Desensitization of β2-Adrenergic Receptors with Mutations of the Proposed G Protein-coupled Receptor Kinase Phosphorylation Sites

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 with a and the other using mutagenizing with In the step, the of the first PCR reactions were using a of the and The was with and into the plasmid The receptor was excised as fragment, blunt-ended, and into The and mutant HA-His6-tagged βAR in pKNH as a for subsequent All other were with PCR reactions using mutagenizing sense and and polymerase PCR, with was to the followed by into cells. The of mutant βAR was to the changes and to that no other were by The mutant βAR designated PKA− had alanine substituted for the serines of PKA consensus sites and be described as and The mutant βAR designated GRK2− was constructed from the PKA− mutant in has threonine 384 and serines 396, 401, and to alanine. The four residues and are 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 of the βAR by The mutant designated was constructed from GRK2− in has threonine 393 and serine 411 to alanine. The six residues and are 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 The HEK 293 cells were in Dulbecco's modified Eagle's containing at in mutant plasmid was by and transfected into the HEK 293 cells using the The after the cells were with in and in containing the after were identified using intact cell binding described To intact cell receptor by cells were in with DMEM, the cells were by and with of reactions were in containing in a of binding was measured with the of 1 The reactions were on for min and by with of 10 The receptor protein was by The were with of the and in a was measured in or with of cells. To βAR in 5 of protein was used reaction containing 2 and 2 and in the or of 1 The reactions were at 30 for min and as described for the intact cell The for epinephrine were determined by of using 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 was to the method of and J. Res. Google Scholar) and et al. Mol. Pharmacol. Google Scholar). was included to The reactions were for min at 30 and as described The reactions included 10 GTPγS, and concentrations of epinephrine from to The were to a with a of using and determined using the were with to cell The cells were with 10 epinephrine or by made to from The epinephrine was in that the of was and 1 were with the at the same was at for and was by of and with 2 of DMEM, receptor was measured with the of 2 of containing 5 designated were on for 1 To cells were with 1 to the To receptor βAR, was to the binding to a of 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, J. 1994; PubMed Scopus Google Scholar). The reactions were by of the binding followed by with DMEM, 2 The cells were into of and in 5 of were in for that were to the were used to The receptor is relative to the in was used to the to for and determine the rate of were into with and were at with 10 epinephrine or for the The was with of 10 of 2 1 1 10 The cells were into 10 and and with in a type The were of and in 1 and at in a for The at the was in and at was by a of the method described by et al. M. PubMed Scopus Google Scholar). were to a protein of and were for 10 min at 30 with 1 1 2 of 30 in a of The was to be to desensitization (3Clark R.B. Friedman J. Dixon R.A.F. Strader C.D. Mol. Pharmacol. 1989; 36: 343-348PubMed Google Scholar, R. J. A., and M. Scholar, J.A. Clark R.B. Friedman J. Dixon R.A.F. Strader C.D. Mol. Pharmacol. Google Scholar). was in with concentrations of epinephrine the The in the reaction was and R.B. Friedman J. J.A. A. 1988; PubMed Scopus Google Scholar). the and were determined with we have previously the expression for be with that for to 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). 1 This the is the measured for and is the is The increase in the in that with desensitization be using the expression for The extent of desensitization be as the of receptor in the desensitized relative to the and do not change desensitization, the be as shown in 2 The expression as the and is using determined and This be to desensitization by the of βAR by and that from in and are presented as the of the of 10 epinephrine-induced desensitization of βARs in HEK 293 of of measured for the WT βAR and the after 2 and 30 min 10 epinephrine were using and were found to be significantly of measured for the WT βAR and the after 2 and 30 min 10 epinephrine were using and were found to be significantly rates of desensitization of the WTβAR and the mutant were using and found to be significantly The of measured for the WT βAR and the after 2 and 30 min 10 epinephrine were using and were found to be significantly The rates of desensitization of the WTβAR and the mutant were using and found to be significantly in a The rates of desensitization and internalization were determined using for several to desensitization and internalization, the be by a it is to a for the of the To phosphorylation of the βAR, cells were in DMEM, for with and for the with 10 epinephrine or The cells were and the were to a two-step using and wheat germ 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 The containing the βAR were with of and for min at with The was and with 5 of and at The was further with of at for a of 10 The was and the βAR with The receptor was on with the of The were and to a for and the were using a and of the were also In the following were to and the of the as the βAR was by analysis using a and a as the 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). we have previously of the for of the of receptor the for and the for of (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). of these using from cell line is shown in two receptor were and shown in were for all subsequent for the of the for epinephrine binding is shown in We found no significant in the for the mutant receptors the WT. The mutant and WT cell for and the for epinephrine The for epinephrine stimulation of the mutant receptors were found to be than that of the WTβAR 2 for the 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, B.S. N. Barber R. Clark R.B. Mol. Pharmacol. 