FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080451044> ?p ?o ?g. }

• W2080451044 endingPage "7642" @default.
• W2080451044 startingPage "7637" @default.
• W2080451044 abstract "Tentative identification of the G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the β2-adrenergic receptor (βAR) was recently reported based on in vitro phosphorylation of recombinant receptor (Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13796–13803). Phosphorylated residues identified for GRK2 were threonine 384 and serines 396, 401, and 407. GRK5 phosphorylated these four residues as well as threonine 393 and serine 411. To determine if mutation of these sites altered desensitization, we have constructed βARs in which the threonines and serines of the putative GRK2 and GRK5 sites were substituted with alanines. These constructs were further modified to eliminate the cAMP-dependent protein kinase (PKA) consensus sites. Mutants βARs were transfected into HEK 293 cells, and standard kinetic parameters were measured following 10 μmepinephrine treatment of cells. The mutant and wild type (WT) receptors were all desensitized 89–94% after 5 min of 10 μmepinephrine stimulation and 96–98% after a 30-min pretreatment. There were no significant changes observed for any of the mutant βARs relative to the WT in the extent of 10 μmepinephrine-induced internalization (77–82% after 30 min). Epinephrine treatment for 1 min induced a rapid increase in the phosphorylation of the GRK5 and PKA− mutant βARs as well as the WT. We conclude that sites other than the GRK2 and GRK5 sites identified by in vitro phosphorylation are involved in mediating the major effects of the in vivoGRK-dependent desensitization of the βAR. Tentative identification of the G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the β2-adrenergic receptor (βAR) was recently reported based on in vitro phosphorylation of recombinant receptor (Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13796–13803). Phosphorylated residues identified for GRK2 were threonine 384 and serines 396, 401, and 407. GRK5 phosphorylated these four residues as well as threonine 393 and serine 411. To determine if mutation of these sites altered desensitization, we have constructed βARs in which the threonines and serines of the putative GRK2 and GRK5 sites were substituted with alanines. These constructs were further modified to eliminate the cAMP-dependent protein kinase (PKA) consensus sites. Mutants βARs were transfected into HEK 293 cells, and standard kinetic parameters were measured following 10 μmepinephrine treatment of cells. The mutant and wild type (WT) receptors were all desensitized 89–94% after 5 min of 10 μmepinephrine stimulation and 96–98% after a 30-min pretreatment. There were no significant changes observed for any of the mutant βARs relative to the WT in the extent of 10 μmepinephrine-induced internalization (77–82% after 30 min). Epinephrine treatment for 1 min induced a rapid increase in the phosphorylation of the GRK5 and PKA− mutant βARs as well as the WT. We conclude that sites other than the GRK2 and GRK5 sites identified by in vitro phosphorylation are involved in mediating the major effects of the in vivoGRK-dependent desensitization of the βAR. Epinephrine stimulation of the β2-adrenergic receptor (βAR) 1The abbreviations used are: βAR, human β2-adrenergic receptor; HA, hemagglutinin; GRK, G protein-coupled receptor kinase; WT, wild type; PKA, cAMP-dependent protein kinase; 125ICYP, [125]iodocyanopindolol; PAGE, polyacrylamide gel electrophoresis; CGP, [3H]CGP-12177; DMEM, Dulbecco's modified Eagle's medium; AT, ascorbic acid/thiourea; WGA, wheat germ agglutinin; PVDF, polyvinylidene difluoride; PCR, polymerase chain reaction; GTPγS, guanosine 5′- 3-O-(thio)triphosphate. 