An Assay Method for Evaluating Chemical Selectivity of Agonists for

The use of resonance energy transfer in high-throughput screening: BRET ... Dawn E. Biazzo-Ashnault , Young-Whan Park , Richard T. Cummings , Victor D...
0 downloads 0 Views 96KB Size
Download PDF
Anal. Chem. 1998, 70, 2345-2352

An Assay Method for Evaluating Chemical Selectivity of Agonists for Insulin Signaling Pathways Based on Agonist-Induced Phosphorylation of a Target Peptide Takeaki Ozawa, Moritoshi Sato, Masao Sugawara, and Yoshio Umezawa*

Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

An optical method for evaluating the physiologically relevant agonist and antagonist selectivity of an insulin signaling pathway based on an insulin-dependent on/off switching of phosphorylation of a target peptide via insulin receptor is described. Insulin receptor serves as a binding for insulin and a given insulin receptor-binding peptide as a target for an insulin-receptor complex. Upon binding of insulin to its receptor, the insulin receptor undergoes autophosphorylation which enables the receptor to have a kinase activity and phosphorylate various substrates. The phosphorylated tyrosine in the substrate was measured with a monoclonal anti-phosphotyrosine antibody. As the target substrate for insulin receptor, a Y939 peptide consisting of 12 amino acid residues derived from insulin receptor substrate 1 (IRS1) was used. The present assay method involves different sequential steps: (1) immobilization of a biotin-coupled Y939 peptide on an avidin coated 96-well plate via biotin-avidin complexation; (2) insulin-dependent phosphorylation of the Y939 peptide by the insulin receptor; (3) enzymatic reaction and absorptiometric assay of the phosphorylated Y939 peptide using the anti-phosphotyrosine antibody labeled with horseradish peroxidase. An insulin-dependent absorbance was observed for insulin concentrations from 1.0 × 10-10 to 1.0 × 10-7 M, and it leveled off. The observed absorbance was explained to be due to an increase in the phosphorylated Y939 peptide caused by insulin and its receptor complexation. No signal was, however, induced by both vanadyl and vanadate ions at concentrations up to 1.0 × 10-4 M; these results and previous intact cell level data taken together led to the conclusion that these ions did not induce phosphorylation of the Y939 peptide. Upon addition of tyrphostin 25, a specific inhibitor for insulin receptor kinase activity, phosphorylation of the Y939 peptide in the presence of 1.0 µM insulin was competitively inhibited over 1.0 × 10-4 M tyrphostin 25. The present system thus exhibited “physiologically more relevant” agonist and antagonist selectivity, the principle of which is based in part on the insulin signal transduction rather than simply relying on the binding assay. The potential use of the present method for evaluating the selectivity of a wide range of S0003-2700(97)01192-X CCC: $15.00 Published on Web 04/28/1998

© 1998 American Chemical Society

agonists and antagonists toward the insulin signaling pathways is discussed. Insulin activity is an important laboratory parameter in the clinical evaluation of several diseases such as diabetes mellitus types I and II, states of impaired glucose tolerance, and insulinproducing tumors (insulinomas), where the insulin secretion released from pancreas β cells is altered.1,2 The introduction of radioimmunoassay (RIA) by Berson and Yalow in 1959 provided a novel method for measuring insulin in plasma and serum.3 In order to improve reliability and practicability, various technologies have appeared in the field of immunoassays.4,5 Variety is seen in the choice of labels (radioactive, enzymatic, fluorescent, etc.), separation methods (precipitation of antigen-antibody complexes, coated solid-phase antibody technology, etc.), detection methods (spectrophotometry, fluorimetry, amperometry, potentiometry, and calorimetry), and principles such as the competitive or noncompetitive immunoassay. These immunoassay techniques have become important analytical methods in clinical chemistry laboratories for the selective detection of drugs or proteins as well as hormones such as insulin at trace levels. In contrast to the binding assay and related techniques represented by immunoassay, some new types of biosensing have emerged that rely on signal transduction mechanisms by intracellular or membrane receptor proteins themselves for generating analytical signals.6-10 Typical examples are ion channel or transporter proteins embedded in lipid bilayer membranes. These sensors utilize the corresponding transmembrane signals, such as ion channel currents or active membrane transport for yielding, (1) Myers, M. G., Jr.; White, M. F. Annu. Rev. Pharmacol. Toxicol. 1996, 36, 615-658. (2) Czech, M. P. Annu. Rev. Nutr. 1995, 15, 441-471. (3) Yalow, R. S.; Berson, S. A. Nature 1959, 184, 1648-1649. (4) Gosling, J. P. Clin. Chem. 1990, 36, 1408-1427. (5) Ishikawa, E.; Hashida, S.; Kohno, T.; Hirota, K. Clin. Chem. Acta 1990, 194, 51-72. (6) Sugawara, M.; Hirano, A.; Rehak, M.; Nakanishi, J.; Kawai, K.; Sato, H.; Umezawa, Y. Biosens. Bioelectron. 1997, 12, 425-439. (7) Radecka, H.; Nakanishi, J.; Hirano, A.; Sugawara, M.; Umezawa, Y. J. Pharm. Biomed. Anal., in press. (8) Minami, H.; Sugawara, M.; Odashima, K.; Umezawa, Y.; Uto, M.; Michaelis, E. K.; Kuwana, T. Anal. Chem. 1991, 63, 2787-2795. (9) Sugao, N.; Sugawara, M.; Minami, H.; Uto, M.; Umezawa, Y. Anal. Chem. 1993, 65, 363-369. (10) Ozawa, T.; Kakuta, M.; Sugawara, M.; Umezawa, Y.; Ikura, M. Anal. Chem. 1997, 69, 3081-3085.

