β-Arrestin-biased Agonism at the β2-Adrenergic Receptor

Classically, the β2-adrenergic receptor (β2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including β2AR, can signal via β-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among β2AR agonists there exist ligands that disproportionately signal via G proteins or β-arrestins and are hence “biased.” Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known β2AR agonists exhibit relative efficacies for β-arrestin-associated activities (β-arrestin membrane translocation and β2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three βAR ligands there is a marked bias toward β-arrestin signaling; these ligands stimulate β-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the α-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for β-arrestin-mediated signaling. Classically, the β2-adrenergic receptor (β2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including β2AR, can signal via β-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among β2AR agonists there exist ligands that disproportionately signal via G proteins or β-arrestins and are hence “biased.” Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known β2AR agonists exhibit relative efficacies for β-arrestin-associated activities (β-arrestin membrane translocation and β2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three βAR ligands there is a marked bias toward β-arrestin signaling; these ligands stimulate β-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the α-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for β-arrestin-mediated signaling. 7TMRs, 5The abbreviations used are:7TMRseven-transmembrane receptorβ2ARβ2-adrenergic receptorAT1aRangiotensin II receptor type 1aCFPcyan fluorescent proteinmCFPmonomeric CFPERKextracellular signal-regulated kinasepERKphosphorylated ERKFRETfluorescence resonance energy transferGRKG protein-coupled receptor kinaseMAPKmitogen-activated protein kinaseCPBN-cyclopentylbutanephrinesiRNAsmall interfering RNAYFPyellow fluorescent proteinmYFPmonomeric YFPIsoisoproterenol. also known as G protein-coupled receptors, are the largest family of proteins involved in the transduction of signals from the extracellular milieu to intracellular effectors (1McCudden C.R. Hains M.D. Kimple R.J. Siderovski D.P. Willard F.S. CMLS Cell. Mol. Life Sci. 2005; 62: 551-577Crossref PubMed Scopus (359) Google Scholar) and account for nearly 2% of all human genes (2Vassilatis D.K. Hohmann J.G. Zeng H. Li F. Ranchalis J.E. Mortrud M.T. Brown A. Rodriguez S.S. Weller J.R. Wright A.C. Bergmann J.E. Gaitanaris G.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4903-4908Crossref PubMed Scopus (604) Google Scholar). Clinically, 7TMR ligands are the single largest class of pharmacologic agents (3Wise A. Gearing K. Rees S. Drug Discov. Today. 2002; 7: 235-246Crossref PubMed Scopus (332) Google Scholar). According to a well established and evolutionarily conserved paradigm, 7TMRs signal through direct activation of heterotrimeric G proteins to promote the activation or inhibition of second messenger-generating enzymes and changes in second messenger-dependent effector activities (4Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4728) Google Scholar). Thus, for example, agonist stimulation of β2AR promotes Gs-mediated activation of adenylyl cyclase, with subsequent cAMP production and cAMP-associated signaling events. Signal termination and 7TMR desensitization result from recruitment of β-arrestin proteins to the cytoplasmic surface of 7TMR following agonist-stimulated receptor phosphorylation by the G protein-coupled receptor kinase (GRK) family of proteins (5Lefkowitz R.J. Rajagopal K. Whalen E.J. Mol. Cell. 2006; 24: 643-652Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 6Lefkowitz R.J. Whalen E.J. Curr. Opin. Cell Biol. 2004; 16: 162-168Crossref PubMed Scopus (245) Google Scholar). seven-transmembrane receptor β2-adrenergic receptor angiotensin II receptor type 1a cyan fluorescent protein monomeric CFP extracellular signal-regulated kinase phosphorylated ERK fluorescence resonance energy transfer G protein-coupled receptor kinase mitogen-activated protein kinase N-cyclopentylbutanephrine small interfering RNA yellow fluorescent protein monomeric YFP isoproterenol. When originally identified, the β-arrestin proteins were believed to play a role only in limiting 7TMR signaling by physically interceding between the receptor and G protein. Recent work has demonstrated, however, that for a variety of 7TMRs, β-arrestin proteins can mediate G protein-independent 7TMR signaling by selectively scaffolding signaling cascade components, including small GTP-binding proteins and members of the MAPK cascade (5Lefkowitz R.J. Rajagopal K. Whalen E.J. Mol. Cell. 2006; 24: 643-652Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 7Lefkowitz R.J. Shenoy S.K. Science. 