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β2-Adrenergic Receptor Desensitization, Internalization, and Phosphorylation in Response to Full and Partial Agonists

1997; Elsevier BV; Volume: 272; Issue: 38 Linguagem: Inglês

10.1074/jbc.272.38.23871

1083-351X

Bridgette January, Anita Seibold, Brenda S. Whaley, R. William Hipkin, Doris Lin, Agnes Schönbrunn, Roger Barber, Richard B. Clark,

Hormonal Regulation and Hypertension

Previous studies indicated that partial agonists cause less desensitization of the β2-adrenergic receptor (βAR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of βAR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the βAR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified βAR behaved identically to the wild type receptor with regard to agonistK d, coupling efficiency, and desensitization. The coupling efficiencies for βAR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0–30 min) of agonist-induced desensitization of βAR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine ≥ fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the βAR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, βAR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11–13-fold increases in the level of βAR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3–4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of βAR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 ± 0.82) was not appreciably different from ephedrine (5.0 ± 0.96) and significantly less than that caused by albuterol (10.4 ± 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of βARK phosphorylation of the βAR. Previous studies indicated that partial agonists cause less desensitization of the β2-adrenergic receptor (βAR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of βAR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the βAR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified βAR behaved identically to the wild type receptor with regard to agonistK d, coupling efficiency, and desensitization. The coupling efficiencies for βAR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0–30 min) of agonist-induced desensitization of βAR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine ≥ fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the βAR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, βAR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11–13-fold increases in the level of βAR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3–4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of βAR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 ± 0.82) was not appreciably different from ephedrine (5.0 ± 0.96) and significantly less than that caused by albuterol (10.4 ± 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of βARK phosphorylation of the βAR. The adenylyl cyclase-coupled βAR 1The abbreviations used are: βAR, β2-adrenergic receptor; βARK, βAR kinase; PKA, cAMP-dependent protein kinase; TACT,N,N′,N′′-triacetylchitotriose;125ICYP, [125I]iodocyanopindolol; HA, hemagglutinin; HA6His, HA- and 6-His-modified receptor; PBS, phosphate-buffered saline. system has served as a model system for the study of the phenomenon of desensitization of G-protein-coupled receptors (1Perkins J.P. Hausdorff W.P. Lefkowitz R.J. Perkins J., P. The β-Adrenergic Receptors. The Humana Press Inc., Clifton, NJ1991: 73-124Google Scholar, 2Dixon R.A.F. Sigal I.S. Strader C.D. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 487-489Crossref PubMed Google Scholar, 3Palczewski K. Benovic J.L. Trends Biol. Sci. 1990; 16: 387-392Abstract Full Text PDF Scopus (187) Google Scholar, 4Clark R.B. J. Cyclic Nucleotide and Protein Phosphor. Res. 1986; 20: 151-209Google Scholar, 5Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (474) Google Scholar). Desensitization is functionally defined as an attenuation of hormonal responsiveness upon agonist stimulation. There are four currently known mechanisms of agonist-induced desensitization that appear to have physiological significance: receptor sequestration/internalization (1Perkins J.P. Hausdorff W.P. Lefkowitz R.J. Perkins J., P. The β-Adrenergic Receptors. The Humana Press Inc., Clifton, NJ1991: 73-124Google Scholar), βAR kinase (βARK) phosphorylation of serines and threonines on the βAR C terminus (3Palczewski K. Benovic J.L. Trends Biol. Sci. 1990; 16: 387-392Abstract Full Text PDF Scopus (187) Google Scholar, 5Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (474) Google Scholar), cAMP-dependent protein kinase (PKA) phosphorylation of βAR serine 261 or 262 (6Yuan N. Friedman J. Whaley B.S. Clark R.B. J. Biol. Chem. 1994; 269: 23032-23038Abstract Full Text PDF PubMed Google Scholar, 7Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar, 8Clark R.B. Friedman J. Dixon R.A.F. Strader C.D. Mol. Pharmacol. 