1The abbreviations used are: βAR, human β2-adrenergic receptor; HA, hemagglutinin; GRK, G protein-coupled receptor kinase; WT, wild type; PKA, cAMP-dependent protein kinase; 125ICYP, [125]iodocyanopindolol; PAGE, polyacrylamide gel electrophoresis; CGP, [3H]CGP-12177; DMEM, Dulbecco's modified Eagle's medium; AT, ascorbic acid/thiourea; WGA, wheat germ agglutinin; PVDF, polyvinylidene difluoride; PCR, polymerase chain reaction; GTPγS, guanosine 5′- 3-O-(thio)triphosphate. in intact cells activates the receptor and rapidly induces its desensitization. The decreased responsiveness of the receptor after stimulation by near-saturating concentrations of epinephrine appears to be caused by rapid cAMP-dependent protein kinase (PKA) and G protein-coupled receptor kinase (GRK) phosphorylation. GRK phosphorylation in turn promotes β-arrestin binding and receptor internalization (1Perkins J.P. Hausdorff W.P. Lefkowitz R.J. Perkins J.P. The β-Adrenergic Receptors. Humana Press Inc., Totowa, NJ1991: 73-124Google Scholar, 2Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-351PubMed Google Scholar). Identification of the specific amino acids phosphorylated by these protein kinases has been the focus of numerous studies. Through the use of several deletion and substitution mutants, the sites for PKA-mediated desensitization of the βAR in intact cells were shown to be serines 261 and 262 in the third intracellular loop PKA consensus site (3Clark R.B. Friedman J. Dixon R.A.F. Strader C.D. Mol. Pharmacol. 1989; 36: 343-348PubMed Google Scholar, 4Yuan N. Friedman J. Whaley B.S. Clark R.B. J. Biol. Chem. 1994; 269: 23032-23038Abstract Full Text PDF PubMed Google Scholar, 5Hausdorff W.P. Bouvier M. 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). For the GRKs, mutagenesis studies indicate the involvement of 11 serines and threonines in the carboxyl terminus (5Hausdorff W.P. Bouvier M. 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, 6Bouvier M. Hausdorff W.P. De Blasi A. O'Dowd B.F. Kobilka B.F. Caron M.G. Lefkowitz R.J. Nature. 1988; 333: 370-373Crossref PubMed Scopus (333) Google Scholar). By utilizing in vitro GRK phosphorylation of recombinant βAR reconstituted into liposomes followed by sequencing of proteolytic fragments of the carboxyl tail, it was found that four sites were phosphorylated by GRK2 (βAR kinase 1), serines 396, 401, and 407, and threonine 384, and six by GRK5 that included the same four phosphorylated by GRK2 and additionally threonine 393 and serine 411 (7Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13796-13803Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). On the basis of this study it was proposed that these amino acids were the sites of GRK-mediated phosphorylation in intact cells; however, the effects of mutating these sites on the desensitization of the βAR in vivo was not addressed. In the studies presented here, we have determined the effects of substitutions of the putative GRK phosphorylation sites identified by the in vitro approach of Fredericks et al. (7Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13796-13803Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) on the desensitization, internalization, and phosphorylation of the respective mutant βARs. The serine or threonine residues tentatively identified as the GRK2 and GRK5 phosphorylation sites were replaced with alanine. To aid our analysis of the effects of these mutations, we also replaced the serine residues of the two consensus PKA phosphorylation sites with alanine to eliminate PKA-mediated desensitization and phosphorylation. The GRK/PKA mutants (designated as GRK2− or GRK5−), as well as a mutant βAR containing only the PKA substitutions (PKA−), were constructed in the WTβAR that had been modified by placement of the hemagglutinin (HA) antigen at the amino terminus and six histidine residues at the carboxyl terminus. We recently established that the desensitization, internalization, and phosphorylation of this double epitope-modified βAR, stably transfected into HEK 293 cells, was indistinguishable from the wild type receptor (8January B. Seibold A. Whaley B. Hipkin R.W. Lin D. Schonbrunn A. Barber R. Clark R.B. J. Biol. Chem. 1997; 272: 23871-23879Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Furthermore, the HEK 293 cell line offers a system in which the effects of overexpressed GRK2 on βAR phosphorylation and internalization have been studied (9Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) and in which endogenous GRK2 expression has been shown (10Menard L. Ferguson S.S.G. Zhang J. Lin F.-T. