρ-TIA noncompetitively inhibited α1B-AR binding (p < 0.005) but was competitive at the α1A-AR (p < 0.05) and α1D-AR (p < 0.01) subtypes (data not shown). Therefore, these observations suggest ρ-TIA acts differently at the α1-AR subtypes as a noncompetitive inhibitor at human α1B-ARs but as a competitive inhibitor at α1A- and α1D-ARs. ρ-TIA Binds to the Human α1B-AR in a Reversible Manner— Although previous experiments suggested that ρ-TIA binds to human α1B-ARs noncompetitively, it is unclear if this binding is also reversible. To examine this, HEK293 cells stably expressing human α1B-ARs were preincubated with increasing concentrations of ρ-TIA for 2 h. As shown in Fig. 3A, preincubation had no effect on ρ-TIA potency for inhibiting 125I-BE binding, demonstrating that ρ-TIA acts in a time-independent manner. In addition, repeated washes slowly reversed ρ-TIA inhibition of 125I-BE binding, supporting the reversibility of this interaction (Fig. 3B). Thus, these data indicate ρ-TIA binds in a noncompetitive, reversible manner to the human α1B-AR, which suggests that ρ-TIA is likely to be an allosteric inhibitor. ρ-TIA Inhibition of NE-stimulated [3H]InsP Formation—Radioligand binding studies suggested that ρ-TIA binds differentially to the human α1-AR subtypes, and we wanted to determine whether similar results could be obtained with functional responses. To address this question, the ability of ρ-TIA in inhibiting NE-stimulated [3H]InsP formation in HEK293 cells stably expressing individual α1-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 ρ-TIA. As shown in Fig. 4, increasing concentrations of ρ-TIA caused parallel rightward shifts in the NE concentration-response curve for α1A- and α1D-ARs, without causing significant decreases in the NE maximal responses. From the concentration-response curves, Schild plots were constructed, and ρ-TIA pA2 values were calculated as –7.0 for α1A-ARs and –6.8 for α1D-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 ρ-TIA binds to a single population of binding sites. In direct contrast, increasing concentrations of ρ-TIA caused sequential decreases in the NE maximal response to α1B-AR activation, indicative of noncompetitive inhibition. These findings further support the hypothesis that ρ-TIA acts as a noncompetitive inhibitor at human α1B-ARs but competitively inhibits human α1A- and α1D-ARs. Role of the α1-AR Amino Terminus in ρ-TIA Binding—Previous studies (38Xie Y.B. Wang H. Segaloff D.L. J. Biol. Chem. 1990; 265: 21411-21414Abstract Full Text PDF PubMed Google Scholar) 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 α1-AR subtypes differ in size and display very little sequence homology (37Hague C. Chen Z. Pupo A.S. Schulte N. Toews M.L. Minneman K.P. J. Pharm. Exp. Ther. 2004; 309: 388-397Crossref PubMed Scopus (63) Google Scholar, 39Pupo A.S. Uberti M.A. Minneman K.P. Eur. J. Pharmacol. 2003; 462: 1-8Crossref PubMed Scopus (44) Google Scholar). To determine whether these domains are important for ρ-TIA binding, we compared the potency of ρ-TIA in inhibiting specific 125I-BE binding to both amino-terminal truncated and full-length α1B- and α1D-ARs. As shown in Fig. 5, ρ-TIA inhibited specific 125I-BE binding to full-length and amino-terminally truncated α1B- and α1D-ARs with similar potency, suggesting that the amino-terminal domains of these α1-AR subtypes are not important for ρ-TIA binding. We did not perform experiments using truncated α1A-ARs, as the α1A-AR has a very short amino terminus (25 amino acids) relative to the α1B-(45 amino acids) and α1D-AR (95 amino acids) subtypes and is unlikely to be involved in ρ-TIA binding given that the amino-terminal regions of α1B- and α1D-ARs were not involved. Effect of Alanine Scanning Mutagenesis on Selectivity of ρ-TIA for α1-AR Subtypes—To examine the importance of individual amino acids in conferring α1-AR subtype selectivity, ρ-TIA was subjected to alanine scanning mutagenesis of non-cysteine residues. ρ-TIA and alanine-substituted analog potencies for inhibiting 125I-BE binding at α1-AR subtypes are displayed in Fig. 6. The majority of the peptide analogs examined retained their α1B-AR selectivity, although it was somewhat lower than that of the parent compound. In a few instances, there was a loss of α1B-AR selectivity (R4A and I8A). A number of analogs (F1A, L7A, and R12A) bound with lower affinity to α1B-ARs than ρ-TIA but had a higher degree of selectivity between the α1B-ARs and α1A- and α1D-AR subtypes. Most interesting, the F18A mutation resulted in an increase in subtype selectivity without a loss in α1B-AR affinity (Figs. 6 and 7). Mutants with high α1B-AR selectivity (F1A, N14A, K16A, and F18A) (Fig. 7) maintained their noncompetitive inhibition at α1B-ARs by progressively decreasing Bmax without affecting KD (Fig. 8). These results suggest that modification of ρ-TIA can increase subtype selectivity without changing its noncompetitive properties in inhibition of α1B-ARs. Among all the alanine-substituted analogs, R4A had the lowest affinity for all three α1-AR subtypes. The R4A mutation was shown previously to impart low affinity binding to hamster α1B-ARs (27Sharpe I.A. Thomas L. Loughnan M. Motin L. Palant E. Croker D.E. Alewood D. Chen S. Graham R.M. Alewood P.F. Adams D.J. Lewis R.J. J. Biol. Chem. 2003; 278: 34451-34457Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), because of a disruption between the positively charged arginine on ρ-TIA and a complementary negatively charged residue on the α1B-AR. In support of these findings, alanine substitution of arginine at positions 12 and 13 resulted in a decrease in ρ-TIA inhibitory potency (Fig. 6). Therefore, these data suggest that the charged nature of the ρ-TIA plays a role in imparting high affinity interactions with the α1-AR subtypes but does not contribute to its subtype selectivity.Fig. 7Effect of selected alanine substitution on ρ-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 α1-AR subtypes are displayed. Symbols represent the mean ± S.E. of four determinations.View Large Image Figure ViewerDownload (PPT)Fig. 8Noncompetitive binding of alanine-substituted ρ-TIA analogs to α1B-ARs.125I-BE saturation binding was performed on HEK293 membranes expressing α1B-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.View Large Image Figure ViewerDownload (PPT) Effect of Amino Acid Subst" @default.
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• W2051530695 date "2004-08-01" @default.
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• W2051530695 title "Subtype-selective Noncompetitive or Competitive Inhibition of Human α1-Adrenergic Receptors by ρ-TIA" @default.
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• W2051530695 doi "https://doi.org/10.1074/jbc.m403703200" @default.
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