1989; 36: 343-348PubMed Google Scholar), and down-regulation of the βAR (1Perkins J.P. Hausdorff W.P. Lefkowitz R.J. Perkins J., P. The β-Adrenergic Receptors. The Humana Press Inc., Clifton, NJ1991: 73-124Google Scholar). Two key factors governing which of these mechanisms predominate are the concentration of agonist and the time of exposure to agonist. Short term exposure to low concentrations of agonist in several different cell lines causes a PKA-mediated desensitization that exhibits a t½ of 1–3 min (9Clark R.B. Kunkel M.W. Friedman J. Goka T.J. Johnson J.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1442-1446Crossref PubMed Scopus (106) Google Scholar, 10Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar). Down-regulation, which does not contribute to rapid desensitization, also occurs in response to low concentrations of agonist (11Proll M.A. Clark R.B. Goka T.J. Barber R. Butcher R.W. Mol. Pharmacol. 1992; 42: 116-122PubMed Google Scholar); however, the t½ is on the order of 50–100 times greater than for the rapid events. In response to high concentrations of full agonists that result in high receptor occupancy, the βAR is rapidly desensitized through βARK-mediated phosphorylation and β-arrestin binding, phosphorylation by PKA, and internalization (1Perkins J.P. Hausdorff W.P. Lefkowitz R.J. Perkins J., P. The β-Adrenergic Receptors. The Humana Press Inc., Clifton, NJ1991: 73-124Google Scholar, 3Palczewski K. Benovic J.L. Trends Biol. Sci. 1990; 16: 387-392Abstract Full Text PDF Scopus (187) Google Scholar, 5Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (474) Google Scholar). Along with these factors that influence the pathways of desensitization, several preliminary studies have indicated that partial βAR agonists may cause less desensitization in intact cells than full agonists. Su et al. (12Su Y.-F. Harden T.K. Perkins J.P. J. Biol. Chem. 1980; 255: 7410-7419Abstract Full Text PDF PubMed Google Scholar) showed that the partial agonists zinterol and soterenol caused a small reduction in βAR desensitization relative to isoproterenol in astrocytoma cells. However, this study did not address which mechanism(s) mediating desensitization were affected. We found that the partial agonist salmeterol had a reduced capacity to desensitize the βAR (13Clark R.B. Allal C. Friedman J. Johnson M. Barber R. Mol. Pharmacol. 1996; 49: 182-189PubMed Google Scholar) and that the partial agonist albuterol caused significantly less internalization than the full agonist epinephrine (14Morrison K.J. Moore R.H. Carsrud N.D.V. Trial J. Millman E.E. Tuvim M. Clark R.B. Barber R. Dickey B.F. Knoll B.J. Mol. Pharmacol. 1996; 50: 692-699PubMed Google Scholar). In summary, it appears that while partial βAR agonists may cause less desensitization, little has been accomplished in terms of defining the desensitization mechanisms altered following treatment of intact cells with partial agonists and in quantitating intact cell desensitization, internalization, and phosphorylation. Further, no attempt has been made to correlate a quantitative measure of the coupling efficiency of partial agonists with these parameters (7Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar). In the present work, we have investigated the desensitization, internalization, and phosphorylation of a double epitope-modified human βAR expressed in HEK-293 cells in response to a series of partial agonists and compared these parameters to the full agonist epinephrine. The agonists we selected provide a 95-fold range of agonist coupling efficiencies. We have found that there is a strong correlation between the coupling efficiencies of agonists and their ability to induce desensitization, internalization, and initial rates of phosphorylation of the βAR. Based on these data, we propose that the reduced desensitization observed with partial agonists results from a decrease in the rate of βARK-mediated phosphorylation and internalization. ATP, alprenolol, β-mercaptoethanol, poly-l-lysine, andN,N′,N′′-triacetylchitotriose (TACT) were obtained from