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 1997; 51: 800-808Crossref PubMed Scopus (213) Google Scholar). Our results demonstrate that the GRK2 substitutions did not significantly alter epinephrine-induced desensitization of the βAR, although a slight reduction of the rate and extent of desensitization was observed with the GRK5 substitutions. Consistent with these observations, we found that the mutant βARs were rapidly phosphorylated and that the rates of internalization were unimpaired. The lack of any major effects on these parameters suggests that the GRK site(s) that mediate the desensitization and subsequent internalization of the βAR do not involve the sites identified by in vitrophosphorylation. The construction of the plasmid containing the HA and six histidine-tagged βAR has been described previously (8January B. Seibold A. Whaley B. Hipkin R.W. Lin D. Schonbrunn A. Barber R. Clark R.B. J. Biol. Chem. 1997; 272: 23871-23879Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). This plasmid is designated here as WTβAR. For construction of the mutant βARs, the HA-His6-tagged βAR was excised from the pBC12B1 plasmid as anNcoI/SalI fragment, made blunt-ended, and ligated into the expression vector pKNH that had beenHindIII-digested and blunt-ended. All mutants were constructed using polymerase chain reaction (PCR) methods. To change the serines at 261 and 262 to alanines (third intracellular loop PKA consensus site), a two-step PCR mutagenesis method was used with the HA-His6 βAR in pBC12B1 as template. In the first step, two independent reactions were carried out, one using a sense mutagenizing oligonucleotide paired with a downstream oligonucleotide, and the other using an antisense mutagenizing oligonucleotide paired with an upstream oligonucleotide. In the second step, the products of the first PCR reactions were amplified using a pair of oligonucleotides nested within the upstream and downstream oligonucleotides. The resulting product was digested with AccI and subcloned into the plasmid pGEM3Z (Promega). The mutagenized receptor was excised as aBamHI/HindIII fragment, blunt-ended, and subcloned into HindIII-digested, blunt-ended pKNH. The S261A and S262A mutant HA-His6-tagged βAR in pKNH served as a template for subsequent mutagenesis. All other mutageneses were performed with single PCR reactions using mutagenizing sense and antisense oligonucleotides and Pfu I polymerase (Stratagene). After PCR, digestion with DpnI (which requires methylated DNA) was performed to remove the non-mutagenized template DNA, followed by transformation into XL1 Blue competent cells. The entire length of each mutant βAR was sequenced to verify the changes and to ensure that no other alterations were introduced by PCR. The mutant βAR designated PKA− had alanine substituted for the serines of both PKA consensus sites and can be described as Ser-261 → Ala, Ser-262 → Ala, Ser-345 → Ala, and Ser-346 → Ala. The mutant βAR designated GRK2− was constructed from the PKA− mutant and, in addition, has threonine 384 and serines 396, 401, and 407 changed to alanine. The four residues Thr-384, Ser-396, Ser-401, and Ser-407 are those identified by Fredericks et al. (7Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13796-13803Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) as the sites of in vitrophosphorylation of the βAR by GRK2. The mutant designated GRK5− was constructed from GRK2− and, in addition, has threonine 393 and serine 411 changed to alanine. The six residues Thr-384, Thr-393, Ser-396, Ser-401, Ser-407, and Ser-411 are those identified by Fredericks et al. (7Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13796-13803Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) as the sites of in vitro phosphorylation of the βAR by GRK5. The HEK 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37 °C in 5% CO2. Each mutant plasmid was linearized by PvuI digestion and transfected into the HEK 293 cells using the CaPO4 method. The day after transfection the cells were shocked with 25% glycerol in DMEM and placed in media containing 0.4 mg/ml G418 the day after shocking. Stable transfectants were identified using an intact cell [125I]iodocyanopindolol (125ICYP) binding assay described below. To measure intact cell receptor number by 125ICYP binding, cells were grown in 12-well dishes. After rinsing with serum-free DMEM, the cells were removed by pipetting up and down with 200–500 μl of serum-free DMEM. Triplicate reactions were performed in DMEM containing ≈200 pm125ICYP, in a total assay volume of 200 μl. Nonspecific