1995; we from the in that the of the mutants were by a of relative to of βARs in HEK 293 for for for in a desensitization of the WT and mutant βARs. the WTβAR PKA− GRK2− or were with or with 10 epinephrine for 2 or 30 were and was in for with the epinephrine concentrations as for The are relative to the for epinephrine of the for after of shown are the of or the of 2 The of that were the of from one To desensitization, HEK 293 cells stably the WT or mutant βARs were with 10 epinephrine for from were and for using a of epinephrine The for desensitization in to 2 and 30 min with 10 epinephrine is shown in These as well as from the other of 10 epinephrine are in The extent of desensitization was as by the in and in as 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 for the and 30-min for the receptors are also shown in with the results of of the for the rates of desensitization. the were determined by the to for the 30-min did not well to the this method of rates of desensitization for of the WT and mutant βARs. The demonstrate that was no significant in the rate or extent of desensitization for the PKA− relative to the WTβAR. was a rate and extent of desensitization for the GRK2− and the the was only significant for the βAR. of the mutant and in to 10 epinephrine was measured by binding and is as the of receptors The rate of internalization was determined by the shown in to for The to did not into the observed for internalization of all the receptor This however, of rates of internalization for of the WT and mutant βARs. The rate of internalization of the WTβAR min was found to be to measured for the GRK2− min the min and the PKA− min βAR The extent of internalization was also with of the WTβAR after 30 min of 10 μmepinephrine with and for the and PKA− mutants, The internalization was not the of receptor or as determined by binding in the and of as described not the WT, and PKA− βARs were for and with or 10 epinephrine for 1 of the βAR was by and of the receptors using the two-step described The receptor was to and the were to as described in is shown in The of the gel after to the is shown in 5 and the of the same is in 5 B. The of phosphorylation for the mutant βARs was to we have previously reported for the WTβAR (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 with the at 1 after 5 min not Our demonstrate that mutant βARs containing alanine substitutions for the tentatively identified by in vitro phosphorylation as the sites of GRK2 or GRK5 phosphorylation (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 and rapid desensitization and We had that these substitutions of the putative GRK and PKA sites eliminate the desensitization of the βAR. Consistent with the desensitization we found that the mutant was rapidly We that sites other than or in to vitro 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) are for in vivo GRK2 or GRK5 phosphorylation and desensitization of the βAR. is that one or of the GRK sites involved in the desensitization of the receptor were in the in vitro study of GRK2 and GRK5 phosphorylation (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). The sequencing of in this study was on a in the of the receptor carboxyl terminus it that serines and and threonine residues in the of the receptor carboxyl terminus are involved in GRK in All 11 of the residues found in the receptor carboxyl from amino to have been as sites of GRK phosphorylation. desensitization and phosphorylation was reported for a mutant βAR containing substitutions at all 11 sites (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). mutagenesis study in which only serines and and threonine were substituted for or alanine that the in vivo site(s) of GRK phosphorylation be these residues W.P. J. Caron M.G. Lefkowitz R.J. A. PubMed Scopus Google Scholar). This study that substitution of the four residues in a mutant βAR in the rapid desensitization, internalization, and phosphorylation. The that the of desensitization, and internalization observed in the βAR containing substitutions at all 11 residues from of a in the mutant with only four substitutions W.P. J. Caron M.G. Lefkowitz R.J. A. PubMed Scopus Google Scholar). however, are with the that serines and and threonine the in vivo sites of GRK phosphorylation. for the we have found the in vitro phosphorylation of the βAR and our studies of the effects of these and following intact cell treatment is that sites be phosphorylated by GRK2 or GRK5 in for this is found in studies of the receptor that vivo and in vitro identification of GRK phosphorylation sites. identification of the sites phosphorylated in vivo by a of the GRK has been described for the receptor by et J.P. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). found that two sites in the receptor carboxyl terminus were phosphorylated by kinase The two serines identified were serine was phosphorylated in to of serine was phosphorylated and only after In to these in vitro phosphorylation identified of of receptor PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). on these studies of at it is to that be vitro and in vivo of the βAR. to is that the numerous substitutions we have made in the GRK2 and GRK5 mutants have in altered the of GRK phosphorylation we have one or sites of GRK the amino changes in the mutants here GRK phosphorylation at other sites. effects are of the of the WTβAR and mutant The desensitization, internalization, and binding of the mutant receptors were to of the and only in were The desensitization we observed for the PKA− and the WTβAR 2 and was also it has been that receptor phosphorylation by PKA is to desensitization in to Hausdorff et al. (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) reported that a mutant βAR containing alanine substitutions for the serines of the two consensus PKA sites decreased desensitization to concentrations of relative to the wild type receptor in cells. et al. L. Bouvier M. B. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) found that a mutant βAR with alanine substitutions for the serines of the PKA site was to desensitization than the wild type receptor in In our results indicate that PKA-mediated receptor phosphorylation is not for desensitization in to These results with studies we with and mutants of the wild type cell line in which we found no in the extent of desensitization relative to the wild type Friedman J. Clark R.B. J. Res. Google Scholar). of the was also to that of the WTβAR. We that our results be caused by of PKA-mediated with the as has been previously Caron M.G. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). be that these To conclude that the sites phosphorylated by GRK2 and GRK5 in vivo do not with the sites identified in vitro 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) that GRK2 and GRK5 are in HEK 293 cells. GRK2 is in HEK 293 cells has been shown by Menard et al. (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) using analysis and by other using PCR as well as and R. GRK5 is or at in HEK 293 cells using The of GRK expression vivo to mediate receptor phosphorylation are The or expression of GRK5 in HEK 293 cells it to determine the in vivo of the sites phosphorylated by GRK5 in GRK2 expression in HEK 293 cells has been the presented here the lack of in vivo for the six residues identified in vitro as sites of GRK2 GRK5 phosphorylation. Identification of the βAR sites phosphorylated in vivo is for a of the of desensitization. the of G protein-coupled receptors have been to analysis of phosphorylation sites in vivo to concentrations in the This however, be for of the of the GRKs, PKA, and other protein kinases that have been in the of the βAR.