Sigma. [α-32P]ATP and [32P]H3PO4 were obtained from NEN Life Science Products. β-Agonists included (−)-epinephrine bitartrate, albuterol hemisulfate, dobutamine hydrochloride, and ephedrine hydrochloride obtained from Research Biochemicals International (RBI). GTP was obtained from Boehringer Mannheim. Nickel-nitrilotriacetic acid-agarose was obtained from Qiagen. Wheat germ agglutinin-agarose was obtained from Vector Laboratories.n-Dodecyl-β-d-maltoside was obtained from Calbiochem. All electrophoresis reagents and standards were obtained from Bio-Rad. [125I]Iodocyanopindolol (125ICYP) was prepared as described previously (16Barovsky K. Brooker G. J. Cyclic Nucleotide Res. 1980; 6: 297-307PubMed Google Scholar) and modified by Hoyer et al. (17Hoyer D.E. Reynolds E. Molinoff P.B. Mol. Pharmacol. 1984; 25: 209-218PubMed Google Scholar). Wild type human βAR cDNA, generously provided by Dr. Robert Lefkowitz (Duke), and a human βAR cDNA, modified by placement of the hemagglutinin (HA) epitope between the second and third amino acids on the N terminus, kindly provided by Dr. Brian Kobilka (Stanford), were cloned into the expression vector pBC12B1 (18von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar). We introduced six histidine residues, followed by a stop codon, into the C terminus just after the last two amino acids (Leu-Leu) of the HA-modified βAR cDNA to construct the βAR that was transfected into HEK cells. The 6-His modification was introduced by the polymerase chain reaction method, and the receptor was sequenced to check for errors in the polymerase chain reaction and verify the alterations. We have determined that the HA and 6-His epitope tags do not significantly modify the ability of this βAR to bind ligand, couple to Gs and activate adenylyl cyclase, or desensitize when exposed to β-agonist (data not shown). HEK-293 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C in 5% CO2. The expression vector pBC12B1 containing either the HA- and 6-His-modified receptor (HA6His) or the wild type receptor (wild type βAR) was transfected into HEK-293 cells, along with the neomycin-expressing plasmid, pSV2neo, by calcium phosphate co-precipitation in a mass ratio of 100:1. Cells were shocked the next day with 25% glycerol for 1 min, and the following day were split into 96-well dishes into selection media, Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and Geneticin at 400 μg/ml. Stable transformants expressing receptor were identified using an intact cell [3H]CGP-12177 binding assay, described below, and cloned by limiting dilution. Several HA6His and wild type βAR clones were isolated, and one of these HA6His clones expressing 2–3 pmol of βAR, was selected for the experiments performed herein. For desensitization experiments, preconfluent HA6His cells in 150-mm dishes were pretreated with various agents or appropriate control buffers in growth medium, and incubated for 0.5–30 min at 37 °C, as described in the figure legends. To prevent oxidation, all βAR agonists were dissolved in aqueous solution containing final concentrations of 0.1 mmascorbate and 1 mm thiourea (AT). Membranes were prepared as described previously (7Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar) except that after pretreatment cells were washed six times with ice cold HME buffer (20 mm Hepes, pH 8, 2 mm MgCl2, 1 mm EDTA, 1 mm benzamidine, 2 mm tetrasodium pyrophosphate, 10 μg/ml trypsin inhibitor, and 0.1 mg/ml bovine serum albumin). Adenylyl cyclase activity in the membrane preparations was measured as described previously (7Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar) with the free Mg2+ concentration set at 0.3 mm. The final concentration of ATP in the assay was 0.1 mm. All incubations were performed at 30 °C. GraphPad software was used to perform dose response curve fitting and to determine EC50and V max values. Surface levels of the βAR were determined by the following procedure. HA6His cells were plated onto 35-mm dishes coated with 10 μg/ml poly-l-lysine. Cells were pretreated with agonist or AT, in triplicate, at 37 °C for 0.5–30 min. Following incubation with agonist, the medium was removed and cells were washed six times with 2 ml of ice-cold phosphate-buffered saline (PBS). 