Norepinephrine- and Epinephrine-induced Distinct β2-Adrenoceptor Signaling Is Dictated by GRK2 Phosphorylation in Cardiomyocytes

Abstract

Agonist-dependent activation of G protein-coupled receptors induces diversified receptor cellular and signaling properties. Norepinephrine (NE) and epinephrine (Epi) are two endogenous ligands that activate adrenoceptor (AR) signals in a variety of physiological stress responses in animals. Here we use cardiomyocyte contraction rate response to analyze the endogenous β2AR signaling induced by Epi or NE in cardiac tissue. The Epi-activated β2AR induced a rapid contraction rate increase that peaked at 4 min after stimulation. In contrast, the NE-activated β2AR induced a much slower contraction rate increase that peaked at 10 min after stimulation. Whereas both drugs activated β2AR coupling to Gs proteins, only Epi-activated receptors were capable of coupling to Gi proteins. Subsequent studies showed that the Epi-activated β2AR underwent a rapid phosphorylation by G protein-coupled receptor kinase 2 (GRK2) and subsequent dephosphorylation on serine residues 355 and 356, which was critical for sufficient receptor recycling and Gi coupling. In contrast, the NE-activated β2ARs underwent slow GRK2 phosphorylation, receptor internalization and recycling, and failed to couple to Gi. Moreover, inhibiting β2AR phosphorylation by βARK C terminus or dephosphorylation by okadaic acid prevented sufficient recycling and Gi coupling. Together, our data revealed that distinct temporal phosphorylation of β2AR on serine 355 and 356 by GRK2 plays a critical role for dictating receptor cellular events and signaling properties induced by Epi or NE in cardiomyocytes. This study not only helps us understand the endogenous agonist-dependent β2AR signaling in animal heart but also offers an example of how G protein-coupled receptor signaling may be finely regulated by GRK in physiological settings. Agonist-dependent activation of G protein-coupled receptors induces diversified receptor cellular and signaling properties. Norepinephrine (NE) and epinephrine (Epi) are two endogenous ligands that activate adrenoceptor (AR) signals in a variety of physiological stress responses in animals. Here we use cardiomyocyte contraction rate response to analyze the endogenous β2AR signaling induced by Epi or NE in cardiac tissue. The Epi-activated β2AR induced a rapid contraction rate increase that peaked at 4 min after stimulation. In contrast, the NE-activated β2AR induced a much slower contraction rate increase that peaked at 10 min after stimulation. Whereas both drugs activated β2AR coupling to Gs proteins, only Epi-activated receptors were capable of coupling to Gi proteins. Subsequent studies showed that the Epi-activated β2AR underwent a rapid phosphorylation by G protein-coupled receptor kinase 2 (GRK2) and subsequent dephosphorylation on serine residues 355 and 356, which was critical for sufficient receptor recycling and Gi coupling. In contrast, the NE-activated β2ARs underwent slow GRK2 phosphorylation, receptor internalization and recycling, and failed to couple to Gi. Moreover, inhibiting β2AR phosphorylation by βARK C terminus or dephosphorylation by okadaic acid prevented sufficient recycling and Gi coupling. Together, our data revealed that distinct temporal phosphorylation of β2AR on serine 355 and 356 by GRK2 plays a critical role for dictating receptor cellular events and signaling properties induced by Epi or NE in cardiomyocytes. This study not only helps us understand the endogenous agonist-dependent β2AR signaling in animal heart but also offers an example of how G protein-coupled receptor signaling may be finely regulated by GRK in physiological settings. GPCRs 3The abbreviations used are: GPCRG protein-coupled receptorNEnorepinephrineEpiepinephrineGRKG protein-coupled receptor kinasePTXpertussis toxinARadrenoceptorOAokadaic acidPKAcAMP-dependent protein kinaseIsoisoproterenolGFPgreen fluorescent proteinβARKctβARK C terminusFLISAfluorescence-linked immunosorbent assayANOVAanalysis of variance. comprise the largest known family of cell-surface receptors and are fundamentally involved in mammalian physiology (1Lefkowitz R.J. Acta Physiol. (Oxf.). 