Analytical Chemistry, Vol. 70, No. 11, June 1, 1998 2345

in some cases, physiologically more relevant signals compared to those with the binding assay approach: The chemical selectivity of agonists and antagonists toward a glutamate receptor displayed by its ion channel has been evaluated from the sum of the total amount of ions6 and fractional calcium ion flux passed through the glutamate receptor.7 The integrated channel currents and fractional calcium ion flux were used respectively as an amplified physiologically more relevant measure of agonist potency to activate the glutamate receptor. The chemical selectivity ratios among three agonists including glutamate ion thus evaluated were found to be ∼1 order of magnitude narrower than that obtained by the binding assay. As to Ca2+ signaling pathways based on a Ca2+-dependent on/off switching of calmodulin in biological cells, the agonist selectivity in terms of ion selectivity has been evaluated for the formation of a calmodulin-Ca2+-target peptide ternary complex.10 By this method, Sr2+ ion has been found to behave as a strong agonist toward the Ca2+ signaling. Novel methods for evaluating chemical selectivities of agonists or antagonists for intracellular or receptor-type proteins are thus increasingly required in addition to sensitive and selective sensing methods of analytes. For the purpose of evaluating for insulin and its agonists physiologically more relevant chemical selectivity based on insulin signaling, it is necessary to know what types of mechanisms occur for the following insulin signal transduction. The signal transduction is initiated by the binding of insulin to its receptor at the membrane surface. The insulin receptor is a tetrameric transmembrane glycoprotein consisting of two MW 120 000 R and two MW 90 000 β subunits.11 The two extracellular R subunits are disulfide-linked and contain the insulin binding sites. Each β subunit is disulfide-linked to an R subunit, spans the membrane, and contains a tyrosine kinase domain in its intracellular portion. The binding of insulin with its receptor stimulates its tyrosine kinase activity, and the tyrosine kinase domain in the β subunit phosphorylates its major substrate protein, insulin receptor substrate 1 (IRS-1).12,13 The IRS-1 contains 21 potential tyrosine and over 30 potential serine/threonine phosphorylation sites, which are recognized by various kinases. The activated insulin receptor phosphorylates at least 9 tyrosines and some methionine residues in IRS-1. The tyrosine-phosphorylated IRS-1 then serves as a docking/effector protein for at least four Src homology 2 (SH2) domain proteins such as phosphatidyl inositol 3-kinase (PI-3 kinase),14 Ras guanine-nucleotide-releasing complex (Grb2-Sos),15 the phosphotyrosine phosphatase (Syp),16 and the adapter protein (Nck)17 and leads to the activation and regulation of these proteins. If the molecular mechanisms of such an insulin-dependent on/ off switching system for transmembrane and intracellular signaling are introduced into a sensing system, then physiologically more (11) Tavare´, J. M.; Siddle, K. Biochim. Biophys. Acta 1993, 1178, 21-39. (12) White, M. F.; Kahn, C. R. J. Biol. Chem. 1994, 269, 1-4. (13) Keller, S. R.; Lienhard, G. E. Trends Cell Biol. 1994, 4, 115-119. (14) Backer, J. M.; Myers, M. G., Jr.; Shoelson, S. E.; Chin, D. J.; Sun, X. J.; Miralpeix, M.; Hu, P.; Margolis, B.; Skolnik, E. Y.; Schlessinger, J.; White, M. F. EMBO J. 1992, 11, 3469-3479. (15) Sun, X. J.; Crimmins, D. L.; Myers, M. G.; Miralpeix, M.; White, M. J. Mol. Cell. Biol. 1993, 12, 7418-7428. (16) Kuhne´, M. R.; Pawson, T.; Lienhard, G. E.; Geng, G.-S. J. Biol. Chem. 1993, 268, 11479-11481. (17) Lee, C. H.; Li, W.; Nishimura, R.; Zhou, M.; Batzer, A. G.; Myers, M. G., Jr.; White, M. F.; Schlessinger, J.; Skolnik, E. Y. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11713-11717.

2346 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

Figure 1. Principle and sequence of steps for the proposed physiologically relevant insulin assay system.

relevant agonist and antagonist selectivity toward insulin signaling pathway may be evaluated. In this paper, a new assay method is described for evaluating agonist and antagonist selectivities for the insulin signaling pathway, which is based on the above-described on/off switching mechanism of the insulin receptor-mediated insulin signaling. The principle is schematically shown in Figure 1. Insulin receptor serves as a primary receptor for insulin and Y939 as a target peptide. The Y939 peptide, a synthetic peptide of 12 amino acid residues, consists of a tyrosine-phosphorylated domain of IRS-1 and of a binding domain of IRS-1 to PI-3 kinase.18,19 The Y939 peptide is immobilized on an avidin-coated 96-well microtiter plate (step 1 in Figure 1). Upon binding of insulin to its receptor, insulin stimulates its receptor kinase activities in a sample solution and phosphorylates the tyrosine residue of the Y939 peptide immobilized on the well surface (step 2 in Figure 1). The sample solution is washed out, and a selective binding protein for the phosphorylated tyrosine, monoclonal anti-phosphotyrosine antibody labeled with horseradish peroxidase, is added and formed a phosphorylated peptide-antibody complex (step 3 in Figure 1). The amount of complex thus formed was detected by enzymatic amplification with horseradish peroxidase that can produce a green-color product with an absorbance at 727 nm capable of being detected by colorimetry. The amount of phosphorylated Y939 peptide thus measured is expected to be a selective and sensitive measure of the extent of the insulin signaling, representing (18) Nishiyama, M.; Wands, J. R. Biochem. Biophys. Res. Commun. 1992, 183, 280-285. (19) Piccione, E.; Case, R. D.; Domchek, S. M.; Hu, P.; Chaudhuri, M.; Backer, J. M.; Schlessinger, J.; Shoelson, S. E. Biochemistry 1993, 32, 3197-3202.