2005; 308: 512-517Crossref PubMed Scopus (1439) Google Scholar). These findings have had a profound impact on our understanding of 7TMR ligand pharmacology. Historically, 7TMR ligands have been classified according to their ability to promote receptor activation (agonists) or inhibit agonist-mediated receptor activation (antagonists). This binary categorization underestimates, however, the true complexity of 7TMR ligand behavior. The identification of inverse agonists, which inhibit ligand-independent (i.e. constitutive) activity of 7TMRs for G protein activation (8Bond R.A. Leff P. Johnson T.D. Milano C.A. Rockman H.A. McMinn T.R. Apparsundaram S. Hyek M.F. Kenakin T.P. Allen L.F. Lefkowitz R.J. Nature. 1995; 374: 272-276Crossref PubMed Scopus (410) Google Scholar), broadened the spectrum of 7TMR ligands to at least three fundamentally distinct classes. In this pharmacologic paradigm, 7TMR ligands were thought to exhibit “correlated efficacies,” stimulating or inhibiting all functions of a receptor to the same extent. Consistent with this were studies performed 2 decades ago in which purified β2AR was reconstituted in a phospholipid vesicle system either with GRK2 or with both Gs and adenylyl cyclase (9Benovic J.L. Staniszewski C. Mayor Jr., F. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1988; 263: 3893-3897Abstract Full Text PDF PubMed Google Scholar). Analysis of the intrinsic abilities of a series of βAR ligands defined as partial agonists for G protein activation demonstrated a near-perfect correlation (coefficient of 0.996) in the ability of each ligand to stimulate G protein-mediated (adenylyl cyclase activation) or β-arrestin-associated (βAR phosphorylation) activities (9Benovic J.L. Staniszewski C. Mayor Jr., F. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1988; 263: 3893-3897Abstract Full Text PDF PubMed Google Scholar). More recent results, however, suggest that some β2AR antagonists are actually agonists for some signaling pathways, thus, for example, stimulating MAPK activation while blocking G protein (10Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Pineyro G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (429) Google Scholar, 11Baker J.G. Hall I.P. Hill S.J. Mol. Pharmacol. 2003; 64: 1357-1369Crossref PubMed Scopus (158) Google Scholar, 12Galandrin S. Bouvier M. Mol. Pharmacol. 2006; 70: 1575-1584Crossref PubMed Scopus (246) Google Scholar). We refer to such selective signal activation as “ligand bias” (13Violin J.D. Lefkowitz R.J. Trends Pharmacol. Sci. 2007; 28: 416-422Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar), but the same phenomenon has also been described as “ligand-directed trafficking” (14Berg K.A. Maayani S. Goldfarb J. Scaramellini C. Leff P. Clarke W.P. Mol. Pharmacol. 1998; 54: 94-104Crossref PubMed Scopus (449) Google Scholar), “protean agonism” (15Kenakin T. Trends Pharmacol. Sci. 1995; 16: 256-258Abstract Full Text PDF PubMed Scopus (100) Google Scholar), “pleuridimensional efficacy” (12Galandrin S. Bouvier M. Mol. Pharmacol. 2006; 70: 1575-1584Crossref PubMed Scopus (246) Google Scholar), and “collateral efficacy” (16Kenakin T. Nat. Rev. Drug Discov. 2005; 4: 919-927Crossref PubMed Scopus (279) Google Scholar). Recent evidence demonstrates that biased ligands can selectively activate β-arrestin signaling without activating G protein signaling. For example, angiotensin II-induced signaling via angiotensin II receptor type 1a (AT1aR) leads to Gαq/11-mediated activation of phospholipase C- and β-arrestin-dependent functions, whereas AT1aR activation by a peptide analogue of angiotensin II (Sar1,Ile4,Ile4angiotensin II, denoted SII) leads to β-arrestin-dependent MAPK activation alone (17Holloway A.C. Qian H. Pipolo L. Ziogas J. Miura S. Karnik S. Southwell B.R. Lew M.J. Thomas W.G. Mol. Pharmacol. 2002; 61: 768-777Crossref PubMed Scopus (203) Google Scholar, 18Kim J. Ahn S. Whalen E.J. H. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, K. Whalen E.J. J.D. Rockman H.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar, H. Ahn S. Shenoy S.K. Karnik S.S. L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar). The of this protein-independent signaling has recently been demonstrated in with of a AT1aR of activating only G protein-independent pathways in response to angiotensin II P. M. J. J. M. J. K. S. T. A. J. J. 2005; PubMed Scopus Google Scholar), as well as in from with K. Whalen E.J. J.D. Rockman H.