2007; 190: 9-19Crossref PubMed Scopus (242) Google Scholar, 2Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 639-650Crossref PubMed Scopus (2125) Google Scholar). This receptor superfamily represents the largest single target for modern drug therapy. A growing body of evidence indicates that divergent efficacies of an activated GPCR are both agonist- and tissue-dependent (3Maudsley S. Martin B. Luttrell L.M. J. Pharmacol. Exp. Ther. 2005; 314: 485-494Crossref PubMed Scopus (161) Google Scholar, 4Eglen R.M. Proc. West Pharmacol. Soc. 2005; 48: 31-34PubMed Google Scholar, 5Kobilka B.K. Biochim. Biophys. Acta. 2007; 1768: 794-807Crossref PubMed Scopus (435) Google Scholar), which presents a great challenge on clinical application when a specific receptor is targeted with drugs. Many of the drug-dependent effects have been attributed to the distinct receptor conformational changes induced by different ligands, leading to different subsequent cellular events and signaling properties (5Kobilka B.K. Biochim. Biophys. Acta. 2007; 1768: 794-807Crossref PubMed Scopus (435) Google Scholar, 6Bissantz C. J. Recept. Signal. Transduct. Res. 2003; 23: 123-153Crossref PubMed Scopus (63) Google Scholar). Thus, there is great interest in elucidating the mechanisms underlying the drug-dependent cellular events in physiologically relevant contexts. G protein-coupled receptor norepinephrine epinephrine G protein-coupled receptor kinase pertussis toxin adrenoceptor okadaic acid cAMP-dependent protein kinase isoproterenol green fluorescent protein βARK C terminus fluorescence-linked immunosorbent assay analysis of variance. Interestingly, βARs, a family of prototypical GPCRs, can be activated by two endogenous ligands NE and Epi. The receptors play critical roles in the regulation of cardiovascular (7Rockman H.A. Koch W.J. Lefkowitz R.J. Nature. 2002; 415: 206-212Crossref PubMed Scopus (788) Google Scholar) and pulmonary function (8Johnson M. Am. J. Respir. Crit. Care Med. 1998; 158: S146-S153Crossref PubMed Scopus (348) Google Scholar), as well as other physiological processes. NE is primarily released from sympathetic nerve terminal on the innervated tissues, whereas Epi is primarily released from adrenal gland to the circulating plasma. The distinct releasing routes suggest preferential activation of a βAR subtype by an individual ligand. This notion is consistent with our recent observations that β1AR and β2AR have distinct subcellular distribution along the adrenergic synapses between sympathetic ganglion neurons and cardiac muscle cells (9Shcherbakova O.G. Hurt C.M. Xiang Y. Dell'Acqua M.L. Zhang Q. Tsien R.W. Kobilka B.K. J. Cell Biol. 2007; 176: 521-533Crossref PubMed Scopus (82) Google Scholar). Meanwhile, it is also speculated that NE and Epi might activate the same βAR subtype to initiate distinct signaling for the same physiological responses such as heart contraction. Evidence supporting this notion is lacking because of strikingly similar properties on the βAR activated by these drugs in vitro. Recently, we have shown that NE and Epi can induce distinct conformational changes on β2ARs (10Swaminath G. Xiang Y. Lee T.W. Steenhuis J. Parnot C. Kobilka B.K. J. Biol. Chem. 2004; 279: 686-691Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar), suggesting that the activated receptors may recruit different molecules for signal transduction and function. Therefore, in this work we study the potential differences of the receptor signaling activated by NE or Epi for physiological responses such as cardiomyocyte contraction. These studies will not only help us understand the physiological implications of receptor signaling activated by these two drugs, but also offer insights into the utility of a large group of drugs that either activate or inhibit βARs in a variety of clinical conditions, including heart failure, hypertension, coronary artery disease, and asthma. In fact, specific drug-dependent signaling properties are proposed to explain the clinical observations under treatment with different β-blockers (11Wisler J.W. DeWire S.M. Whalen E.J. Violin J.D. Drake M.T. Ahn S. Shenoy S.K. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 16657-16662Crossref PubMed Scopus (489) Google Scholar). Whereas carvedilol has been used as an effective long term therapy for heart failure, other drugs in the same class have failed the clinical trials (12Bristow M.R. Gilbert E.M. Abraham W.T. Adams K.F. Fowler M.B. Hershberger R.E. Kubo S.H. Narahara K.A. Ingersoll H. Krueger S. Young S. Shusterman N. Circulation. 