physiologically more relevant selectivity for agonists and metal ions such as vanadium ion. EXPERIMENTAL SECTION Materials. Insulin (human) was purchased from Peptide Institute, Inc. (Osaka, Japan). (()-5-[4-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-yl-methoxy)benzyl]-2,4-thiazolidinedione (CS045, troglitazone) was kindly provided as a gift from Sankyo Co. (Tokyo, Japan). Chemically synthesized HPLC-purified Y939 peptide, which consists of the amino acid sequence of SEEYMNMDLGPC (expressed by one-letter abbreviations), was purchased from Biologica Co. (Nagoya, Japan). This Y939 peptide differs from its native amino acid sequence in IRS-1 by addition of a cysteine on the C-terminal end of the peptide. [γ-32P]ATP (∼110 TBq/mmol) and (3-[125I]iodotyrosylA14) insulin (∼74 TBq/ mmol) were purchased from Amersham Co. (Buckingham, U.K.). Anti-phosphotyrosine monoclonal antibody (cell line PY20) labeled with horseradish peroxidase was purchased from Takara Shuzo Co. (Kyoto, Japan). 6-[N′-[2-(N-Maleimido)ethyl]-N-piperazinylamido]hexyl-D-biotinamide hydrochloride (biotin-PEAC5-maleimide) was obtained from Dojindo Laboratories (Kumamoto, Japan). Polyoxyethylene(20) sorbitan monolaurate (Tween 20), polyoxyethylene(10) octylphenyl ether (Triton X-100), and N′-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-biphenylamine sodium salt (DA-64) were obtained from Wako Pure Chemical Industries (Osaka, Japan). N-(2-Hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). [3,4,5-Trihydroxybenzylidene]-malononitrile (Tyrphostin 25) and random copolymer of glutamic acid:tyrosine to be 4:1 ((Glu80Tyr20)n) was purchased from Sigma Chemical Co. (St. Louis, MO). Other salts and solvents used were all of the highest purity available. All aqueous solutions were prepared with Milli-Q grade (>18.2 MΩ resistance) water obtained with a Milli-Q Plus system (Millipore Corp., Bedford, MA). Preparation of Purified Insulin Receptor. Insulin receptor was extracted and purified from human placenta as previously described.20 After preparation of placental membranes from one piece of fresh normal human placenta by differential centrifugation, a suspension containing the membrane proteins was mixed with the same volume of a 50 mM Tris-HCl buffer, pH 7.4, containing 2% Triton X-100 and protein inhibitors (2 mM BAEE, 1 mg/mL pepstatin, 1 mg/mL aprotinin, 1 mg/mL leupeptin, and 0.1 mM PMSF) for 45 min at 4 °C with stirring. A clear supernatant was obtained by centrifugation at 100 000g for 90 min at 4 °C and was transferred to 40 mL of a WGA-Sepharose affinity column. The fractions including the protein were pooled and applied to 6.3 mL of an insulin-Sepharose column. After thorough washing of the column with 50 mM Tris-HCl buffer, pH 7.4, containing 1.0 M NaCl, 0.1% Triton X-100 and 0.1 mM PMSF, the insulin receptor was eluted with 50 mM acetate buffer (pH 5.0), containing 1.0 M NaCl and 0.1% Triton X-100. The active fractions assessed by 125Iinsulin binding activity were collected and concentrated by pressured dialysis using a Diaflo ultrafiltration membrane PM-30 (Amicon Inc., Beverly, MA). The purified insulin receptor was stored in a freezer at -80 °C until needed. The purity of the receptor was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The

purified receptor gave two bands of MW 135 000 and 90 000, which correspond to R and β subunits of insulin receptor, respectively.20 The kinase activities of the purified insulin receptor obtained were assessed by using random copolymers of (Glu80Tyr20)n serving as a substrate for insulin receptor kinase according to the method described previously.21 The purified insulin receptor was preincubated with 0.48 µM insulin at 22 °C for 1 h in 630 µL of a solution (solution A) containing 25 µM ATP and 5.0 mM MnCl2. The random copolymer of (Glu80Tyr20)n was dissolved in a buffer solution (solution B) containing 0.1% Triton X-100, 5.0 mM MnCl2, 10 mM MgCl2, 50 µCi [γ-32P]ATP, and 50 mM HEPES buffer (pH 7.4) to its final concentration of 0.5 mg/mL. A 30 µL portion of solution B was taken into a microtube. Phosphorylation of (Glu80Tyr20)n was initiated by adding 15 µL of the preincubated insulin receptor solution into the microtube, and the solution was incubated for 5, 30, 60, or 100 min. After each incubation time, the sample was applied to filter paper (Whatman 3MM). The filter paper was washed 6 times with 10% trichloroacetic acid and then with acetone and finally air-dried. Radioactivity was counted with a liquid scintillation counter. The time-dependent incorporation of 32P in the random copolymers is shown in Figure 2. The magnitude of phosphorylated random copolymers incubated with insulin was ca. 4 times higher than that without insulin at each time, illustrating the active preparation of insulin receptor by the present purification method. The stability of the purified insulin receptor was determined by the kinase activity of insulin receptor at various time intervals after elution of the insulin-Sepharose column. There was no apparent change for the kinase activity up to 1 month, which was evaluated from the magnitude of phosphorylated (Glu80Tyr20)n incubated with insulin and its receptor. The insulin receptor prepared by the present method was thus found to be sufficiently stable. Preparation of Biotin-Y939 Peptide Conjugate. A Y939 peptide was biotinylated by reaction of a maleimide group of biotin-PEAC5-maleimide with a thiol group of cysteine intro-

(20) Yamaguchi, Y.; Choi, S.; Sakamoto, Y.; Itakura, K. J. Biol. Chem. 1983, 258, 5045-5049.