binding was measured with the addition of 1 μm alprenolol. The reactions were incubated on ice for 50 min and terminated by dilution with 2.5 ml of ice-cold 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2. The 125ICYP-bound receptor protein was isolated by filtration through Whatman GF/C filters. The filters were rinsed three times with 2.5 ml of the Tris/MgCl2 buffer and counted in a Beckman 4000 Gamma counter. Protein was measured in duplicate or triplicate with 100 μl of cells. To measure βAR levels in membranes, 5 μg of membrane protein was used per reaction containing 0.1 mm phentolamine, 40 mm Hepes, pH 7.2, 2 mm EDTA, 0.2 mm ascorbate, and 2 mmthiourea, and ≈200 pm125ICYP in the presence or absence of 1 μm alprenolol. The reactions were incubated at 30 °C for 50 min and terminated as described for the intact cell binding. The Kd values for epinephrine were determined by displacement of 125ICYP using methods previously described (8January B. Seibold A. Whaley B. Hipkin R.W. Lin D. Schonbrunn A. Barber R. Clark R.B. J. Biol. Chem. 1997; 272: 23871-23879Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The 125ICYP was prepared according to the method of Barovsky and Brooker (11Barovsky K. Brooker G. J. Cyclic Nucleotide Res. 1980; 6: 297-307PubMed Google Scholar) and Hoyer et al. (12Hoyer D.E. Reynolds E. Molinoff P.B. Mol. Pharmacol. 1984; 25: 209-218PubMed Google Scholar). Alprenolol (1 μm) was included to measure nonspecific binding. The reactions were incubated for 50 min at 30 °C and stopped as described above. The reactions included 40–50 pm125ICYP, 10 μm GTPγS, and concentrations of epinephrine ranging from 0.1 to 100 μm. The data were fit to a one-component competition sigmoidal curve with a Hill coefficient of −1 using GraphPad software and Kd values determined using the Cheng-Prusoff formulation. Cells were plated onto 60-mm dishes coated with poly-l-lysine (Sigma) to improve cell adhesion. The cells were pretreated with 10 μm epinephrine or carrier by additions made directly to growth medium from 100× stock solutions. The 100× epinephrine stock (1 mm) was prepared in 100× AT carrier, such that the final concentration of AT was 0.1 mm ascorbate and 1 mm thiourea, pH 7. Controls were treated with the AT carrier at the same final concentration. Pretreatment was performed at 37 °C for various times and was stopped by removal of media and 6 washes with 2 ml of ice-cold serum-free DMEM, pH 7. Surface receptor number was then measured with the addition of 2 ml of serum-free DMEM containing 5 nm [3H]CGP-12177, designated CGP hereafter. Incubations were on ice for 1 h. To measure nonspecific binding, cells were incubated with 1 μm alprenolol added to the CGP mix. To measure total receptor number, including internalized βAR, digitonin was added to the binding mix (including alprenolol controls) to a final concentration of 0.2% as described previously (8January B. Seibold A. Whaley B. Hipkin R.W. Lin D. Schonbrunn A. Barber R. Clark R.B. J. Biol. Chem. 1997; 272: 23871-23879Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 13Slowiejko D.M. Levey A.I. Fisher S.K. J. Neurochem. 1994; 62: 1795-1803Crossref PubMed Scopus (20) Google Scholar). The reactions were stopped by removal of the binding mix followed by 3 washes with ice-cold DMEM, 2 ml each. The cells were scraped into 0.75 ml of trypsin and counted in 5 ml of scintillation fluid. Measurements were performed in triplicate for each time point. Additional plates that were washed identically to the experimental plates were used to measure protein. The surface receptor number is expressed relative to the AT-treated control in each experiment. GraphPad software was used to fit the data to an equation for monoexponential decay and determine the apparent rate of internalization. Cells were plated into 150-mm dishes coated with poly-l-lysine and were pretreated at 37 °C with 10 μm epinephrine or AT carrier for the indicated times. The pretreatment was stopped with 6 washes of 10 ml of ice-cold HME buffer (20 mm Hepes, pH 8.0, 2 mmMgCl2, 1 mm EDTA, 1 mm benzamidine, 10 μg/ml trypsin inhibitor, 0.1 mg/ml bovine serum albumin). The washed cells were scraped into HME plus 10 μg/ml leupeptin, 20 mm tetrasodium pyrophosphate, and 0.1 μmokadaic acid and homogenized with 7 strokes in a type B Dounce homogenizer. The