1.25 ml of serum-free Dulbecco's modified Eagle's medium containing hydrophilic antagonist [3H]CGP-12177 at a concentration of 5 nm was added to each plate and incubated for 1 h on ice, which we determined was sufficient for maximum radioligand binding. Nonspecific binding was measured by the addition of 1 μm alprenolol to the [3H]CGP-12177. Radioligand binding was terminated by three washes with 2 ml of ice-cold PBS. The cells were removed by the addition of 750 μl of 0.25% trypsin, 0.02% EDTA, PBS. [3H]CGP binding was quantitated by scintillation counting. To determine the amount of βAR expressed on the cell surface under control conditions, [3H]CGP binding was performed in the presence and absence of 0.2% digitonin (19Slowiejko D.M. Levey A., I. Fisher S.K. J. Neurochem. 1994; 62: 1795-1803Crossref PubMed Scopus (20) Google Scholar). This method was also used to measure total receptor levels after agonist pretreatment. The K d value for agonist binding to the βAR was determined by displacement of 20–40 pm125ICYP in the presence of 10 μm GTP in membranes (6Yuan N. Friedman J. Whaley B.S. Clark R.B. J. Biol. Chem. 1994; 269: 23032-23038Abstract Full Text PDF PubMed Google Scholar, 7Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar). Nonspecific binding was assessed by inclusion of 1 μm alprenolol, and data analysis was performed using GraphPad software. TheK d for all of the agonists was identical (within the limits of experimental error, ±30%) for cells overexpressing either the wild type or HA6His βAR in the parental HEK- 293 cells (data not shown). Receptor number (B max) and the K d for hydrophobic antagonist 125ICYP were determined by Scatchard analysis of 125ICYP binding to membranes as described previously (6Yuan N. Friedman J. Whaley B.S. Clark R.B. J. Biol. Chem. 1994; 269: 23032-23038Abstract Full Text PDF PubMed Google Scholar, 7Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar) with the following modifications. The concentration of 125ICYP was varied from 1 to 200 pm, and radioligand was diluted in HE buffer (20 mm Hepes, pH 8, 1 mm EDTA) plus 50 mm phentolamine, 0.1 mm ascorbate, and 1 mm thiourea. βAR levels in the parental HEK cell line were 5–10 fmol/mg of membrane protein, and in the HA6His cells they were between 2 and 3 pmol/mg. HA6His cells were grown to confluency in 100-mm dishes coated with 10 μg/ml poly-l-lysine. Cells were rinsed three times with 5 ml of phosphate-free buffer (40 mm Hepes, 5.4 mm KCl, 122 mm NaCl, 0.8 mm MgSO4, 1.8 mm CaCl2, 5.4 mm glucose, pH 7.4) followed by labeling with approximately 0.5 mCi of [32P]H3PO4 in phosphate-free Dulbecco's modified Eagle's medium for 3 h. 32P uptake, monitored by measuring extracellular 32P, reached a maximum by 2 h and averaged 92% by 3 h. Cells were then treated with concentrations of agonists yielding >90% occupancy for varying times. Agonist incubations were stopped by three washes with ice-cold PBS. Cells were then scraped in PBS containing 10 μg/ml leupeptin and 100 nm okadaic acid, and pelleted by centrifugation at 600 × g for 5 min. Cell pellets were solubilized by resuspension with a 23-gauge needle in solubilization buffer (20 mm Hepes, pH 7.4, 0.8%n-dodecyl-β-d-maltoside, 150 mmNaCl, 5 mm EDTA, 3 mm EGTA, 20 mmtetrasodium pyrophosphate, 0.1 mmNa3VO4, 10 μg/ml benzamidine, 10 μg/ml leupeptin, 10 μg/ml trypsin inhibitor, 100 nm okadaic acid, and 14 mm β-mercaptoethanol) and incubated for 1 h at 4 °C on a rocking apparatus. The solubilized extracts were cleared by centrifugation at 100,000 × g for 30 min. The solubilized extracts were subjected to a two-step purification procedure. First, extracts were partially purified by utilizing the βAR C-terminal 6-His extension. Nickel-nitrilotriacetic acid-agarose was prepared by one wash with 100 mm NiSO4followed by two washes with nickel column buffer (0.05%n-dodecyl-β-d-maltoside, 20 mmHepes, pH 7.4, 150 mm NaCl). Solubilized extracts were then loaded onto columns containing 250 μl of packed nickel resin, which allowed us to achieve maximum βAR binding to the resin (>90%). Columns were washed with 70 volumes of nickel column buffer containing 5 mm imidazole. Washes were followed by competitive elution of the βAR with a step gradient of imidazole (1.5 ml/fraction), which extended from 10 to 50 mm. Our βAR elution peak fractions extended from 25 to 45 mm imidazole with approximately 80% specific recovery (as determined by 125ICYP binding). Fractions eluted in 35–50 mm imidazole were collected and frozen overnight before proceeding to the next purification step. Further purification was achieved by lectin chromatography using wheat germ agglutinin-agarose that was first prepared by two 10-min washes with nickel column buffer. 