(21) Braun, S.; Raymond, W. E.; Racker, E. J. Biol. Chem. 1984, 259, 20512054.

Figure 2. Time course of phosphorylation of (Glu80Tyr20)n by insulin receptor. The polymer substrate ((Glu80Tyr20)n, 0.1 mg/mL) was incubated at 37 °C for the indicated periods of time with [γ-32P]ATP and insulin receptor in the presence (") and absence (O) of 1.0 µM insulin. The phosphorylated substrate was applied to Whatman 3MM paper squares and washed with 10% trichloroacetic acid. The radioactivity of 32P incorporated into the substrate of (Glu80Tyr20)n was detected by liquid scintillation counting. The results are the means and standard deviations of three separate experiments (mean ( SD).

Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

2347

duced at the C-terminal end of the Y939 peptide as described previously.22 In brief, 1.0 mg of Y939 peptide (7.2 × 10-7 mol) was dissolved in 160 µL of PBS buffer (150 mM NaCl, 3.0 mM KCl, 10 mM phosphate buffer, pH 7.2), and 2.5 mg of biotinPEAC5-maleimide (3.6 × 10-6 mol) dissolved in 40 µL of dimethyl sulfoxide was added dropwise to the Y939 peptide solution with gentle stirring. The reaction mixture was left for 12 h at 25 °C. The reactants were purified by reverse phase HPLC performed on a 801-SC system (Japan Spectroscopic Co., Tokyo, Japan), equipped with a Kaseisorb LC ODS-300-5 column (4.6 φ × 250 mm) and a UV detector (UV-970, Japan Spectroscopic Co., Tokyo, Japan), using a linear gradient of 0-80% acetonitrile in 0.05% trifluoroacetic acid, at a constant flow rate of 1.0 mL/min over 60 min. The eluents were monitored by UV absorbance at 220 nm. Ten milliliters of an eluent containing biotinylated peptide was collected and lyophilized. To evaluate the ratio of covalently immobilized biotin molecules to the Y939 peptide, the amounts of the Y939-immobilized biotin and the Y939 peptide in the eluent were determined as follows. After lyophilization, the biotin-Y939 conjugate was dissolved in 1.0 mL of a PBS buffer and its absorbance at 280 nm was measured. Assuming that the observed absorbance is due to a tyrosine residue in a Y939 peptide, total amount of Y939 peptide was obtained as 4.4 × 10-7 mol from the absorbance and molar absorptivity for tyrosine of 1.34 × 103 M-1 cm-1. The amount of Y939-conjugated D-biotin was evaluated by a spectrophotometric assay method as described elsewhere.23 The assay gave 2.8 × 10-7 mol of Y939-conjugated biotin. Hence, 63 (mol/mol) % of Y939 peptide was found to be biotinylated. The relatively low yield of biotinylated Y939 peptide may be due to disulfide bond formation to a certain extent in Y939 peptide: In the presence of oxygen, it is likely that the sulfhydryl group of the cysteine residue in a Y939 peptide molecule reacts to form disulfide bonds between two Y939 peptides to be molecules, causing the cysteine residue of Y939 peptide to be partly unavailable for the reaction with the biotin-PEAC5-maleimide. Assay Procedure. Concentration of Insulin Receptor. Kinase activities of purified insulin receptor were found to change from one extraction to another. In order to control this kinase activity so that it would to be the same throughout the experiment, the concentration of the purified insulin receptor was adjusted by dilution before the receptor was applied for measurements in the present method. For this purpose, a solution was prepared in a microtube, consisting of 1.0 µM insulin, 0.05% Triton X-100, 50 µM ATP, 1.7 mM MnCl2, and 50 mM HEPES/NaOH (pH 7.4), to which 1/100 volume of the insulin receptor solution obtained after pressured dialysis was added. The mixture was incubated for 10 min, and kinase reaction, enzymatic reactions, and absorptiometric assay proceeded according to the procedure (steps 1-3) described below. In most cases, the absorbance of DA-64 thus measured was larger than 0.1. On the basis of the observed absorbance, the stock solution of insulin receptor was diluted to give its concentration corresponding to an absorbance of 0.1. By thus adjusting the concentration of the receptor, it was possible to measure 100-200 sample solutions from one human placenta. (22) Hashida, S.; Imagawa, M.; Inoue, S.; Ruan, K.-H.; Ishikawa, I.; Ueno, T. J. Appl. Biochem. 1984, 6, 56-63. (23) Green, N. M. Biochem. J. 1965, 94, 23c.