homogenates were layered onto step gradients of 23 and 43% sucrose prepared in HE buffer (20 mm Hepes, pH 8.0, 1 mm EDTA) and centrifuged at 25,000 rpm in a Beckman SW28.1 rotor for 35 min. The fraction at the 23/43% interface was removed, flash-frozen in liquid nitrogen, and stored at −80 °C. Adenylyl cyclase activity was assayed by a modification of the method described by Salomon et al. (14Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3367) Google Scholar). Membranes were diluted to a final protein concentration of 0.2–0.4 mg/ml and were incubated for 10 min at 30 °C with 40 mm Hepes, pH 7.7, 1 mm EDTA, 1.34 mm MgCl2, 8 mm creatine phosphate, 16 units/ml creatine kinase, 100 μm ATP, 1 μm GTP, 0.1 mm 1-methyl-3-isobutylxanthine, 2 μCi of [α-32P]ATP (NEN Life Science Products, 30 Ci/mmol) in a total volume of 100 μl. The final free Mg2+concentration was calculated to be 0.3 mm to optimize desensitization measurements (3Clark R.B. Friedman J. Dixon R.A.F. Strader C.D. Mol. Pharmacol. 1989; 36: 343-348PubMed Google Scholar, 15Clark, R. B., Friedman, J., Johnson, J. A., and Kunkel, M. W. FASEB J., 1, 289–297.Google Scholar, 16Johnson J.A. Clark R.B. Friedman J. Dixon R.A.F. Strader C.D. Mol. Pharmacol. 1990; 38: 289-293PubMed Google Scholar). Each point was assayed in triplicate, with 6–8 concentrations of epinephrine bracketing the EC50. The [32P]cAMP produced in the reaction was purified over Dowex and alumina columns (17Clark 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 (105) Google Scholar). the Vmax and EC50 values were determined with GraphPad software. As we have previously shown, the expression for coupling efficiency can be combined with that for Vmax to give Equation 1 (8January B. Seibold A. Whaley B. Hipkin R.W. Lin D. Schonbrunn A. Barber R. Clark R.B. J. Biol. Chem. 1997; 272: 23871-23879Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). (k1)r=(Vmax)(Kd)/(V100)(EC50)Equation 1 This equation describes the coupling capacity, (k1)r, whereVmax is the maximum adenylyl cyclase activity measured for saturating agonist concentrations, and V100 is the theoretical value whenk1 is infinite. The increase in EC50and the decrease in Vmax that occurs with desensitization can be quantitated using the expression for coupling capacity. The extent of desensitization can be expressed as the ratio of receptor coupling capacity in the desensitized relative to naive state. Since the Kd and V100values do not change upon desensitization, the ratio can be expressed as shown in Equation 2. (k1r)D/(k1r)N=(Vmax)D(EC50)N/(Vmax)N(EC50)DEquation 2 The expression (k1 r)D/(k1 r)Nis defined as the fraction activity remaining and is quantitated using experimentally determined EC50 and Vmax values. This calculation can be converted to percent desensitization by multiplying the fraction of βAR activity remaining by 100 and subtracting that value from 100. Desensitization data in Fig. 3 and Table II are presented as the mean of the fraction activity remaining ± S.E.Table IICharacterization of 10 μm epinephrine-induced desensitization of βARs expressed in HEK 293 cellsCell lineTime of pretreatment2 min30 mint1/2fraction activity remainingminWT βAR0.13 ± 0.02 (3)0.024 ± 0.003 (3)0.38 ± 0.04 (3)PKA−0.16 ± 0.05 (2)0.027 ± 0.003 (3)0.45 ± 0.06 (3)GRK2−0.21 ± 0.05 (3)0.04 ± 0.02 (3)0.56 ± 0.07 (3)GRK5−0.21 ± 0.02 (5)2-2001The fraction activity remaining (extent of desensitization) measured for the WT βAR and the GRK5−mutant after 2 and 30 min 10 μm epinephrine pretreatment were compared using an unpaired t test and were found to be significantly different, p < 0.05.0.042 ± 0.004 (5)2-2001The fraction activity remaining (extent of desensitization) measured for the WT βAR and the GRK5−mutant after 2 and 30 min 10 μm epinephrine pretreatment were compared using an unpaired t test and were found to be significantly different, p < 0.05.0.65 ± 0.06 (4)2-bThe apparent rates of desensitization of the WTβAR and the GRK5− mutant were compared using an unpaired t test and found to be significantly different, p < 0.05.2-2001 The fraction activity remaining (extent of desensitization) measured for the WT βAR and the GRK5−mutant after 2 and 30 min 10 μm epinephrine pretreatment were compared using an unpaired t test and were found to be significantly different, p < 0.05.2-b The