1996; 94: 2807-2816Crossref PubMed Scopus (1189) Google Scholar). Studies in cardiac tissue are of prime interest because adrenergic signaling properties are not only manipulated with β-blockers in managing heart failure but are also linked to the progression of this disease (13Bristow J. Hersherger R. Port J. Minobe W. Rasmussen R. Mol. Pharmacol. 1989; 35: 295-303PubMed Google Scholar). Here we analyzed the NE- or Epi-activated β2AR signaling for physiological contraction response on primary cardiomyocytes. We have for the first time uncovered that agonist-dependent phosphorylation of β2AR receptor on Ser-355 and -356 by GRK2 plays a critical role to differentiate the receptor signaling activated by Epi or NE to regulate cardiomyocyte contraction rate response. Cell Culture and Recombinant Adenoviruses—Spontaneous beating neonatal cardiac myocytes were prepared from hearts of 1-day-old wild type, β1AR-knock-out (β1AR-KO), or β1β2AR-knock-out (β1β2AR-KO) mouse pups as published previously (14Devic E. Xiang Y. Gould D. Kobilka B. Mol. Pharmacol. 2001; 60: 577-583PubMed Google Scholar). Neonatal myocytes were infected with viruses at a multiplicity of infection as indicated in the text after being cultured for 24 h. Recombinant adenoviruses expressing FLAG-tagged human or murine β2ARs have been described previously (15Wang Y. Lauffer B. Von M. Kobilka B. Xiang Y. Mol. Pharmacol. 2007; PubMed Scopus Google Scholar). The receptor were in myocytes by and as described Y. Kobilka B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). expressing were a from Koch and were a 2 with receptors were by or receptor were with as described (10Swaminath G. Xiang Y. Lee T.W. Steenhuis J. Parnot C. Kobilka B.K. J. Biol. Chem. 2004; 279: 686-691Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) in the myocytes expressing the indicated were for 2 with 10 or isoproterenol The recycling of β2AR was by after 10 min of drug to receptor for an or of contraction from expressing either the endogenous or the indicated was with or the use of pertussis toxin as described previously (14Devic E. Xiang Y. Gould D. Kobilka B. Mol. Pharmacol. 2001; 60: 577-583PubMed Google Scholar). In okadaic acid was min of Epi. of β2AR or with to the C terminus of the β2AR and to the serine of β2AR were from Neonatal were for 2 to of epinephrine or norepinephrine for different myocytes were with for min drugs. The were and in 2 and The were at for The were on and with the β2AR at and revealed with a at with The were and with β2AR at to showed signal after the The signals from the β2AR were first for β2AR and either as increase or of the as indicated in the and were In neonatal the Epi-activated β2ARs similar of to activated by isoproterenol not internalization Y. Kobilka B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and sufficient recycling and In contrast, NE-activated β2ARs much slower internalization and recycling and which to an of the receptor We have previously that agonist-dependent β2AR internalization and recycling is for the receptor to coupling from Gs to Gi to the contraction rate Y. Kobilka B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We the potential in β2AR contraction rate response after the endogenous receptors were with Epi or NE in the β2ARs were activated by a of the contraction rate response an increase by a the rate the The response is because of coupling of the receptor to Gs and Gi Y. Kobilka B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Epi and NE induced a contraction rate and the contraction rate increase was by a and Moreover, contraction rate at 4 were much at 10 and These data suggest that both NE- and Epi-activated β2ARs couple to the to regulate contraction and couple to distinct G protein to regulate contraction rate in neonatal cardiomyocytes. 