2348 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

Step 1: Immobilization of Y939 Peptide on a 96-Well Microtiter Plate. All 96 wells on a microtiter plate were incubated with 150 µL each of avidin solution (0.1 mg/mL avidin in PBS buffer) over 18 h at 4 °C. After the immobilization of avidin on the well, each well of the plate was washed with a PBS-T buffer solution (0.05% Tween 20 in PBS buffer). To fill up space between the immobilized avidin molecules with bovine serum albumin (BSA) molecules, a 200 µL portion of a BSA solution (1.0% bovine serum albumin in PBS buffer) was added to each well and the plate was left for 4 h at 4 °C. The BSA solution was discarded, and each well was washed four times with the PBS-T solution. After washing with the PBS-T solution, 150 µL of biotin-Y939 conjugates (1.0 µg/mL in the PBS-T solution) was added to each well and biotin-avidin complexation was carried out for 1 h at 4 °C. An excess of the conjugates was discarded, and the well was washed four times with the PBS-T solution. Step 2: Kinase Reaction. A sample solution was prepared in a microtube, consisting of 0.05% Triton X-100, 50 µM ATP, 1.7 mM MnCl2, a given concentration of insulin receptor (vide supra), 50 mM HEPES/NaOH (pH 7.4), and each concentration of insulin. The sample solution was incubated in a microtube at 25 °C for 10 min, and 150 µL portion of the sample solution thus prepared was introduced in a given well. For obtaining a background absorbance, the sample solution without insulin was also introduced in one of the wells. The plate was shaken on a mixer (Tomy Seiko Co., Tokyo, Japan) for 1 h at 37 °C to react the insulin-receptor complex with the Y939 peptide immobilized on the well surface. The plate was washed three times with the PBS-T solution and then two times with a TBS solution (20 mM NaCl, 3.0 mM KCl, and 20 mM Tris/HCl, pH 7.4) for step 3. Step 3: Enzymatic Reaction and Absorptiometric Assay. After dilution of monoclonal anti-phosphotyrosine antibody labeled with horseradish peroxidase in the TBS solution to its final concentration of 0.5 µg/mL, 150 µL of the solution was added into each well. After standing for 2 h at room temperature without shaking, the plate was washed six times with a TBS-T (0.05% Tween 20 in the TBS buffer) solution. To evaluate the amount of the antibody specifically bound to the phosphorylated Y939 peptide, 175 µL of a DA-64 solution (0.4 mg/mL of DA-64 in 2.0% H2O2, 50 mM citric acid, and 100 mM Na2HPO4) was added to each well and was incubated with shaking for ca. 1 h at 37 °C in the dark. After the enzymatic reaction, the DA-64 solution was subjected to absorbance measurement: a 150 µL portion of the DA-64 solution was diluted with 1.0 mL of Milli-Q water, and the absorbance of the solution in a 1.0 mL cuvette was measured at 727 nm against water with a Shimadzu (Kyoto, Japan) UV-240 spectrometer. Calculation of Insulin-Dependent Signals and Statistical Analysis. For obtaining net insulin-dependent signals (absorbance), a background absorbance obtained by introducing a buffer solution without insulin (see step 2) was subtracted from the observed absorbance in the presence of a given concentration of insulin. Changes in absolute value of this net absorbance were not negligible from one microtiter plate to another, seemingly because of unexpected alteration in the experimental conditions such as concentration of antibody and/or a decrease in activities for insulin receptor kinase. In order for the net absorbances obtained from one microtiter plate to be comparable to those obtained from another, the measured net absorbance for a given

concentration of insulin was normalized against absorbance for 1.0 µM insulin in terms of B/Bmax × 100, where B is the absorbance for each concentration of insulin and Bmax that for 1.0 µM insulin. All measurements for samples were made in triplicate, and the results were expressed as means ( SD. P values were determined by an unpaired Student’s t-test to compare mean values, and P < 0.05 was considered statistically significant. Responses for Vanadium Ions, Tyrphostin 25, and Troglitazone. Responses for vanadate and vanadyl ions of the present sensing system were evaluated by adding each concentration of vanadium ions into a solution consisting of 0.05% Triton X-100, 50 µM ATP, 1.7 mM MnCl2, 50 mM HEPES/NaOH (pH 7.4), and a given concentration of insulin receptor. Note that the solution does not contain insulin. The concentration dependence of vanadate (as a sodium salt) was examined up to 1.0 × 10-4 M. For vanadate at a concentration of 1.0 × 10-4 M vanadate, its major species at pH 7.4 is H2VO4-.24,25 In the case of an antagonist, tyrphostin 25, its response was examined in the presence of a constant concentration (1.0 µM) of insulin; the insulin solution was added to a solution containing 0.05% Triton X-100, 50 µM ATP, 1.7 mM MnCl2, 50 mM HEPES/ NaOH (pH 7.4), and a given concentration of insulin receptor in a microtube. The mixture was incubated for 10 min at 25 °C, and then each concentration of antagonist dissolved in ethanol solution was added to the solution. The final concentration of ethanol was 5.0 (v/v)% of the sample solution. Incubation of the mixture including tyrphostin 25 was performed for 10 min at 25 °C, and then the solution was applied to given wells of the microtiter plate. To examine the effect of troglitazone on kinase activities of the insulin receptor, 20 nM and 20 µM solutions of troglitazone dissolved in 1.0% BSA solution were prepared, and 7.5 µL portions were applied to wells of interest immediately after introduction of the solution containing 0.05% Triton X-100, 50 µM ATP, 1.7 mM MnCl2, a given concentration of insulin receptor, 50 mM HEPES/ NaOH (pH 7.4), and different concentrations of insulin. As a reference absorbance, 1.0% BSA solution without troglitazone was also applied to given wells under otherwise identical conditions. Final concentrations of troglitazone were 0 M, 1.0 nM, and 1.0 µM, respectively. RESULTS AND DISCUSSION Upon binding of insulin with its receptor in biological insulin signaling pathways, the activated insulin receptor is known to phosphorylate at least 9 tyrosine residues in IRS-1.12,13 Y939 peptide used in the present study contains a tyrosine residue at a position of 939 from the N terminal of IRS-1. There is evidence obtained through in vitro experiments that the other synthetic peptides corresponding to amino acid sequences surrounding those tyrosine residues in IRS-1 interact with an insulin-receptor complex having sequence-dependent kinetic constants.26 If these peptides instead of Y939 peptide are immobilized on a microtiter plate, we may observe selective interaction of each peptide with the insulin-receptor complex. (24) Brito, F.; Ingri, N.; Sille´n, L. G. Acta Chem. Scand. 1964, 18, 1557-1558. (25) Brito, F. Acta Chem. Scand. 1967, 21, 1968-1969. (26) Shoelson, S. E.; Chatterjee, S.; Chaudhuri, M.; White, M. F. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 2027-2031.