apparent rates of desensitization of the WTβAR and the GRK5− mutant were compared using an unpaired t test and found to be significantly different, p < 0.05. Open table in a new tab The apparent rates of desensitization and internalization were determined using GraphPad software for monoexponential decay. With several mechanisms contributing to desensitization and internalization, the data cannot be explained by a simple monoexponential decay. However it is useful to give a t½ for the sum of the total process. To measure phosphorylation of the βAR, confluent cells were washed three times in phosphate-free DMEM, incubated for 3 h with [32P]H3PO4 (0.5–1.0 mCi/100-mm dish), and pretreated for the indicated times with either 10 μm epinephrine or AT carrier. The cells were solubilized, and the extracts were subjected to a two-step purification using nickel nitrilotriacetic acid-agarose and wheat germ agglutinin-agarose (WGA) as described previously (8January B. Seibold A. Whaley B. Hipkin R.W. Lin D. Schonbrunn A. Barber R. Clark R.B. J. Biol. Chem. 1997; 272: 23871-23879Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) with the following modifications. The nickel nitrilotriacetic acid eluent fractions containing the βAR were mixed with 100 μl of WGA (packed volume) and incubated for 90 min at 4 °C with rocking. The WGA/βAR was collected and washed with 5 ml of nickel column buffer (0.05%n-dodecyl-β-d-maltoside, 20 mmHepes, pH 7.4, and 150 mm NaCl) at 4 °C. The WGA was further washed twice with 400 μl of 0.5% sodium dodecyl sulfate (SDS) at 37 °C for a total incubation time of 10 min. The WGA pellet was collected and the βAR eluted with SDS sample buffer (50 mm Tris, pH 6.8, 2% SDS, 0.025% bromphenol blue, 6m urea, 14.3 mm β-mercaptoethanol). The receptor was resolved on 7.5% SDS-polyacrylamide gels with the addition of pre-stained low molecular weight standards (Bio-Rad). The gels were dried and exposed to a phosphorscreen for 2–24 h, and the data were analyzed using a Molecular Dynamics Storm PhosphorImager model 860 and Imagequant software. Autoradiograms of the dried gels were also obtained (24–48 h). In some experiments the gels following SDS-PAGE were transferred to 0.22-micron PVDF membranes, and the identity of the radiolabeled band as the βAR was confirmed by Western analysis using a primary anti-HA polyclonal antibody (Babco) and a horseradish peroxidase-conjugated goat anti-rabbit (Bio-Rad) as the secondary antibody as described previously (8January B. Seibold A. Whaley B. Hipkin R.W. Lin D. Schonbrunn A. Barber R. Clark R.B. J. Biol. Chem. 1997; 272: 23871-23879Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). As we have previously shown, determination of the coupling efficiency for agonist activation of adenylyl cyclase requires the measurement of receptor levels, the low affinity Kd for agonist binding, and the EC50 for activation of adenylyl cyclase (8January B. Seibold A. Whaley B. Hipkin R.W. Lin D. Schonbrunn A. Barber R. Clark R.B. J. Biol. Chem. 1997; 272: 23871-23879Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). A summary of these determinations using membranes prepared from each cell line is shown in Table I. At least two clones expressing each receptor were examined, and those shown in Table I were selected for all subsequent experiments since they expressed reasonably similar levels. A representative experiment for the determination of the low affinity Kd for epinephrine binding is shown in Fig. 1. We found no significant differences in the Kd values for the mutant receptors versus the WT. The mutant and WT cell lines displayed similar values for basal activity and the Vmax for epinephrine stimulation. The EC50 values for epinephrine stimulation of the mutant receptors were found to be consistently 4–6.5 times higher than that of the WTβAR (see Fig. 2 for data summary). Using the formulations we described previously (8January B. Seibold A. Whaley B. Hipkin R.W. Lin D. Schonbrunn A. Barber R. Clark R.B. J. Biol. Chem. 1997; 272: 23871-23879Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 19Whaley B.S. Yuan N. Barber R. Clark R.B. Mol. Pharmacol. 