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). studies of β2AR that a β2AR of signaling via Gs activation of the MAPK cascade via β-arrestins S.K. M.T. K. S. Lefkowitz R.J. J. 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Rev. 2007; PubMed Scopus Google Scholar). the to 7TMR ligand and is on G protein-dependent activities such as second we to a that would to the ability of a ligand to G protein-dependent activities from distinct we to the 7TMR β2AR as a system both we have that a β2AR was of G signaling and a of well ligands that have been demonstrated to G protein-dependent activities at β2AR are Using a of we a series of ligands in of agonists in of for β-arrestin ligand bias at and were from was by was by the and to be to that by for all tested. was from were used at as as and and as well as these are described J.D. Lefkowitz R.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). the cAMP of cAMP was from and is described J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar, J.D. J. Lefkowitz R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was a of of J.D. A.C. Science. 2002; PubMed Scopus Google Scholar). Cell were in and For was used at for and for and was used at for and for were as described with β2AR surface by of of protein J.D. Lefkowitz R.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). all were to were with in and in the on a to were on a with a by CFP and were through a at a and for CFP and for YFP was from a a and a were from and were in were between and to signal and AMP protein efficacy was by of the second cAMP was the J.D. J. Lefkowitz R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), an of J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). is an protein by to both CFP and cAMP to changes that a in G protein efficacy was as the in the YFP the following ligand This both the and of G protein efficacy was as the of β-arrestin recruitment to We this by of the by between and (13Violin J.D. Lefkowitz R.J. Trends Pharmacol. Sci. 2007; 28: 416-422Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). was by and a a for and J.D. A.C. Science. 2002; PubMed Scopus Google Scholar). In the these proteins in the their is to result in in the of This can to by M. J. Cell Biol. 1998; PubMed Scopus Google Scholar), but is used to a of membrane When from the surface this of leads to a in in to the of that is Thus, this assay a of receptor and can in be to the of protein that to or from the RNA of with RNA and were from in a and The human was to relative to the S. T.R. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar). RNA was as the as the S. T.R. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar). For that were on were with of the the were and or for with were the of was in or were for in to stimulation with the ligand for were by by at for For each an of was for protein of were on or β-arrestin and to membrane for according to and were by with MAPK Cell and protein were with as described H. J.L. C. J. Caron M.G. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was performed with and was by a according to the was used for the recent that 7TMRs are of G we a of described and partial agonists of G protein activity at β2AR for activities as the for our β2AR the ability of each to cAMP production via the β2AR was in an of the cAMP J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). This highly sensitive for the comparison of both the relative of cAMP in response to ligand stimulation and the of the at which cAMP production in the βAR agonist in a but cAMP as a series of βAR partial The response was by of with the βAR or and a for of with an to findings J.D. J. Lefkowitz R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The was the same of the response was as the the or the response the response both the and of cAMP we that as the of G protein The in β-arrestin-dependent signaling is thought to be 7TMR phosphorylation by by β-arrestin translocation from the to the phosphorylated 7TMR (5Lefkowitz R.J. Rajagopal K. Whalen E.J. Mol. Cell. 2006; 24: 643-652Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). We used a of as an of β-arrestin-dependent signaling. We have that the of β-arrestin and the of β-arrestin is sensitive to changes in J.D. Lefkowitz R.