Figure 3. Insulin-dependent tyrosine phosphorylation of Y939 peptide by addition of sample solutions including insulin receptor in the absence (IR(+) + Insulin(-)) or presence (IR(+) + Insulin(+)) of 1.0 µM insulin, or by the addition of PBS buffer (IR(-) + Insulin(-)). After immobilization of Y939 peptide on the well surface, sample solutions containing each concentration of insulin were added to the well and incubated for 1 h. The amount of phosphorylated Y939 was evaluated by enzyme immunoassay with a horseradish peroxidase label. A little difference in absorbance was found between (IR(+) + Insulin(-)) and (IR(-) + Insulin(-)), but a significantly large difference was found between (IR(+) + Insulin(+)) and (IR(+) + Insulin(-)). The results are the means and standard deviations of three separate experiments (mean ( SD).

(i) Response for Insulin. When aqueous insulin receptor solutions including 50 µM ATP, 1.7 mM Mn2+, and 1.0 µM insulin were added into the well on which a biotin-Y939 conjugate was immobilized, the Y939 peptide was phosphorylated by insulin and its receptor complex. The amount of phosphorylated Y939 peptide was evaluated by immunoassay with an enzyme label: a monoclonal anti-phosphotyrosine antibody labeled with horseradish peroxidase was introduced into the well, which selectively bound to the phosphorylated Y939 peptide and produced Bindschedler’s green products from DA-64 with an absorption maximum at 727 nm. Also, in order to evaluate the background absorbance, a PBS buffer without both insulin and insulin receptor instead of the sample solutions was added into the well under otherwise identical experimental conditions: When this PBS buffer was added to the Y939-immobilized well, absorbance of the DA-64 at 727 nm was 0.006 ( 0.001 (IR(-) + Insulin (-) in Figure 3), while for the cases of the insulin receptor-containing solutions in the absence and presence of insulin, absorbance of DA-64 at 727 nm was 0.013 ( 0.003 (IR(+) + Insulin(-)) and 0.180 ( 0.008 (IR(+) + Insulin(+)), respectively, of which the absorbance for IR(+) + Insulin(+) is significantly larger than the background absorbance. The absorbance without insulin (IR(+) + Insulin(-)) was subtracted from the absorbance (IR(+) + Insulin(+)) for obtaining an absorbance specific for the insulin concentrations. Figure 4 shows the dependence of insulin concentration on absorbance produced by the anti-phosphotyrosine antibody through the enzymatic reaction of horseradish peroxidase. The absorbance was expressed in terms of B/Bmax × 100 (see Experimental Section) as a function of the logarithm of the concentration of insulin. The absorbance increased with increasing concentration of insulin from 1.0 × 10-10 to 1.0 × 10-7 M and leveled off. At insulin concentration levels lower than 1.0 × 10-11 M, no change in the absorbance was observed. The median effective value (ED50 values) for phosphorylation of Y939 peptide was 2.2 × 10-8 M. Comparing the ED50 value with those (1-4 nM) of insulin required for kinase reaction by Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

2349

Figure 4. Dependence of the amount of phosphorylated Y939 peptide on the concentration of insulin ("), vanadate (O), and vanadyl (]), each of which was dissolved in 0.05% Triton X-100, 50 mM ATP, 1.7 mM MnCl2, 50 mM HEPES buffer (pH 7.4), and a given concentration of insulin receptor. The concentration of insulin and the vanadium ions ranged from 1.0 × 10-13 to 1.0 × 10-6 M and from 1.0 × 10-11 to 1.0 × 10-4 M, respectively.

insulin receptor to endo- and exogenous substrates such as IRS-1 and random copolymers of (Glu80Tyr20)n in vitro,27,28 the ED50 value in the present system was ∼10 times higher than that for in vitro experiments. The higher ED50 value may be due to biotin-Y939 peptide’s being directly immobilized on the surface, which possibly hindered sterically the interaction between insulin receptor and Y939 peptide. The detection limit for insulin, defined as the concentration that gives an absorbance of 3 times the standard deviation of the background signal, was 1.0 × 10-10 M. The detection limit for the present insulin sensing system was comparable to that of competitive immunoassay for insulin29 but inferior to that of enzyme-linked two-site immunoassay for insulin.30,31 The precision of the measured values for varying concentrations of insulin was evaluated in terms of coefficient of variation (% CV) defined as the standard deviation expressed as percentage of the mean value. The CV values from 1.0 × 10-11 to 1.0 × 10-6 M insulin were 39.0%, 73.8%, 28.9%, 29.4%, 28.6%, and 23.4%, respectively. It should be noted that the background absorbance with the PBS buffer (IR(-) + Insulin(-)) was 0.003 ( 0.000, almost identical to that obtained upon addition of PBS buffer without immobilization of biotin-Y939 peptide (data not shown). This result indicates that phosphotyrosine antibody did not nonspecifically adsorb on the biotin-Y939 peptide in the present experimental conditions. Compared to this absorbance, the absorbance for the sample solution in the absence of insulin (IR(+) + Insulin(-)) was slightly higher; this is due to kinase reaction by insulin receptor in the absence of insulin. As shown in Figure 2, a timedependent moderate increase is also observed in phosphorylation by insulin receptor in the absence of insulin when a random copolymer of (Glu80Tyr20)n was used as a substrate for insulin (27) Wilden P. A.; Broadway, D. E. J. Cell. Physiol. 1995, 163, 9-18. (28) Lima, F. B.; Thies, R. S.; Garvey, W. T. Endocrinology 1991, 128, 24152426. (29) Schultz, N. M.; Huang, L.; Kennedy, R. T. Anal. Chem. 1995, 67, 924929. (30) Anderson, L.; Dinesen, B.; Jørgensen, P. N.; Poulsen, F.; Røder, M. E. Clin. Chem. 1993, 39, 578-582. (31) Kratzsch, J.; Ackermann, W.; Keilacker, H.; Besch, W.; Keller, E. Exp. Clin. Endocrinol. 1990, 95, 229-236.