100 μl of packed resin was added to the combined 35–50 mm imidazole nickel column fractions and allowed to incubate for 90 min at 4 °C on a rocking apparatus. Wheat germ pellets were then washed three times with 50 volumes of nickel column buffer for 10 min at 4 °C. At this point, aliquots were taken from selected experiments to quantitate receptor level by125ICYP binding. Elution of the βAR was achieved by incubation with 3 mm TACT in 0.5% SDS for 1 h at 37 °C, followed by a second TACT/SDS elution for 30 min at 37 °C. The eluates were combined and mixed with an equal volume of 2 × SDS sample buffer (100 mm Tris, pH 6.8, 4% SDS, 45% glycerol, 0.05% bromphenol blue, 6 m urea, 143 mm β-mercaptoethanol) and incubated for 15 min at 60 °C. SDS-polyacrylamide gel electrophoresis was performed using 7.5% polyacrylamide gels. Gels were dried, and 32P was quantitated by using a Molecular Dynamics PhosphorImager model 860 and ImageQuant software. We were able to convert gel 32P area/volume quantitation by ImageQuant software to cpm by spotting and quantitating a known amount of 32P. Within an individual experiment where independent samples were processed in triplicate the S.E. was minimal. For example, in one experiment, the 32P cpm value for a 5-min treatment with 10 μm epinephrine (n = 3) was 265 ± 8.5. To confirm the identity of the 32P-labeled βAR, after SDS-polyacrylamide gel electrophoresis the protein was transferred to a polyvinylidene difluoride membrane and subjected to 32P quantitation followed by immunoblot analysis as described (15Hipkin R.W. Friedman J. Clark R.B. Eppler C.M. Schonbruun A. J. Biol. Chem. 1997; 272: 13869-13876Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) using a primary rabbit polyclonal anti-HA antibody from Berkeley Antibody Company (Babco) and a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Immunoblot and 23P images of the βAR were identical in molecular weight, confirming the identity of the βAR and also demonstrating that total receptor levels did not change with agonist pretreatment. We have verified experimentally that the following equation describes relative coupling efficiency of the agonist stimulation of adenylyl cyclase over a 1000-fold range of βAR expression levels (6Yuan N. Friedman J. Whaley B.S. Clark R.B. J. Biol. Chem. 1994; 269: 23032-23038Abstract Full Text PDF PubMed Google Scholar, 7Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar). k1k−1=Kd−EC50(EC50)rEquation 1 EC50 is the concentration of agonist required for half-maximal stimulation of adenylyl cyclase activity,K d is the binding constant for the agonist and βAR, k 1 is the rate constant for activation of adenylyl cyclase through βAR/Gs coupling induced by agonist binding, k −1 is the rate constant for inactivation of adenylyl cyclase, and r is total receptor number. Because k −1 is a first order rate constant that is independent of receptor and agonist levels, it is assumed to be invariant, and for all calculations it has been set to 1. Therefore, any changes in thek 1/k −1 ratio can be attributed to k 1, and k 1can be defined as coupling efficiency. This equation was used to calculate the coupling efficiencies given in Table I.Table ICoupling efficiencies of βAR agonistAgonistK d (+GTP)EC50V maxCoupling efficiencyCalculatedPercentage of epinephrinenmnmpmol/min/mg%Epinephrine653 ± 1231.7 ± 0.257 ± 4.90.16100Fenoterol126 ± 80.79 ± 0.353 ± 5.10.0742Albuterol528 ± 9928 ± 3.148 ± 4.30.0084.9Dobutamine697 ± 5265 ± 1137 ± 4.60.0042.5Ephedrine2830 ± 466565 ± 10131 ± 3.60.0021.1The EC50 and V max values for the activation of adenylyl cyclase activity in HA6His cells by β-agonists are listed and are the mean ± S.E., n = 3. Membranes were prepared from HA6His cells as described under "Experimental Procedures." HA6His cells expressed 2300 fmol of βAR/mg of membrane protein. The K d values were calculated from agonist displacement of 125ICYP and are the mean of two experiments with the exception of albuterol (n = 6). The EC50 and V maxvalues were determined by dose responses of agonist stimulation of adenylyl cyclase (n = 3). The coupling efficiencies were calculated using Equation 1. Open table in a new tab The EC50 and V max values for the activation of adenylyl cyclase activity in HA6His cells by β-agonists are listed and are the mean ± S.E., n = 3. Membranes were prepared from HA6His cells as described under "Experimental