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google thus, in efficacy as changes in the of β-arrestin we used the of β-arrestin recruitment to with of and were with the same of βAR ligands in the described for in these ligands demonstrated a of we that a marked β-arrestin efficacy only G protein We this bias by a of βAR including a number that are used The of each ligand at a than a of efficacies for G protein activation and β-arrestin in a correlation that ligands were for G protein-dependent and β-arrestin-dependent ligands were for β-arrestin translocation to β2AR than for cAMP G protein efficacy nearly identical to that of the partial agonist but β-arrestin efficacy greater than that of the agonist isoproterenol. β-arrestin recruitment than relative to the G protein agonist which demonstrate bias in ability to promote cAMP production or β-arrestin was only as for G protein but for β-arrestin ligands in were also to have β-arrestin These were and of and of these activities was demonstrated by blocking all with the and by the of of blocking with the We also the bias among the ligands by a the of β-arrestin efficacy to G protein efficacy This to which ligands biased and are of for a we a of the of bias and exhibit only bias a bias than this Thus, we that and are ligands and as such in with unbiased translocation to 7TMRs in response to ligand stimulation receptor phosphorylation at cytoplasmic and by the family of R.J. Whalen E.J. Curr. Opin. Cell Biol. 2004; 16: 162-168Crossref PubMed Scopus (245) Google Scholar). of efficacy for functions, we the of phosphorylation of β2AR S.K. M.T. K. S. Lefkowitz R.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) were at from to and were for receptor phosphorylation with an for β2AR phosphorylated on and to phosphorylation by J. F. Mol. Pharmacol. 2004; PubMed Scopus Google Scholar, E.J. A. K. J.D. M. J.R. Rockman H.A. Lefkowitz R.J. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). The of β2AR phosphorylation that by whereas ligands with partial agonist activity for both G protein and β-arrestin activities and much than that by The of phosphorylation of β2AR are in These show with on β-arrestin is a agonist for receptor phosphorylation by partial for G protein activity. leads to both β-arrestin recruitment and β2AR phosphorylation we that also promote β2AR an or of β2AR would be a for this we a receptor assay in which was in with YFP by the of both and J.D. A.C. Science. 2002; PubMed Scopus Google Scholar). these YFP is nearly to the with from the of to a in the of J.D. A.C. Science. 2002; PubMed Scopus Google Scholar). This is by of the but the agonist stimulation This to a of agonist with a the in with a well established for the inhibition of S.S. Lefkowitz R.J. W.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar), β2AR in response to ligands demonstrated to be for β-arrestin recruitment such as and in both and β2AR than the and with efficacy for β-arrestin recruitment stimulation with in β2AR and the extent to which β2AR the ability of a ligand to promote either G or β-arrestin-dependent we the of β2AR for ligands from with cAMP and with the of β-arrestin recruitment in the correlation of the β2AR and β-arrestin recruitment for a of a of ligand with a single receptor J. F. Mol. Pharmacol. 2004; PubMed Scopus Google Scholar). This that ligand and receptor and that efficacy from the of to the of efficacy in a efficacy However, correlation of the β2AR and cAMP response is with this the biased ligand is disproportionately for β2AR relative to efficacy for cAMP This is with a single of the receptor and that distinct receptor can be by potential our of ligand bias is that we used both and to ligand Signal and signal are to we a single to that is biased toward In our studies of we both G protein-dependent and β-arrestin-dependent to MAPK activation S.K. M.T. K. S. Lefkowitz R.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, we used activation of the MAPK to the β-arrestin bias of were with and in the of either or We that in the of was by whereas for stimulation was by these demonstrate that and of their of G β-arrestin-dependent signaling pathways to activate ERK (i.e. there is a greater of β-arrestin signaling to the ERK these the β-arrestin bias of relative to and demonstrate that the bias leads to in a signaling kinase these the of signal transduction in this the of β-arrestin recruitment and the cAMP to β-arrestin bias among a of the ability of 7TMRs to through G protein activation in response to ligand has been an of evidence demonstrates that 7TMRs can also G protein-dependent or β-arrestin-dependent The is with these recent J. Ahn S. Whalen E.J. H. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, H. Ahn S. Shenoy S.K. Karnik S.S. L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar, S.K. M.T. K. S. Lefkowitz R.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, Ahn S. J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, M. Ahn S. S. Lefkowitz R.