2350 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

receptor. This strongly suggests that insulin-independent phosphorylation of Y939 peptide by insulin receptor proceeds to a certain extent, though its level is still much lower than that of the kinase reaction by insulin receptor in the presence of insulin (vide supra). (ii) Responses for Vanadium Ions. The effect of vanadate and vanadyl ions (as sodium salts) on the tyrosine-kinase activity of insulin receptor was examined as described in the Experimental Section. The results obtained are shown in Figure 4. No change in the absorbance was observed even at vanadyl and vanadate ion concentrations up to 1.0 × 10-4 M. These results indicate that the vanadyl and vanadate ions did not activate insulin receptortyrosine kinase, inducing no absorbance signals. An insulin-like effect of vanadate ion has been found to stimulate glucose uptake into adipocytes32,33 and hepatocytes in rats,34 glycogen synthesis in rats35,36 and glycolysis.32,33 Several groups, however, have reported that no significant increase was observed in the phosphotyrosine content of the insulin receptor in vanadate-treated cells or tissues,37-41 though tyrosine phosphorylation of IRS-1 is an essential event for activating the glucose uptake and its metabolism. Furthermore, it has been revealed that vanadate can be reduced intracellularly to vanadyl in those cell types.42,43 From these observations, it has been concluded that both vanadate and vanadyl ions are incapable of activating insulin receptor-tyrosine kinase, but each ion affects insulin receptor-independent pathways such as inhibition of phosphotyrosine phosphatase (PTPase) by vanadate and/or increases in cytosolic (nonreceptor) protein-tyrosine kinase (cytPTK) activities by vanadyl.44-46 Considering the vanadium ion effects, the results obtained by the present sensing system are in accordance with the ones reported as above. In view of the above considerations, it is concluded that the present method provides a simple new approach for evaluating “physiologically more relevant” agonist selectivity for the insulin signaling pathway. (iii) Response for Tyrphostin 25. In order to show a possibility of evaluating antagonist selectivities by our present sensing system, tyrphostin 25, which is a specific inhibitor for kinase activities of insulin receptor, was added to a sample solution containing 1.0 µM insulin under otherwise identical conditions. The results obtained are shown in Figure 5. The magnitude of absorbance decreased with increasing concentrations of tyrphostin (32) Dubyak, G. R.; Kleinzeller, A. J. Biol. Chem. 1980, 255, 5306-5312. (33) Shechter, Y.; Karlish, S. J. D. Nature 1980, 284, 556-558. (34) Miralpeix, M.; Gil, J.; Rosa, J. L.; Carreras, J.; Bartrons, R. Life Sci. 1989, 44, 1491-1497. (35) Tamura, S.; Brown, T. A.; Dubler, R. E.; Larner, J. Biochem. Biophys. Res. Commun. 1983, 113, 80-86. (36) Strout, H. V.; Vicario, P. P.; Saperstein, R.; Slater, E. E. Endocrinology 1989, 124, 1918-1924. (37) D’Onofrio, F.; Le, M. Q.; Chiassion, J. L.; Srivastava, A. K. FEBS Lett. 1994, 340, 269-275. (38) Shisheva, A.; Shechter, Y. J. Biol. Chem. 1993, 268, 6463-6469. (39) Shisheva, A.; Shechter, Y. FEBS Lett. 1992, 300, 93-96. (40) Mooney, R. A.; Bordwell, K. L.; Luhowskyj, S.; Casnellie, J. E. Endocrinology 1989, 124, 422-429. (41) Green, A. Biochem. J. 1986, 238, 663-669. (42) Willsky, G. R.; White, D. A.; McCabe, B. C. J. Biol. Chem. 1984, 259, 1327313281. (43) Degani, H.; Gochin, M.; Karlish, S. J. D.; Shechter, Y. Biochemistry 1981, 20, 5795-5799. (44) Sekar, N.; Li, J.; Shechter, Y. Crit. Rev. Biochem. Mol. Biol. 1996, 31, 339359. (45) Shechter, Y.; Shisheva, A. Endeavour 1993, 17, 27-31. (46) Shechter, Y. Diabetes 1990, 39, 1.

Figure 5. Inhibitory effect of tyrphostin 25 on the amount of phosphorylated Y939 peptide. Tyrphostin 25 was dissolved in a sample solution containing 1.0 µM insulin, 0.05% Triton X-100, 50 µM ATP, 1.7 mM MnCl2, 50 mM HEPES buffer (pH 7.4), and a given concentration of insulin receptor. The sample solution was added to each of the Y939-immobilized wells and incubated for 1 h. The amount of phosphorylated Y939 peptide was measured by enzyme immunoassay with a horseradish peroxidase label.