10 isoproterenol or NE-activated endogenous β2AR showed distinct contraction rate responses in the and treatment the but not contraction rate increase of the both Epi and NE effects on contraction rate of the and the contraction rate increase was by a specific and Epi and NE induced contraction rate responses by β2AR lacking the FLAG-tagged mouse was in the and with Epi or NE to induce contraction rate treatment not the contraction rate responses induced by Epi and NE The contraction response the of beating from different time were different between Epi and NE and between Epi and Epi by in of 10 both and contraction rate responses the induced by the The time of contraction rate induced by 10 NE or Epi were different This was not because of the activation of by NE or Epi because myocytes with these two drugs not on contraction not The of the of contraction rate in NE- or myocytes a role of the coupling in the contraction responses Y. Kobilka B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, only the not the contraction rate response was by inhibiting the Gi protein with a Gi and Therefore, only Epi-activated β2AR sufficient coupling to Gi proteins. This is consistent with the internalization and recycling of β2ARs Epi supporting the notion that agonist-dependent receptor is for the receptor coupling to Gi in cardiomyocytes. This notion is by an a β2AR that the of β2AR has been shown for receptor recycling after internalization Y. Kobilka B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the β2AR lacking this was in myocytes and activated by Epi or the receptor contraction rate responses were not to treatment and The agonist-dependent GPCR internalization and recycling is regulated by receptor phosphorylation by the GRK family and dephosphorylation by C. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We agonist-dependent phosphorylation on β2AR in cardiomyocytes. Epi and NE induced a phosphorylation of serine residues 355 and 356 of β2AR in 10 β2AR with Epi a rapid increase of phosphorylation on these serine residues that peaked at min by a min after drug A and In contrast, β2AR with 10 NE a much slower increase in phosphorylation of the serine residues that peaked at min and at min after drug treatment A and the that by NE for the slower phosphorylation of the we Epi to a to 10 NE in of to activate Gs for The cellular induced by Epi a increase a of which was to 10 NE in Epi induced a rapid receptor phosphorylation at Ser-355 and The time of the receptor phosphorylation induced by of Epi that induced by 10 of Epi and In inhibiting with okadaic acid dephosphorylation of Ser-355 and -356 on the activated β2ARs at min of Epi treatment but not the phosphorylation of these residues on the NE-activated receptors the NE-activated β2ARs myocytes were with for min being and NE-activated receptors dephosphorylation on Ser-355 and -356 after of drugs, which was but by with C and The failure of treatment to the receptor phosphorylation indicates that other may be involved in the β2AR dephosphorylation in cardiac Together, Epi and NE induced distinct temporal phosphorylation of Ser-355 and -356 on β2AR in cardiomyocytes. These data are consistent with the β2AR contraction rate responses under of NE and Epi and We the of these Ser-355 and -356 on receptor and signaling in by inhibiting receptor dephosphorylation with treatment the recycling of the Epi-activated β2ARs after which in an of the receptors A and In the myocytes by treatment contraction rate the contraction rate response whereas treatment the contraction rate increase induced by treatment the effects of on inhibiting Gi signaling C and of Gi protein with both contraction rate and the contraction rate min of with Epi. with the on the contraction rate were and the on contraction rate increase the of was suggesting a Gi Meanwhile, treatment only both the contraction rate increase and the contraction rate increase induced by NE and These data with the data from suggest that treatment the Epi-activated β2AR dephosphorylation and subsequent recycling after The receptors are at which in coupling to Gi protein in cardiomyocytes. These observations are consistent with the notion that the agonist-dependent receptor is for the β2AR coupling to Gi in cardiomyocytes. the effects of phosphorylation of Ser-355 and -356 on receptor and signaling in we used a to the agonist-dependent GRK2 phosphorylation W. H. R. R. C. Lefkowitz R. PubMed Scopus Google Scholar). both and GRK2 phosphorylation of β2AR in in a A and a by adenoviruses not or GRK2 phosphorylation of β2AR in C and at but not the the β2AR internalization and recycling, which in an of the receptors Meanwhile, the β2AR internalization in the not the contraction rate response induced by NE suggesting that the contraction rate response induced by NE is from β2AR internalization after In