1995; 45: 481-489Google Scholar), we calculated from the data in Table I that the coupling efficiencies of the mutants were reduced by a factor of 2–4-fold relative to WTβAR.Table ICharacterization of βARs expressed in HEK 293 cellsCell lineBasal adenylyl cyclaseVmax for epinephrineEC50 for epinephrineReceptor levelKd for epinephrinepmol cAMP/min/mgpmol cAMP/min/mgnmfmol/mgnmWT βAR8.3 ± 1.2 (5)75.5 ± 8.6 (5)3.5 ± 0.3 (5)3363 ± 118 (5)339 ± 49 (4)PKA−8.6 ± 0.8 (6)119.9 ± 11.6 (6)15.7 ± 1.2 (6)2311 ± 220 (6)503 ± 46 (3)GRK2−7.3 ± 0.9 (7)91.1 ± 11.4 (7)17.1 ± 2.0 (7)1532 ± 167 (6)374 ± 24 (3)GRK5−4.9 ± 0.78 (7)84.8 ± 10.1 (7)22.9 ± 5.0 (7)2497 ± 399 (6)489 ± 37 (3) Open table in a new tab Figure 2Epinephrine-induced desensitization of the WT and mutant βARs. Cells expressing the WTβAR (A), PKA− (B), GRK2− (C), or GRK5− (D) were pretreated with either carrier (○) or with 10 μm epinephrine for 2 (•) or 30 (□) min. Membranes were prepared, and adenylyl cyclase was assayed in triplicate for each experiment with the various epinephrine concentrations as indicated for each experiment. The data are normalized relative to the Vmax for epinephrine of the control sample (100%) for each experiment after subtraction of basal. Data shown are the mean ± S.E. of 3–5 experiments or the mean ± range of 2 experiments. The absence of error bars indicates data points that were the average of triplicate determinations from one experiment.View Large Image Figure ViewerDownload (PPT) To assess desensitization, HEK 293 cells stably expressing the WT or mutant βARs were pretreated with 10 μm epinephrine for various times from 0.5–30 min. Following pretreatment, membranes were prepared and assayed for epinephrine-stimulated adenylyl cyclase activity using a range of epinephrine concentrations. The data summary for desensitization in response to 2 and 30 min pretreatment with 10 μm epinephrine is shown in Fig. 2. These data as well as data from the other time points of 10 μm epinephrine pretreatment are summarized in Fig. 3. The extent of desensitization was quantitated as fraction activity remaining by measuring the right-shift in EC50 and decrease in Vmax as described under “Experimental Procedures” (8January B. Seibold A. Whaley B. Hipkin R.W. Lin D. Schonbrunn A. Barber R. Clark R.B. J. Biol. Chem. 1997; 272: 23871-23879Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The fraction activity remaining for the 2- and 30-min time points for the various receptors are also shown in Table II, along with the results of calculations of the t½ values for the apparent rates of desensitization. the t½ values were determined by fitting the data to an equation for monoexponential decay. Although the 30-min data did not fit well to the theoretical curve, this method allowed calculation of approximate apparent rates of desensitization for comparison of the WT and mutant βARs. The data demonstrate that there was no significant difference in the apparent rate or extent of desensitization for the PKA− relative to the WTβAR. Although there was a slightly reduced apparent rate and extent of desensitization for the GRK2− and the GRK5−, the decrease was only significant for the GRK5− βAR. Internalization of the mutant and WTβARs in response to 10 μm epinephrine was m" @default.
• W2080451044 created "2016-06-24" @default.
• W2080451044 creator A5015270071 @default.
• W2080451044 creator A5023333880 @default.
• W2080451044 creator A5029364998 @default.
• W2080451044 creator A5053422043 @default.
• W2080451044 creator A5087891732 @default.
• W2080451044 date "1998-03-01" @default.
• W2080451044 modified "2023-09-30" @default.
• W2080451044 title "Desensitization of β2-Adrenergic Receptors with Mutations of the Proposed G Protein-coupled Receptor Kinase Phosphorylation Sites" @default.
• W2080451044 cites W1482398833 @default.
• W2080451044 cites W1493055704 @default.
• W2080451044 cites W1563035586 @default.
• W2080451044 cites W1603981767 @default.
• W2080451044 cites W1976390463 @default.
• W2080451044 cites W1993755473 @default.
• W2080451044 cites W1995605077 @default.
• W2080451044 cites W2024040954 @default.
• W2080451044 cites W2029313038 @default.
• W2080451044 cites W2034929403 @default.
• W2080451044 cites W2059247616 @default.
• W2080451044 cites W2061725106 @default.
• W2080451044 cites W2083704518 @default.
• W2080451044 cites W2087167617 @default.
• W2080451044 cites W2137676735 @default.
• W2080451044 cites W2151775257 @default.
• W2080451044 cites W2169503005 @default.