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, S. T.R. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar) and a that can be for the identification of 7TMR ligands that promote signaling. by Kenakin (16Kenakin T. Nat. Rev. Drug Discov. 2005; 4: 919-927Crossref PubMed Scopus (279) Google Scholar), all 7TMRs are of a of distinct each of which can to the activation of distinct intracellular signaling The of these distinct in be by the of such that distinct receptor are or in response to a of a of by their ability to stimulate G protein-dependent activities at β2AR, is in with the that biased 7TMR signaling can be by Thus, the majority of ligands demonstrated efficacies for G and β-arrestin-associated at least three ligands a bias for β-arrestin-associated activity. studies of purified to an of ligands also this S. S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Pharmacol. 2007; PubMed Scopus Google Scholar). In recent studies the of a variety of βAR ligands to activate adenylyl cyclase and MAPK signaling pathways at β2AR activation the extent to which MAPK signaling was independent of G protein activation and the of this response to β-arrestin were (12Galandrin S. Bouvier M. Mol. Pharmacol. 2006; 70: 1575-1584Crossref PubMed Scopus (246) Google Scholar). to these findings is that to the true efficacy of a ligand for a the of effector signaling pathways in response to the 7TMR ligands have been described according to their relative abilities to G protein-dependent pathways (i.e. as agonists, partial agonists, or inverse However, our as well as the work of other suggest that the efficacy of each ligand for G protein-mediated as well as protein-mediated pathways also be It is that have been described among members of a 7TMR ligand class to these protein but this an and we ligands that demonstrated a bias toward β-arrestin-dependent we ligands that a G protein bias relative to β-arrestin activity. However, there is to the of ligands that promote 7TMR with a selective bias toward G protein activation. studies only ligands demonstrated to have βAR activity (i.e. of a of ligands in some with a G protein bias relative to It is to however, that in a recent in which a of βAR ligands was used to biased signaling at both the and were that adenylyl cyclase activity relative to MAPK activation (12Galandrin S. Bouvier M. Mol. Pharmacol. 2006; 70: 1575-1584Crossref PubMed Scopus (246) Google Scholar), a result that is with our of the findings by our is that the three ligands a absent from all of the ligands and all an ethyl on the is identical to for the of the ethyl whereas is identical to for the of an ethyl in the of an ethyl to in a in G protein with in the β-arrestin translocation of an ethyl to the of the of β-arrestin recruitment while on adenylyl cyclase activity. Thus, the of the same motif on ligands in β-arrestin bias at β2AR relative to the the for this bias is the of in β2AR to be the of ligands for β2AR, of the ligands in understanding the for highly biased ligands for this all 7TMR ligands currently used as agents in have been their ability to 7TMR G protein For example, βAR antagonists currently used in for the of and and following were their ability to βAR activation of G It is to however, that βAR antagonists with a ability to inhibit βAR G protein but with the to stimulate β-arrestin-dependent signaling pathways, have to for recent from our demonstrate that a βAR with efficacy in the of to be among in in the ability to promote β-arrestin-mediated signaling while G protein activity Whalen E.J. J.D. M.T. Shenoy S.K. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). 7TMR ligands that promote biased signaling as demonstrated for have efficacy for the and of other human is at The potential for ligands to have that from ligands without bias is however, by studies of the angiotensin receptor This angiotensin peptide promotes and in K. Whalen E.J. J.D. Rockman H.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar) in the of G protein signaling. other such as the analogue M. Ahn S. S. Lefkowitz R.J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) or of the three ligands have in to be be for nearly 7TMR that is a of The recent that 7TMRs are of G signaling has in our understanding of 7TMR signal transduction Thus, is that with the of for the of both and G protein-dependent we were to ligands that activities at our of 7TMR ligands was on the efficacy of the ligand for of G protein our for the complexity of 7TMR signaling that ligand the of other signaling or effector in response to