contrast, the contraction rate response induced by Epi also the effects of on the contraction rate increase These data suggest that inhibiting agonist-dependent phosphorylation of β2AR by GRK2 receptor and the Epi-activated receptor coupling to Gi in a β2AR and the receptor signaling contraction rate response in neonatal cardiomyocytes. and were in or GRK2 phosphorylation at serine 355 and 356 was by in an analysis of the of in A by as of infected or GRK2 phosphorylation at serine 355 and 356 was by but not the analysis of the of in C by as of infected β2AR internalization and recycling after Epi and of receptor in β2AR internalization after NE stimulation. the contraction rate increase by Epi-activated β2AR signaling in myocytes but the of on contraction rate In contrast, not the contraction rate increase by NE-activated β2AR signaling in myocytes The contraction response the of beating from different time were different between Epi and Epi by in In this we have shown that distinct β2AR phosphorylation by GRK2 plays a critical role in NE- and receptor signaling to regulate physiological cardiomyocyte contraction rate and cellular efficacies have been on a growing of GPCRs, including receptor M. 2004; PubMed Scopus Google Scholar), receptor Whalen E.J. Violin J.D. Premont R.T. H.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and M. S. G. M. G. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). These efficacies divergent signaling activated by the same GPCR under different drug Lefkowitz R.J. H.A. J. 2005; PubMed Scopus Google Scholar) as well as divergent cellular after receptor internalization M. Sci. 2003; PubMed Scopus Google Scholar). In the of and failed to activate the β2AR coupling to Gs protein but are capable of the protein kinase signaling in M. S. G. M. G. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). These studies suggest that diversified signaling can be activated by the same GPCR in physiological contexts. Meanwhile, different clinical from long term therapy of heart failure the that different β-blockers can induce distinct cellular effects in M. 1998; PubMed Scopus Google Scholar). Epi and NE are two endogenous ligands that activate the family in studies suggest that NE- and Epi-activated human β2AR can couple to distinct G protein signaling when in mouse heart U. Mol. Pharmacol. 2004; PubMed Scopus Google Scholar). these in cellular and mechanisms underlying the physiological of βAR signaling activated by NE and Epi Here we analyzed the signaling induced by Epi and NE in cardiac tissue by the effects on cardiomyocyte contraction rate response. We have shown for the first time that NE- and Epi-activated β2ARs induce distinct cellular signals in physiological cardiomyocyte contraction whereas both drugs induced β2AR coupling to Gs only Epi-activated receptors were capable of coupling to Gi because of sufficient recycling in cardiomyocytes. Subsequent studies showed that a rapid GRK2 phosphorylation and subsequent dephosphorylation of the Epi-activated β2AR were critical for sufficient receptor recycling and Gi coupling. These data suggest that NE and Epi induce different signaling and properties of β2AR in animal by Epi at different a rapid contraction rate increase the contraction rate underwent an the of receptor and coupling. In contrast, when activated by NE at different the receptor a much slower contraction rate increase that peaked 10 min after drug This increase is in because of a slow of the NE-activated receptor by GRK2 phosphorylation which in a coupling to Gs The in receptor signaling and GRK2 phosphorylation was not because of the in these two In fact, with a to 10 NE in of of Gs to increase also induced a rapid receptor phosphorylation an that is consistent with the receptor phosphorylation at Ser-355 and in cells J. E. Mol. Pharmacol. 2004; PubMed Scopus Google Scholar). In our the phosphorylation was on human β2AR in mouse different signaling and properties between human and mouse β2ARs (15Wang Y. Lauffer B. Von M. Kobilka B. Xiang Y. Mol. Pharmacol. 2007; PubMed Scopus Google Scholar, J. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), these two receptors recent studies on β2ARs in mouse that these two receptors are similar in Gs and Gi for contraction and agonist-dependent internalization and recycling (15Wang Y. Lauffer B. Von M. Kobilka B. Xiang Y. Mol. Pharmacol. 