25 over 1.0 × M, and it leveled off at concentrations higher than 4.0 × 10-3 M. At 1.0 × 10-5 M tyrphostin 25, no inhibitory effect was observed. The effect of inhibition by tyrphostin 25 on the insulin receptor kinase toward (Glu80Tyr20)n as a substrate has been studied extensively by measuring 32P Cerenkov radiation incorporated in the substrates by the receptor kinase reactions.47 It was found that tyrphostin 25 has an affinity toward the substrate site of the insulin receptor kinase domain and competes with its substrates. Our result, together with reported ones, concludes that the tyrphostin 25 binds competitively with Y939 peptide at the receptor kinase domain and inhibits its kinase reaction with the peptide. This also demonstrates that the present sensing system exhibits “physiologically relevant” antagonist selectivity for the insulin signaling pathway. (iv) Effect of Troglitazone on Insulin Signaling Pathway. Troglitazone is a new oral hypoglycemic agent for non-insulin dependent diabetes mellitus (NIDDM).48 Studies with rat fat cells and rat-1 fibroblasts transfected with human insulin receptor have suggested that hyperglycemia reduces the insulin receptor kinase activity and troglitazone in the presence of insulin is capable of increasing the reduced tyrosine kinase activity.49,50 To examine whether troglitazone directly affects insulin receptor kinase activity, we examined the effect of troglitazone on phosphorylation of Y939 peptide by the insulin receptor extracted from normal human placenta. The results are shown in Figure 6. Upon addition of 1.0% BSA solution without troglitazone into a sample solution containing each concentration of insulin as a reference signal, phosphorylated Y939 peptide started to increase from an insulin concentration of 2.5 × 10-10 M and then sharply increased over 2.5 × 10-9 M. Upon addition of 20 nM or 20 µM troglitazone 10-4

(47) Gazit, A.; Yaish, P.; Gilon, C.; Levitzki, A. J. Med. Chem. 1989, 32, 23442352. (48) Yoshioka, T.; Fujita, T.; Kanai, T.; Aizawa, Y.; Kurumada, T.; Hasegawa, K. J. Med. Chem. 1989, 32, 421-428. (49) Mu ¨ llaer, H. K.; Kellerer, M.; Ermel, B.; Mu ¨ hlho¨fer, A.; Obermaier-Kusser, B.; Vogt, B.; Haring, H. U. Diabetes 1991, 40, 1440-1447. (50) Kellerer, M.; Kroder, G.; Tippmer, S.; Berti, L.; Kiehn, R.; Mosthaf, L.; Ha¨ring, H. U. Diabetes 1994, 43, 447-453.

Figure 6. Dose-response curve of phosphorylation of Y939 peptide by insulin-receptor complex in the presence of 1.0 nM (O) and 1.0 µM (]) troglitazone and in its absence ("). Troglitazone dissolved in 1.0% BSA solution was added to each well after sample solution was added to each well immobilized on the Y939 peptide. Phosphorylated Y939 peptide was analyzed by enzyme immunoassay with a horseradish label.

dissolved in 1.0% BSA solution to the sample solutions to final concentrations of 1.0 nM and 1.0 µM, phosphorylated Y939 peptide similarly increased with increasing concentration of insulin. The P values for each concentration of insulin were 0.59, 0.87, 0.23, 0.14, 0.82 and 0.82, for 1.0 nM troglitazone and 0.53, 0.66, 0.46, 0.50, 0.84, and 0.61 for 1.0 µM troglitazone, respectively, showing that there is no statistically significant difference between the signals in the absence and in the presence of troglitazone. These results conclude that troglitazone does not enhance the kinase activities of the insulin receptor in the concentration range examined. The human insulin receptor is known to exist in two functionally different isoforms which differ by the absence (type A) or presence (type B) of 12 amino acids in the COOH terminus of the R-subunit.51 There is evidence that, in the case of human placenta of normal subjects, the two isoforms are equally expressed, but the expression of type B isoform, of which binding ability and kinase activity are 2 times lower than those of the type A isoform, is significantly increased with increasing glucose levels and body mass indexes (defined as [weight]/[height]2).51 Mosthaf et al. have reported that the altered expression of insulin receptor types A and B correlates with NIDDM subjects.52,53 Our result obtained with normal human placenta shows that troglitazone does not enhance the kinase activities of both type A and type B isoforms; lower kinase activity of the type B form of insulin receptor does not increase by incubation of insulin receptor with troglitazone. This implies that troglitazone does not act on the altered expression of the isoforms with NIDDM but may regulate intracellular post-insulin receptor signaling pathways. CONCLUSION A new method for evaluating agonist and antagonist selectivity in the insulin signaling pathway was developed based on the extent of phosphorylation of its target Y939 peptide upon selective (51) Sesti, G.; D’Alfonso, R.; Punti, M. D. V.; Frittitta, L.; Liu, Y. Y.; Borboni, P.; Longhi, R.; Montemurro, A.; Lauro, R. Diabetologia 1995, 38, 445-453. (52) Mosthaf, L.; Eriksson, J.; Ha¨ring, H. U.; Groop, L.; Wide´n, E.; Ullrich, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 2633-2635. (53) Mosthaf, L.; Vogt, B.; Ha¨ring, H. U.; Ullrich, A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4728-4730.

Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

2351

recognition of insulin by insulin receptor. The insulin-switched phosphorylation of Y939 peptide was detected by anti-phosphotyrosine antibody labeled with horseradish peroxidase. The amount of phosphorylated Y939 peptide depended on insulin concentration, the result of which confirmed the sequential transduction mechanism based on the insulin signaling pathway. The present assay method also revealed that troglitazone does not directly act on both insulin receptor type A and type B. The present approach may provide a means of screening agonistlike drugs for the insulin signaling pathway from hundreds of candidate pharmaceutical samples; there has so far been found no drug that directly increases insulin receptor kinase activities. The general applicability of the present approach could be improved if the insulin receptor was extracted from cultured cells expressing a large amount of insulin receptor.

2352 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

ACKNOWLEDGMENT We are grateful to Prof. Yoshinori Kuwabara, Department of Obstetrics and Gynecology, Juntendo University School of Medicine (Tokyo, Japan), for kindly providing us with sample tissue of human placenta needed for extracting insulin receptor. We thank D. Kuboshima for his experimental help in insulin receptor isolation. We are indebted to Prof. Asaya Kobashi, School of Science, The University of Tokyo, for direction of the use of radioisotopes. This work was financially supported from Grants for Scientific Research by the Ministry of Education, Science and Culture, Japan. Received for review October 27, 1997. Accepted March 19, 1998. AC971192S