Procedures." HA6His cells expressed 2300 fmol of βAR/mg of membrane protein. The K d values were calculated from agonist displacement of 125ICYP and are the mean of two experiments with the exception of albuterol (n = 6). The EC50 and V maxvalues were determined by dose responses of agonist stimulation of adenylyl cyclase (n = 3). The coupling efficiencies were calculated using Equation 1. As seen from the equation for coupling efficiency, the ability of an agonist to stimulate adenylyl cyclase activation is dependent on both the coupling efficiency (k 1) and receptor number (r) on the cell surface. The total capacity of the βAR to activate adenylyl cyclase may be expressed ask 1 r, which we define as thecoupling capacity of the agonist-bound βAR at a given receptor density. We have previously shown (7Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar) that coupling capacity is defined by the equation (k 1)r= (V max)(K d) /(V 100)(EC50), where V max is the maximum adenylyl cyclase activity observed for saturating concentrations of agonist, andV 100 is the theoretical value representing adenylyl cyclase activity when k 1 is infinite. The change in (k 1)r that occurs as desensitization proceeds can be used as a measure of the extent of desensitization. This is expressed as the ratio of (k 1)r for the desensitized receptor (D) to the naive receptor (N). BecauseV 100 and K d are the same for the desensitized and naive receptor, (k 1 r) D /(k 1 r) N can be expressed in the following form, which we define as thefraction of βAR activity remaining (7Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar, 20Whaley B.S. Yuan N. Barber R. Clark R.B. Pharmacol. Commun. 1995; 6: 203-210Google Scholar). *k1r*D*k1r*N=*Vmax*D*EC50*N*Vmax*N*EC50*DEquation 2 This calculation can be converted to percentage of desensitization by multiplying the fraction of βAR activity remaining by 100 and subtracting that value from 100. This formulation is essentially identical to the empirical equation previously reported by Pippig et al. (21Pippig S. Andexinger S. Daniel K. Puzicha M. Caron M.G. Lefkowitz R.J. Lohse M.J. J. Biol. Chem. 1993; 268: 3201-3208Abstract Full Text PDF PubMed Google Scholar) for the quantitative evaluation of desensitization. Fig. 1 Agraphically depicts the ability of various agonists to stimulate adenylyl cyclase activity and shows typical V maxand EC50 values. Fig. 1 B shows data from Fig.1 A normalized to the individual agonistK d. This normalization makes it easier to visualize the EC50/K d ratio for each agonist (15Hipkin R.W. Friedman J. Clark R.B. Eppler C.M. Schonbruun A. J. Biol. Chem. 1997; 272: 13869-13876Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar);i.e. for poorer agonists the EC50 approaches theK d, and the EC50/K d ratio approaches 1 (7Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar, 15Hipkin R.W. Friedman J. Clark R.B. Eppler C.M. Schonbruun A. J. Biol. Chem. 1997; 272: 13869-13876Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Values for coupling efficiency give a quantitative measure of the ability of the agonist-bound βAR to stimulate adenylyl cyclase activity. The ranges ofK d values, EC50 values,V max values, and coupling efficiencies determined for the agonists used in this study are given in TableI. Our calculations indicate that the coupling efficiency for epinephrine stimulation of the double epitope-modified βAR is 0.16, and this value is identical to that found for the unmodified wild type receptor (data not shown), demonstrating that the double epitope modifications do not alter coupling efficiency. The coupling efficiencies determined for the other agonists relative to epinephrine were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. These agonists provided a 95-fold range of coupling efficiencies and were used to determine the relationship of coupling efficiency to agonist-induced desensitization, internalization, and phosphorylation. To determine the rate of desensitization, HA6His cells were pretreated for various times (0–30 min) with a concentration of each agonist that was calculated to give >90% occupancy of the βAR. Following the pretreatments, membranes were prepared, and the dose responses for epinephrine stimulation of adenylyl cyclase were measured. Fig. 2 A shows the result of a typical experiment in which HA6His cells were pretreated with 10 μm epinephrine from 1 to 5 min. The pretreatment caused a progressive increase in