2007; PubMed Scopus Google Scholar). Here the between the endogenous mouse contraction and the human β2AR phosphorylation under the between these two We have previously that Epi and NE can induce distinct conformational changes on β2AR (10Swaminath G. Xiang Y. Lee T.W. Steenhuis J. Parnot C. Kobilka B.K. J. Biol. Chem. 2004; 279: 686-691Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). the in the GRK2 phosphorylation of β2AR may be to the Epi-activated receptors a GRK2 or as GRK2 the NE-activated it may be to the of cellular to the β2ARs that regulate the phosphorylation of the phosphorylation of a GPCR has been in receptor and subsequent internalization R.T. Rev. Physiol. 2007; PubMed Scopus Google Scholar). The receptors to a protein for The GPCRs from and dephosphorylation for recycling C. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). data the phosphorylation plays a role for the agonist-dependent and The dephosphorylation of the β2ARs activated by NE and Epi to be C and Thus, in a when the β2ARs are activated by the rapid GRK2 phosphorylation to Gs coupling and which is by sufficient dephosphorylation recycling and Gi coupling in β2AR dephosphorylation by okadaic acid the receptor coupling to Gi. the phosphorylation of β2AR with the receptor internalization and subsequent recycling, which the contraction rate and the coupling for contraction rate These data are consistent with recent that GRK2 and regulate protein coupling J. Ahn S. Whalen E.J. E. H. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, E. Ahn S. J. W. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar), but not the role of and in β2AR cellular signaling and in cardiomyocytes. In fact, the receptor phosphorylation by other may to the internalization of Epi-activated β2ARs after GRK2 is by In contrast, the NE-activated β2ARs a slow but GRK2 phosphorylation, which may to a Gs slow receptor and Gi coupling. is not that of GRK2 phosphorylation by has a on the contraction rate response by NE These data also that the contraction response is not on the slower receptor phosphorylation by GRK2 and subsequent the NE-activated β2ARs are of and to sufficient and Gi our data suggest that the β2AR phosphorylation by GRK2 may play a role in β2AR These not the that other cellular are involved in the GRK phosphorylation of β2AR under NE which may regulate receptor signaling for contraction rate on the recycling GPCRs can be into two rapid and slow recycling receptors R.J. Shenoy S.K. 2005; PubMed Scopus Google Scholar). The slow recycling receptors with in which are for signaling a of R.J. Shenoy S.K. 2005; PubMed Scopus Google Scholar). Therefore, it will be to the NE-activated β2ARs with to regulate signaling in cardiomyocytes. Meanwhile, it to be the after by are targeted to for The slow recycling of the NE-activated βAR also a of receptor at of sympathetic synapses in animal such as heart failure and are by of βAR including and of receptors (8Johnson M. Am. J. Respir. Crit. Care Med. 1998; 158: S146-S153Crossref PubMed Scopus (348) Google Scholar, Lee PubMed Scopus Google Scholar, N. J. Med. 1996; PubMed Scopus Google Scholar, R.J. H.A. Koch W.J. Circulation. PubMed Scopus Google Scholar), and much evidence to GRK as a R.J. H.A. Koch W.J. Circulation. PubMed Scopus Google Scholar, G. Koch W.J. Exp. PubMed Scopus Google Scholar, Koch W.J. H.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.L. Koch W.J. J. Mol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Am. J. Respir. Cell Mol. Biol. 1998; PubMed Scopus Google Scholar). 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Moreover, the may the different roles of GRK2 phosphorylation of βARs in physiological settings. studies along this as well as studies with myocytes will on the effects of GRK regulation of βAR signaling in physiological and In we revealed the critical role of GRK2 phosphorylation underlying the distinct cellular events and signaling properties of β2AR induced by Epi or NE in cardiomyocytes. This the to the physiological of these two endogenous ligands to These data not only help us understand the physiological and of βAR activation by Epi and NE in but also the utility of the of βAR and GRK in the treatment of a of in both cardiovascular and pulmonary We of the Xiang for critical and for and Koch for the We Kobilka for the of this with