• W2080451044 doi "https://doi.org/10.1074/jbc.273.13.7637" @default.
• W2080451044 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9516468" @default.
• W2080451044 hasPublicationYear "1998" @default.
• W2080451044 type Work @default.
• W2080451044 sameAs 2080451044 @default.
• W2080451044 citedByCount "75" @default.
• W2080451044 countsByYear W20804510442012 @default.
• W2080451044 countsByYear W20804510442013 @default.
• W2080451044 countsByYear W20804510442014 @default.
• W2080451044 countsByYear W20804510442015 @default.
• W2080451044 countsByYear W20804510442018 @default.
• W2080451044 countsByYear W20804510442019 @default.
• W2080451044 countsByYear W20804510442020 @default.
• W2080451044 countsByYear W20804510442021 @default.
• W2080451044 countsByYear W20804510442022 @default.
• W2080451044 crossrefType "journal-article" @default.
• W2080451044 hasAuthorship W2080451044A5015270071 @default.
• W2080451044 hasAuthorship W2080451044A5023333880 @default.
• W2080451044 hasAuthorship W2080451044A5029364998 @default.
• W2080451044 hasAuthorship W2080451044A5053422043 @default.
• W2080451044 hasAuthorship W2080451044A5087891732 @default.
• W2080451044 hasBestOaLocation W20804510441 @default.
• W2080451044 hasConcept C11960822 @default.
• W2080451044 hasConcept C135285700 @default.
• W2080451044 hasConcept C169331234 @default.
• W2080451044 hasConcept C170493617 @default.
• W2080451044 hasConcept C185592680 @default.
• W2080451044 hasConcept C2779779143 @default.
• W2080451044 hasConcept C33235085 @default.
• W2080451044 hasConcept C40465926 @default.
• W2080451044 hasConcept C46688247 @default.
• W2080451044 hasConcept C55493867 @default.
• W2080451044 hasConcept C78976303 @default.
• W2080451044 hasConcept C86803240 @default.
• W2080451044 hasConcept C95444343 @default.
• W2080451044 hasConcept C97029542 @default.
• W2080451044 hasConcept C98274493 @default.
• W2080451044 hasConceptScore W2080451044C11960822 @default.
• W2080451044 hasConceptScore W2080451044C135285700 @default.
• W2080451044 hasConceptScore W2080451044C169331234 @default.
• W2080451044 hasConceptScore W2080451044C170493617 @default.
• W2080451044 hasConceptScore W2080451044C185592680 @default.
• W2080451044 hasConceptScore W2080451044C2779779143 @default.
• W2080451044 hasConceptScore W2080451044C33235085 @default.
• W2080451044 hasConceptScore W2080451044C40465926 @default.
• W2080451044 hasConceptScore W2080451044C46688247 @default.
• W2080451044 hasConceptScore W2080451044C55493867 @default.
• W2080451044 hasConceptScore W2080451044C78976303 @default.
• W2080451044 hasConceptScore W2080451044C86803240 @default.
• W2080451044 hasConceptScore W2080451044C95444343 @default.
• W2080451044 hasConceptScore W2080451044C97029542 @default.
• W2080451044 hasConceptScore W2080451044C98274493 @default.
• W2080451044 hasIssue "13" @default.
• W2080451044 hasLocation W20804510441 @default.
• W2080451044 hasOpenAccess W2080451044 @default.
• W2080451044 hasPrimaryLocation W20804510441 @default.
• W2080451044 hasRelatedWork W1945832201 @default.
• W2080451044 hasRelatedWork W1979038001 @default.
• W2080451044 hasRelatedWork W2034451512 @default.
• W2080451044 hasRelatedWork W2039711939 @default.
• W2080451044 hasRelatedWork W2060207881 @default.
• W2080451044 hasRelatedWork W2166414244 @default.
• W2080451044 hasRelatedWork W2216806740 @default.
• W2080451044 hasRelatedWork W2467964952 @default.
• W2080451044 hasRelatedWork W2485064844 @default.
• W2080451044 hasRelatedWork W91561007 @default.
• W2080451044 hasVolume "273" @default.
• W2080451044 isParatext "false" @default.
• W2080451044 isRetracted "false" @default.
• W2080451044 magId "2080451044" @default.
• W2080451044 workType "article" @default.